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• 1.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Extending the Reach of Computational Approaches to Model Enzyme Catalysis2017Doctoral thesis, comprehensive summary (Other academic)

Recent years have seen tremendous developments in methods for computational modeling of (bio-) molecular systems. Ever larger reactive systems are being studied with high accuracy approaches, and high-level QM/MM calculations are being routinely performed. However, applying high-accuracy methods to large biological systems is computationally expensive and becomes problematic when conformational sampling is needed. To address this challenge, classical force field based approaches such as free energy perturbation (FEP) and empirical valence bond calculations (EVB) have been employed in this work. Specifically:

1. Force-field independent metal parameters have been developed for a range of alkaline earth and transition metal ions, which successfully reproduce experimental solvation free energies, metal-oxygen distances, and coordination numbers. These are valuable for the computational study of biological systems.

2. Experimental studies have shown that the epoxide hydrolase from Solanum tuberosum (StEH1) is not only an enantioselective enzyme, but for smaller substrates, displays enantioconvergent behavior. For StEH1, two detailed studies, involving combined experimental and computational efforts have been performed: We first used trans-stilbene oxide to establish the basic reaction mechanism of this enzyme. Importantly, a highly conserved and earlier ignored histidine was identified to be important for catalysis. Following from this, EVB and experiment have been used to investigate the enantioconvergence of the StEH1-catalyzed hydrolysis of styrene oxide. This combined approach involved wildtype StEH1 and an engineered enzyme variant, and established a molecular understanding of enantioconvergent behavior of StEH1.

3. A novel framework was developed for the Computer-Aided Directed Evolution of Enzymes (CADEE), in order to be able to quickly prepare, simulate, and analyze hundreds of enzyme variants. CADEE’s easy applicability is demonstrated in the form of an educational example.

In conclusion, classical approaches are a computationally economical means to achieve extensive conformational sampling. Using the EVB approach has enabled me to obtain a molecular understanding of complex enzymatic systems. I have also increased the reach of the EVB approach, through the implementation of CADEE, which enables efficient and highly parallel in silico testing of hundreds-to-thousands of individual enzyme variants.

1. Force Field Independent Metal Parameters Using a Nonbonded Dummy Model
Open this publication in new window or tab >>Force Field Independent Metal Parameters Using a Nonbonded Dummy Model
2014 (English)In: Journal of Physical Chemistry B, ISSN 1520-6106, E-ISSN 1520-5207, Vol. 118, no 16, p. 4351-4362Article in journal (Refereed) Published
##### Abstract [en]

The cationic dummy atom approach provides a powerful nonbonded description for a range of alkaline-earth and transition-metal centers, capturing both structural and electrostatic effects. In this work we refine existing literature parameters for octahedrally coordinated Mn2+, Zn2+, Mg2+, and Ca2+, as well as providing new parameters for Ni2+, Co2+, and Fe2+. In all the cases, we are able to reproduce both M2+-O distances and experimental solvation free energies, which has not been achieved to date for transition metals using any other model. The parameters have also been tested using two different water models and show consistent performance. Therefore, our parameters are easily transferable to any force field that describes nonbonded interactions using Coulomb and Lennard-Jones potentials. Finally, we demonstrate the stability of our parameters in both the human and Escherichia coli variants of the enzyme glyoxalase 1 as showcase systems, as both enzymes are active with a range of transition metals. The parameters presented in this work provide a valuable resource for the molecular simulation community, as they extend the range of metal ions that can be studied using classical approaches, while also providing a starting point for subsequent parametrization of new metal centers.

##### National Category
Physical Chemistry
##### Identifiers
urn:nbn:se:uu:diva-225523 (URN)10.1021/jp501737x (DOI)000335113600010 ()
##### Funder
Swedish National Infrastructure for Computing (SNIC), 2013/26-1 Available from: 2014-06-23 Created: 2014-06-04 Last updated: 2018-12-03Bibliographically approved
2. Expanding the catalytic triad in epoxide hydrolases and related enzymes
Open this publication in new window or tab >>Expanding the catalytic triad in epoxide hydrolases and related enzymes
2015 (English)In: ACS Catalysis, ISSN 2155-5435, E-ISSN 2155-5435, Vol. 5, no 10, p. 5702-5713Article in journal (Refereed) Published
##### Abstract [en]

Potato epoxide hydrolase 1 exhibits rich enantio- and regioselectivity in the hydrolysis of a broadrange of substrates. The enzyme can be engineered to increase the yield of optically pureproducts, as a result of changes in both enantio- and regioselectivity. It is thus highly attractive inbiocatalysis, particularly for the generation of enantiopure fine chemicals and pharmaceuticals.The present work aims to establish the principles underlying the activity and selectivity of theenzyme through a combined computational, structural, and kinetic study, using the substratetrans-stilbene oxide as a model system. Extensive empirical valence bond simulations have beenperformed on the wild-type enzyme together with several experimentally characterized mutants.We are able to computationally reproduce the differences in activities between differentstereoisomers of the substrate, and the effects of mutations in several active-site residues. Inaddition, our results indicate the involvement of a previously neglected residue, H104, which iselectrostatically linked to the general base, H300. We find that this residue, which is highlyconserved in epoxide hydrolases and related hydrolytic enzymes, needs to be in its protonatedform in order to provide charge balance in an otherwise negatively-charged active site. Our datashow that unless the active-site charge balance is correctly treated in simulations, it is notpossible to generate a physically meaningful model for the enzyme that can accurately reproduceactivity and selectivity trends. We also expand our understanding of other catalytic residues,demonstrating in particular the role of a non-canonical residue, E35, as a “backup-base” in theabsence of H300. Our results provide a detailed view of the main factors driving catalysis andregioselectivity in this enzyme, and identify targets for subsequent enzyme design efforts.

##### National Category
Biochemistry and Molecular Biology
Biochemistry
##### Identifiers
urn:nbn:se:uu:diva-260232 (URN)10.1021/acscatal.5b01639 (DOI)000362391500006 ()
##### Funder
EU, FP7, Seventh Framework Programme, 306474Swedish Research Council, 621-2011-6055, 621-2010-5145Swedish National Infrastructure for Computing (SNIC), 2015/16-12 Available from: 2015-08-18 Created: 2015-08-18 Last updated: 2017-12-04Bibliographically approved
3. Conformational Diversity and Enantioconvergence in Potato Epoxide Hydrolase 1
Open this publication in new window or tab >>Conformational Diversity and Enantioconvergence in Potato Epoxide Hydrolase 1
2016 (English)In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 14, no 24, p. 5639-5651Article in journal (Refereed) Published
##### Abstract [en]

Potato epoxide hydrolase 1 (StEH1) is a biocatalytically important enzyme that exhibits rich enantio-and regioselectivity in the hydrolysis of chiral epoxide substrates. In particular, StEH1 has been demonstrated to enantioconvergently hydrolyze racemic mixes of styrene oxide (SO) to yield (R)-1-phenylethanediol. This work combines computational, crystallographic and biochemical analyses to understand both the origins of the enantioconvergent behavior of the wild-type enzyme, as well as shifts in activities and substrate binding preferences in an engineered StEH1 variant, R-C1B1, which contains four active site substitutions (W106L, L109Y, V141K and I155V). Our calculations are able to reproduce both the enantio-and regioselectivities of StEH1, and demonstrate a clear link between different substrate binding modes and the corresponding selectivity, with the preferred binding modes being shifted between the wild-type enzyme and the R-C1B1 variant. Additionally, we demonstrate that the observed changes in selectivity and the corresponding enantioconvergent behavior are due to a combination of steric and electrostatic effects that modulate both the accessibility of the different carbon atoms to the nucleophilic side chain of D105, as well as the interactions between the substrate and protein amino acid side chains and active site water molecules. Being able to computationally predict such subtle effects for different substrate enantiomers, as well as to understand their origin and how they are affected by mutations, is an important advance towards the computational design of improved biocatalysts for enantioselective synthesis.

##### National Category
Biochemistry and Molecular Biology
##### Identifiers
urn:nbn:se:uu:diva-282015 (URN)10.1039/C6OB00060F (DOI)000378933400042 ()27049844 (PubMedID)
##### Funder
Swedish National Infrastructure for Computing (SNIC), 25/2-10EU, European Research Council, 306474;283570Swedish Research Council, 621-2011-6055Carl Tryggers foundation , CTS13:104 Available from: 2016-04-01 Created: 2016-04-01 Last updated: 2017-11-30Bibliographically approved
4. CADEE: Computer-Aided Directed Evolution of Enzymes
Open this publication in new window or tab >>CADEE: Computer-Aided Directed Evolution of Enzymes
2017 (English)In: IUCrJ, ISSN 0972-6918, E-ISSN 2052-2525, Vol. 4, no 1, p. 50-64Article in journal (Refereed) Published
##### Abstract [en]

The tremendous interest in enzymes as biocatalysts has led to extensive work in enzyme engineering, as well as associated methodology development. Here, a new framework for computer-aided directed evolution of enzymes (CADEE) is presented which allows a drastic reduction in the time necessary to prepare and analyze in silico semi-automated directed evolution of enzymes. A pedagogical example of the application of CADEE to a real biological system is also presented in order to illustrate the CADEE workflow.

##### Keywords
computational directed evolution, computational enzyme design, distributed computing, empirical valence bond, triosephosphate isomerase
##### National Category
Structural Biology Bioinformatics (Computational Biology) Theoretical Chemistry
##### Identifiers
urn:nbn:se:uu:diva-314218 (URN)10.1107/S2052252516018017 (DOI)000392925800007 ()
##### Funder
EU, FP7, Seventh Framework Programme, 306474Knut and Alice Wallenberg FoundationThe Royal Swedish Academy of SciencesSwedish Research Council, 2015-04928Swedish National Infrastructure for Computing (SNIC), 2015/16-12 Available from: 2017-01-31 Created: 2017-01-31 Last updated: 2018-01-13Bibliographically approved
• 2.
Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
CADEE: Computer-Aided Directed Evolution of Enzymes2017In: IUCrJ, ISSN 0972-6918, E-ISSN 2052-2525, Vol. 4, no 1, p. 50-64Article in journal (Refereed)

The tremendous interest in enzymes as biocatalysts has led to extensive work in enzyme engineering, as well as associated methodology development. Here, a new framework for computer-aided directed evolution of enzymes (CADEE) is presented which allows a drastic reduction in the time necessary to prepare and analyze in silico semi-automated directed evolution of enzymes. A pedagogical example of the application of CADEE to a real biological system is also presented in order to illustrate the CADEE workflow.

• 3. Andersen, Birgit
Karolinska Institutet.
A recruited protease is involved in catabolism of pyrimidines2008In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 379, no 2, p. 243-250Article in journal (Refereed)

In nature, the same biochemical reaction can be catalyzed by enzymes having fundamentally different folds, reaction mechanisms and origins. For example, the third step of the reductive catabolism of pyrimidines, the conversion of N-carbamyl-beta-alanine to beta-alanine, is catalyzed by two beta-alanine synthase (beta ASase, EC 3.5.1.6) subfamilies. We show that the "prototype" eukaryote beta ASases, such as those from Drosophila melanogaster and Arabidopsis thaliana, are relatively efficient in the conversion of N-carbamyl-beta A compared with a representative of fungal beta ASases, the yeast Saccharomyces kluyveri beta ASase, which has a high K(m) value (71 mM). S. kluyveri beta ASase is specifically inhibited by dipeptides and tripeptides, and the apparent K(i) value of glycyl-glycine is in the same range as the substrate K(m). We show that this inhibitor binds to the enzyme active center in a similar way as the substrate. The observed structural similarities and inhibition behavior, as well as the phylogenetic relationship, suggest that the ancestor of the fungal beta ASase was a protease that had modified its profession and become involved in the metabolism of nucleic acid precursors.

• 4.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Computational Analysis of Molecular Recognition Involving the Ribosome and a Voltage Gated K+ Channel2009Doctoral thesis, comprehensive summary (Other academic)

Over the last few decades, computer simulation techniques have been established as an essential tool for understanding biochemical processes. This thesis deals mainly with the application of free energy calculations to ribosomal complexes and a cardiac ion channel.

The linear interaction energy (LIE) method is used to explore the energetic properties of the essential process of codon–anticodon recognition on the ribosome. The calculations show the structural and energetic consequences and effects of first, second, and third position mismatches in the ribosomal decoding center.

Recognition of stop codons by ribosomal termination complexes is fundamentally different from sense codon recognition. Free energy perturbation simulations are used to study the detailed energetics of stop codon recognition by the bacterial ribosomal release factors RF1 and RF2. The calculations explain the vastly different responses to third codon position A to G substitutions by RF1 and RF2. Also, previously unknown highly specific water interactions are identified.

The GGQ loop of ribosomal RFs is essential for its hydrolytic activity and contains a universally methylated glutamine residue. The structural effect of this methylation is investigated. The results strongly suggest that the methylation has no effect on the intrinsic conformation of the GGQ loop, and, thus, that its sole purpose is to enhance interactions in the ribosomal termination complex.

A first microscopic, atomic level, analysis of blocker binding to the pharmaceutically interesting potassium ion channel Kv1.5 is presented. A previously unknown uniform binding mode is identified, and experimental binding data is accurately reproduced. Furthermore, problems associated with pharmacophore models based on minimized gas phase ligand conformations are highlighted.

Generalized Born and Poisson–Boltzmann continuum models are incorporated into the LIE method to enable implicit treatment of solvent, in an effort to improve speed and convergence. The methods are evaluated and validated using a set of plasmepsin II inhibitors.

1.
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2. Energetics of stop codon recognition on the ribosome
Open this publication in new window or tab >>Energetics of stop codon recognition on the ribosome
##### Identifiers
urn:nbn:se:uu:diva-101412 (URN)
Available from: 2009-04-26 Created: 2009-04-26 Last updated: 2010-01-14
3. Does glutamine methylation affect the intrinsic conformation of the universally conserved GGQ motif in ribosomal release factors?
Open this publication in new window or tab >>Does glutamine methylation affect the intrinsic conformation of the universally conserved GGQ motif in ribosomal release factors?
2009 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, no 15, p. 3483-3489Article in journal (Refereed) Published
##### Abstract [en]

The GGQ motif is the only universally conserved feature of ribosomal class 1 release factors. Mutational experiments and structural studies have suggested that the glutamine residue of the GGQ motif Q 185 in human eRF1 numbering) is critical for catalysis of the termination   reaction on the ribosome. Furthermore, it has been established that Q185 is NE methylated in prokaryotes as well as eukaryotes, and that methylation significantly enhances the catalytic activity. It is, however, not known whether this methylation affects the intrinsic   structure of the free release factor, which could be important for its interaction with the ribosome. In this work, we report molecular dynamics simulations, starting from 25 different NMR structures of human eRF1, in addressing this problem. The results show that there is   no such structural effect on the free release factor caused by the NE methylation of Q185, suggesting that its role is intimately associated with the ribosome environment.

##### Place, publisher, year, edition, pages
Washington, DC, USA: American Chemical Society, 2009
##### National Category
Biological Sciences
##### Identifiers
urn:nbn:se:uu:diva-101411 (URN)10.1021/bi900117r (DOI)000265170200025 ()
Available from: 2009-04-26 Created: 2009-04-26 Last updated: 2017-12-13Bibliographically approved
4. Ligand binding to the voltage-gated Kv1.5 potassium channel in the open state - Docking and computer simulations of a homology model
Open this publication in new window or tab >>Ligand binding to the voltage-gated Kv1.5 potassium channel in the open state - Docking and computer simulations of a homology model
2008 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 94, no 3, p. 820-831Article in journal (Refereed) Published
##### Abstract [en]

The binding of blockers to the human voltage-gated Kv1.5 potassium ion channel is investigated using a three-step procedure consisting of homology modeling, automated docking, and binding free energy calculations from molecular dynamics simulations, in combination with the linear interaction energy method. A reliable homology model of Kv1.5 is constructed using the recently published crystal structure of the Kv1.2 channel as a template. This model is expected to be significantly more accurate than earlier ones based on less similar templates. Using the three-dimensional homology model, a series of blockers with known affinities are docked into the cavity of the ion channel and their free energies of binding are calculated. The predicted binding free energies are in very good agreement with experimental data and the binding is predicted to be mainly achieved through nonpolar interactions, whereas the relatively small differences in the polar contribution determine the specificity. Apart from confirming the importance of residues V505, I508, V512, and V516 for ligand binding in the cavity, the results also show that A509 and P513 contribute significantly to the nonpolar binding interactions. Furthermore, we find that pharmacophore models based only on optimized free ligand conformations may not necessarily capture the geometric features of ligands bound to the channel cavity. The calculations herein give a detailed structural and energetic picture of blocker binding to Kv1.5 and this model should thus be useful for further ligand design efforts.

##### National Category
Biochemistry and Molecular Biology
##### Identifiers
urn:nbn:se:uu:diva-101410 (URN)10.1529/biophysj.107.112045 (DOI)000252243200011 ()17905851 (PubMedID)
Available from: 2009-04-26 Created: 2009-04-26 Last updated: 2017-12-13Bibliographically approved
5. Continuum solvation models in the linear interaction energy method
Open this publication in new window or tab >>Continuum solvation models in the linear interaction energy method
2006 In: Journal of Physical Chemistry B, ISSN 1520-6106, Vol. 110, no 24, p. 12034-12041Article in journal (Refereed) Published
##### Identifiers
urn:nbn:se:uu:diva-97212 (URN)
Available from: 2008-04-29 Created: 2008-04-29Bibliographically approved
• 5. Aquila, A.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
The linac coherent light source single particle imaging road map2015In: Structural Dynamics, Vol. 2, no 4, article id 041701Article in journal (Refereed)

Intense femtosecond x-ray pulses from free-electron laser sources allow the imag-ing of individual particles in a single shot. Early experiments at the Linac CoherentLight Source (LCLS) have led to rapid progress in the field and, so far, coherentdiffractive images have been recorded from biological specimens, aerosols, andquantum systems with a few-tens-of-nanometers resolution. In March 2014, LCLSheld a workshop to discuss the scientific and technical challenges for reaching theultimate goal of atomic resolution with single-shot coherent diffractive imaging. This paper summarizes the workshop findings and presents the roadmap towardreaching atomic resolution, 3D imaging at free-electron laser sources.

• 6. Aquila, Andrew
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Physical Chemistry. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
Time-resolved protein nanocrystallography using an X-ray free-electron laser2012In: Optics Express, ISSN 1094-4087, E-ISSN 1094-4087, Vol. 20, no 3, p. 2706-2716Article in journal (Refereed)

We demonstrate the use of an X-ray free electron laser synchronized with an optical pump laser to obtain X-ray diffraction snapshots from the photoactivated states of large membrane protein complexes in the form of nanocrystals flowing in a liquid jet. Light-induced changes of Photosystem I-Ferredoxin co-crystals were observed at time delays of 5 to 10 µs after excitation. The result correlates with the microsecond kinetics of electron transfer from Photosystem I to ferredoxin. The undocking process that follows the electron transfer leads to large rearrangements in the crystals that will terminally lead to the disintegration of the crystals. We describe the experimental setup and obtain the first time-resolved femtosecond serial X-ray crystallography results from an irreversible photo-chemical reaction at the Linac Coherent Light Source. This technique opens the door to time-resolved structural studies of reaction dynamics in biological systems.

• 7. Arnlund, David
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Physical Chemistry. Linac Coherent Light Source, SLAC National Accelerator Laboratory, Menlo Park, California, USA..
Visualizing a protein quake with time-resolved X-ray scattering at a free-electron laser2014In: Nature Methods, ISSN 1548-7091, E-ISSN 1548-7105, Vol. 11, no 9, p. 923-926Article in journal (Refereed)

We describe a method to measure ultrafast protein structural changes using time-resolved wide-angle X-ray scattering at an X-ray free-electron laser. We demonstrated this approach using multiphoton excitation of the Blastochloris viridis photosynthetic reaction center, observing an ultrafast global conformational change that arises within picoseconds and precedes the propagation of heat through the protein. This provides direct structural evidence for a 'protein quake': the hypothesis that proteins rapidly dissipate energy through quake-like structural motions.

• 8. Barends, Thomas R. M.
Structure and mechanism of a bacterial light-regulated cyclic nucleotide phosphodiesterase2009In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 459, p. 1015-1018Article in journal (Refereed)

The ability to respond to light is crucial for most organisms. BLUF is a recently identified photoreceptor protein domain that senses blue light using a FAD chromophore. BLUF domains are present in various proteins from the Bacteria, Euglenozoa and Fungi. Although structures of single-domain BLUF proteins have been determined, none are available for a BLUF protein containing a functional output domain; the mechanism of light activation in this new class of photoreceptors has thus remained poorly understood. Here we report the biochemical, structural and mechanistic characterization of a full-length, active photoreceptor, BlrP1 (also known as KPN_01598), from Klebsiella pneumoniae. BlrP1 consists of a BLUF sensor domain and a phosphodiesterase EAL output domain which hydrolyses cyclic dimeric GMP (c-di-GMP). This ubiquitous second messenger controls motility, biofilm formation, virulence and antibiotic resistance in the Bacteria. Crystal structures of BlrP1 complexed with its substrate and metal ions involved in catalysis or in enzyme inhibition provide a detailed understanding of the mechanism of the EAL-domain c-di-GMP phosphodiesterases. These structures also sketch out a path of light activation of the phosphodiesterase output activity. Photon absorption by the BLUF domain of one subunit of the antiparallel BlrP1 homodimer activates the EAL domain of the second subunit through allosteric communication transmitted through conserved domain-domain interfaces.

• 9.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
Automated liquid-handling systems for submicroliter crystallization2007In: Protein Crystallization Strategies for Structural Genomics / [ed] Naomi E. Chayen, La Jolla, California: International University Line , 2007, p. 57-73Chapter in book (Other academic)
• 10.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
Succeeding with seeding: some practical advice2007In: Evolving Methods for Macromolecular Crystallography / [ed] Read, Randy J.; Sussman, Joel L., 2007, p. 1-10Conference paper (Refereed)

Seeding is a powerful and versatile method for optimizing crystal growth conditions. This article discusses, from a practical point of view, what seeding is, the selection and transfer of seeds, and into what conditions they should be transferred. The most common causes of failures in seeding experiments are also analyzed.

• 11.
Uppsala University, Disciplinary Domain of Science and Technology.
The RAPID crystallization strategy for structure-based inhibitor design2009In: From Molecules to Medicines: Structure of Biological Macromolecules and Its Relevance in Combating New Diseases and Bioterrorism / [ed] J. Sussman and P. Spadon, Dordrecht, The Netherlands: Springer , 2009, p. 11-19Chapter in book (Other academic)
• 12.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Enzymes in the Mycobacterium tuberculosis MEP and CoA Pathways Targeted for Structure-Based Drug Design2012Doctoral thesis, comprehensive summary (Other academic)

Tuberculosis, caused by the pathogenic bacteria Mycobacterium tuberculosis, is one of the most widespread and deadly infectious diseases today. Treatment of tuberculosis relies on antibiotics that were developed more than 50 years ago. These are now becoming ineffective due to the emergence of antibiotic resistant strains of the bacteria.

The aim of the research in this thesis was to develop new antibiotics for tuberculosis treatment. To this end, we targeted enzymes from two essential biosynthetic pathways in M. tuberculosis for drug development. The methylerythritol phosphate (MEP) pathway synthesizes a group of compounds called isoprenoids. These compounds have essential roles in all living organisms. The fact that humans utilize a different pathway for isoprenoid synthesis makes the MEP pathway enzymes attractive targets for drug development. We have determined the structures of two essential enzymes from this pathway by X-ray crystallography: 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) and 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase (IspD). These are the first structures of these enzymes from M. tuberculosis. Additionally, structures of the IspD enzyme from the related bacteria Mycobacterium smegmatis were determined. We have characterized these enzymes and evaluated the efficiency of a number of inhibitors of the DXR enzyme by biochemical methods. Crystal structures of DXR in complex with some of these inhibitors were also determined.

The second pathway of interest for drug development is the universal pathway for Coenzyme A biosynthesis. Enzymes in this pathway have essential roles in all living organisms. However, the bacterial enzymes have little similarity to the human homologues. We have determined a number of structures of the M. tuberculosis pantothenate kinase (PanK), the regulatory enzyme of this pathway, in complex with two new classes of inhibitory compounds, and evaluated these by biochemical methods.

The structures and biochemical characterization of these enzymes provide us with detailed information about their functions and broadens our knowledge of these bacteria. Biochemical and structural information about new inhibitors of these enzymes serve as a starting point for future development of antibiotics against tuberculosis.

1. The 1.9 Å resolution structure of Mycobacterium tuberculosis 1-deoxy-D-xylulose 5-phosphate reductoisomerase, a potential drug target
Open this publication in new window or tab >>The 1.9 Å resolution structure of Mycobacterium tuberculosis 1-deoxy-D-xylulose 5-phosphate reductoisomerase, a potential drug target
2006 (English)In: Acta Crystallogr. Section D Biol. Crystallogr., ISSN 0907-4449, Vol. 62, p. 807-813Article in journal (Refereed) Published
##### National Category
Natural Sciences
##### Identifiers
urn:nbn:se:uu:diva-96292 (URN)
Available from: 2007-10-19 Created: 2007-10-19 Last updated: 2015-04-02
2. Structural and functional studies of mycobacterial IspD enzymes
Open this publication in new window or tab >>Structural and functional studies of mycobacterial IspD enzymes
2011 (English)In: Acta Crystallographica Section D: Biological Crystallography, ISSN 0907-4449, E-ISSN 1399-0047, Vol. 67, p. 403-414Article in journal (Refereed) Published
##### Abstract [en]

A number of pathogens, including the causative agents of tuberculosis and malaria, synthesize isopentenyl diphosphate via the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway rather than the classical mevalonate pathway found in humans. As part of a structure-based drug-discovery program against tuberculosis, IspD, the enzyme that carries out the third step in the MEP pathway, was targeted. Constructs of both the Mycobacterium smegmatis and the Mycobacterium tuberculosis enzymes that were suitable for structural and inhibitor-screening studies were engineered. Two crystal structures of the M. smegmatis enzyme were produced, one in complex with CTP and the other in complex with CMP. In addition, the M. tuberculosis enzyme was crystallized in complex with CTP. Here, the structure determination and crystallographic refinement of these crystal forms and the enzymatic characterization of the M. tuberculosis enzyme construct are reported. A comparison with known IspD structures allowed the definition of the structurally conserved core of the enzyme. It indicates potential flexibility in the enzyme and in particular in areas close to the active site. These well behaved constructs provide tools for future target-based screening of potential inhibitors. The conserved nature of the extended active site suggests that any new inhibitor will potentially exhibit broad-spectrum activity.

##### National Category
Biological Sciences
##### Identifiers
urn:nbn:se:uu:diva-154127 (URN)10.1107/S0907444911006160 (DOI)000290235200002 ()21543842 (PubMedID)
Available from: 2011-05-26 Created: 2011-05-26 Last updated: 2017-12-11Bibliographically approved
3. Design, Synthesis, and X-ray Crystallographic Studies of alpha-Aryl Substituted Fosmidomycin Analogues as Inhibitors of Mycobacterium tuberculosis 1-Deoxy-D-xylulose 5-Phosphate Reductoisomerase
Open this publication in new window or tab >>Design, Synthesis, and X-ray Crystallographic Studies of alpha-Aryl Substituted Fosmidomycin Analogues as Inhibitors of Mycobacterium tuberculosis 1-Deoxy-D-xylulose 5-Phosphate Reductoisomerase
2011 (English)In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 54, no 14, p. 4964-4976Article in journal (Refereed) Published
##### Abstract [en]

The natural antibiotic fosmidomycin acts via inhibition of 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), an essential enzyme in the non-mevalonate pathway of isoprenoid biosynthesis. Fosmidomycin is active on Mycobacterium tuberculosis DXR (MtDXR), but it lacks antibacterial activity probably because of poor uptake. alpha-Aryl substituted fosmidomycin analogues have more favorable physicochemical properties and are also more active in inhibiting malaria parasite growth. We have solved crystal structures of MtDXR in complex with 3,4-dichlorophenyl substituted fosmidomycin analogues; these show important differences compared to our previously described forsmidomycin-DXR complex. Our best inhibitor has an IC(50) = 0.15 mu M on MtDXR but still lacked activity in a mycobacterial growth assay (MIC > 32 mu g/mL). The combined results, however, provide insights into how DXR accommodates the new inhibitors and serve as an excellent starting point for the design of other novel and more potent inhibitors, particularly against pathogens where uptake is less of a problem, such as the malaria parasite.

##### National Category
Biochemistry and Molecular Biology Other Basic Medicine
##### Identifiers
urn:nbn:se:uu:diva-156614 (URN)10.1021/jm2000085 (DOI)000292892300003 ()21678907 (PubMedID)
Available from: 2011-08-07 Created: 2011-08-04 Last updated: 2018-01-12Bibliographically approved
4. Structural studies on Mycobacterium tuberculosis DXR in complex with the antibiotic FR-900098
Open this publication in new window or tab >>Structural studies on Mycobacterium tuberculosis DXR in complex with the antibiotic FR-900098
2012 (English)In: Acta Crystallographica Section D: Biological Crystallography, ISSN 0907-4449, E-ISSN 1399-0047, Vol. 68, p. 134-143Article in journal (Refereed) Published
##### Abstract [en]

A number of pathogens, including the causative agents of tuberculosis and malaria, synthesize the essential isoprenoid precursor isopentenyl diphosphate via the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway rather than the classical mevalonate pathway that is found in humans. As part of a structure-based drug-discovery program against tuberculosis, DXR, the enzyme that carries out the second step in the MEP pathway, has been investigated. This enzyme is the target for the antibiotic fosmidomycin and its active acetyl derivative FR-900098. The structure of DXR from Mycobacterium tuberculosis in complex with FR-900098, manganese and the NADPH cofactor has been solved and refined. This is a new crystal form that diffracts to a higher resolution than any other DXR complex reported to date. Comparisons with other ternary complexes show that the conformation is that of the enzyme in an active state: the active-site flap is well defined and the cofactor-binding domain has a conformation that brings the NADPH into the active site in a manner suitable for catalysis. The substrate-binding site is highly conserved in a number of pathogens that use this pathway, so any new inhibitor that is designed for the M. tuberculosis enzyme is likely to exhibit broad-spectrum activity.

##### Keywords
tuberculosis, DXR, IspC, MEP pathway
##### National Category
Medical and Health Sciences Biochemistry and Molecular Biology
##### Identifiers
urn:nbn:se:uu:diva-169337 (URN)10.1107/S0907444911052231 (DOI)000299469100006 ()
Available from: 2012-03-05 Created: 2012-02-28 Last updated: 2017-12-07Bibliographically approved
5. Structural and biochemical characterization of compounds inhibiting Mycobacterium tuberculosis Pantothenate Kinase
Open this publication in new window or tab >>Structural and biochemical characterization of compounds inhibiting Mycobacterium tuberculosis Pantothenate Kinase
2013 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, no 25, p. 18260-18270Article in journal (Refereed) Published
##### Abstract [en]

Mycobacterium tuberculosis, the bacterial causative agent oftuberculosis, currently affects millions of people. The emergence of drug-resistant strains makes development of new antibiotics targeting the bacterium a global health priority. Pantothenate kinase, a key enzyme in the universal biosynthesis of the essential cofactor CoA, was targeted in this study to find new tuberculosis drugs. The biochemicalcharacterizations of two new classes of compounds that inhibitpantothenate kinase from M. tuberculosis are described, along with crystal structures of their enzyme-inhibitor complexes. These represent the first crystal structures of this enzyme with engineered inhibitors. Both classes of compounds bind in the active site of the enzyme, overlapping with the binding sites of the natural substrate and product, pantothenateand phosphopantothenate, respectively. One class of compounds also interferes with binding of the cofactor ATP. The complexes were crystallized in two crystal forms, one of which is in a new space group for this enzyme and diffracts to the highest resolution reported for anypantothenate kinase structure. These two crystal forms allowed, for the first time, modeling of the cofactor-binding loop in both open and closed conformations. The structures also show a binding mode of ATP different from that previously reported for the M. tuberculosis enzyme but similar to that in the pantothenate kinases of other organisms.

##### Keywords
Tuberculosis, Mycobacterium tuberculosis, drug development, pantothenate kinase, PanK
##### National Category
Structural Biology Biochemistry and Molecular Biology
##### Research subject
Molecular Biology; Biochemistry
##### Identifiers
urn:nbn:se:uu:diva-179056 (URN)10.1074/jbc.M113.476473 (DOI)000320721900030 ()
##### Funder
Swedish Research Council Available from: 2012-08-06 Created: 2012-08-06 Last updated: 2017-12-07Bibliographically approved
• 13.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. AstraZeneca India Private Limited. AstraZeneca India Private Limited. AstraZeneca India Private Limited. AstraZeneca India Private Limited. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
Structural and biochemical characterization of compounds inhibiting Mycobacterium tuberculosis Pantothenate Kinase2013In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 288, no 25, p. 18260-18270Article in journal (Refereed)

Mycobacterium tuberculosis, the bacterial causative agent oftuberculosis, currently affects millions of people. The emergence of drug-resistant strains makes development of new antibiotics targeting the bacterium a global health priority. Pantothenate kinase, a key enzyme in the universal biosynthesis of the essential cofactor CoA, was targeted in this study to find new tuberculosis drugs. The biochemicalcharacterizations of two new classes of compounds that inhibitpantothenate kinase from M. tuberculosis are described, along with crystal structures of their enzyme-inhibitor complexes. These represent the first crystal structures of this enzyme with engineered inhibitors. Both classes of compounds bind in the active site of the enzyme, overlapping with the binding sites of the natural substrate and product, pantothenateand phosphopantothenate, respectively. One class of compounds also interferes with binding of the cofactor ATP. The complexes were crystallized in two crystal forms, one of which is in a new space group for this enzyme and diffracts to the highest resolution reported for anypantothenate kinase structure. These two crystal forms allowed, for the first time, modeling of the cofactor-binding loop in both open and closed conformations. The structures also show a binding mode of ATP different from that previously reported for the M. tuberculosis enzyme but similar to that in the pantothenate kinases of other organisms.

• 14.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
Computational Modelling of Ligand Complexes with G-Protein Coupled Receptors, Ion Channels and Enzymes2014Doctoral thesis, comprehensive summary (Other academic)

Accurate predictions of binding free energies from computer simulations are an invaluable resource for understanding biochemical processes and drug action. The primary aim of the work described in the thesis was to predict and understand ligand binding to several proteins of major pharmaceutical importance using computational methods.

We report a computational strategy to quantitatively predict the effects of alanine scanning and ligand modifications based on molecular dynamics free energy simulations. A smooth stepwise scheme for free energy perturbation calculations is derived and applied to a series of thirteen alanine mutations of the human neuropeptide Y1 G-protein coupled receptor and a series of eight analogous antagonists. The robustness and accuracy of the method enables univocal interpretation of existing mutagenesis and binding data. We show how these calculations can be used to validate structural models and demonstrate their ability to discriminate against suboptimal ones. Site-directed mutagenesis, homology modelling and docking were further used to characterize agonist binding to the human neuropeptide Y2 receptor, which is important in feeding behavior and an obesity drug target.  In a separate project, homology modelling was also used for rationalization of mutagenesis data for an integron integrase involved in antibiotic resistance.

Blockade of the hERG potassium channel by various drug-like compounds, potentially causing serious cardiac side effects, is a major problem in drug development. We have used a homology model of hERG to conduct molecular docking experiments with a series of channel blockers, followed by molecular dynamics simulations of the complexes and evaluation of binding free energies with the linear interaction energy method. The calculations are in good agreement with experimental binding affinities and allow for a rationalization of three-dimensional structure-activity relationships with implications for design of new compounds. Docking, scoring, molecular dynamics, and the linear interaction energy method were also used to predict binding modes and affinities for a large set of inhibitors to HIV-1 reverse transcriptase. Good agreement with experiment was found and the work provides a validation of the methodology as a powerful tool in structure-based drug design. It is also easily scalable for higher throughput of compounds.

1. Mutagenesis of human neuropeptide Y/peptide YY receptor Y2 reveals additional differences to Y1 in interactions with highly conserved ligand positions
Open this publication in new window or tab >>Mutagenesis of human neuropeptide Y/peptide YY receptor Y2 reveals additional differences to Y1 in interactions with highly conserved ligand positions
2010 (English)In: Regulatory Peptides, ISSN 0167-0115, E-ISSN 1873-1686, Vol. 163, no 1-3, p. 120-129Article in journal (Refereed) Published
##### Abstract [en]

Neuropeptide Y (NPY) and peptide YY (PYY) share similar to 70% of their 36 amino acids and bind to the same three human receptor subtypes, Y1, Y2 and Y5, even though these receptors only share similar to 30% sequence identity Based on our previous investigation of human Y1 we describe here a mutagenesis study of three corresponding positions in human Y2, i e Tyr(2 64), Val(6 58) and Tyr(7 31) Pharmacological characterization was performed with the four peptide agonists PYY, NPY, PYY(3-36) and NPY(13-36) as well as the non-peptide antagonist BIIE0246 Results from mutants where Tyr(2 64) has been substituted by Ala suggest that Tyr(2 64) is involved in the interaction with all investigated ligands whereas position Tyr(7 31) seems to be more important for interaction with the truncated peptide PYY(3-36) than with intact NPY Surprisingly, substitution of Tyr(7 31) with His, the corresponding residue in Y1, resulted in total loss of binding of iodinated porcine PYY The third position. Val(6 58), did not influence binding of any of the ligands. These findings differ from those obtained for Y1 where Ala substitution resulted in lost or changed binding for each of the three positions. Although Tyr(2 64) and Tyr(7 31) in Y2 are involved in ligand binding, their interactions with the peptide ligands seem to be different from the corresponding positions in Y1 This suggests that the receptor-ligand interactions have changed during evolution after Y1 and Y2 arose from a common ancestral receptor.

##### Keywords
Site-directed mutagenesis, G-protein coupled receptor, Three dimensional model, Evolution
##### National Category
Medical and Health Sciences
##### Identifiers
urn:nbn:se:uu:diva-135756 (URN)10.1016/j.regpep.2010.04.011 (DOI)000280050000018 ()
##### Note

Manuscript title: Investigation of receptor-ligand interactions of the human neuropeptide Y receptor Y2 by site-directed mutagenesis: comparison with the structurally divergent Y1 subtype

Available from: 2010-12-08 Created: 2010-12-08 Last updated: 2017-12-11Bibliographically approved
2. Mutagenesis and Computational Modeling of Human G‑Protein-Coupled Receptor Y2 for Neuropeptide Y and Peptide YY
Open this publication in new window or tab >>Mutagenesis and Computational Modeling of Human G‑Protein-Coupled Receptor Y2 for Neuropeptide Y and Peptide YY
2013 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 52, no 45, p. 7987-7998Article in journal (Refereed) Published
##### Abstract [en]

Neuropeptide Y and peptide YY receptor type 2 (Y2) is involved in appetite regulation and several other physiological processes. We have investigated the structure of the human Y2 receptor. Computational modeling of receptor–agonist interactions was used as a guide to design a series of receptor mutants, followed by binding assays using full-length and truncated peptide agonists and the Y2-specific antagonist BIIE0246. Our model suggested a hydrogen bond network among highly conserved residues Thr2.61, Gln3.32, and His7.39, which could play roles in ligand binding and/or receptor structure. In addition, the C-terminus of the peptide could make contact with residues Tyr5.38 and Leu6.51. Mutagenesis of all these positions, followed by binding assays, provides experimental support for our computational model: most of the mutants for the residues forming the proposed hydrogen bond network displayed reduced peptide agonist affinities as well as reduced hPYY3-36 potency in a functional assay. The Ala and Leu mutants of Gln3.32 and His7.39 disrupted membrane expression of the receptor. Combined with the modeling, the experimental results support roles for these hydrogen bond network residues in peptide binding as well as receptor architecture. The reduced agonist affinity for mutants of Tyr5.38 and Leu6.51 supports their role in a binding pocket surrounding the invariant tyrosine at position 36 of the peptide ligands. The results for antagonist BIIE0246 suggest several differences in interactions compared to those of the peptides. Our results lead to a new structural model for NPY family receptors and peptide binding.

##### Place, publisher, year, edition, pages
American Chemical Society (ACS), 2013
##### National Category
Natural Sciences Medical and Health Sciences
##### Identifiers
urn:nbn:se:uu:diva-154994 (URN)10.1021/bi400830c (DOI)000330017700012 ()
Available from: 2011-08-04 Created: 2011-06-14 Last updated: 2019-01-03Bibliographically approved
3. Computational prediction of alanine scanning and ligand binding energetics in G-protein coupled receptors
Open this publication in new window or tab >>Computational prediction of alanine scanning and ligand binding energetics in G-protein coupled receptors
2014 (English)In: PloS Computational Biology, ISSN 1553-734X, E-ISSN 1553-7358, Vol. 10, no 4, p. e1003585-Article in journal (Refereed) Published
##### Abstract [en]

Site-directed mutagenesis combined with binding affinity measurements is widely used to probe the nature of ligand interactions with GPCRs. Such experiments, as well as structure-activity relationships for series of ligands, are usually interpreted with computationally derived models of ligand binding modes. However, systematic approaches for accurate calculations of the corresponding binding free energies are still lacking. Here, we report a computational strategy to quantitatively predict the effects of alanine scanning and ligand modifications based on molecular dynamics free energy simulations. A smooth stepwise scheme for free energy perturbation calculations is derived and applied to a series of thirteen alanine mutations of the human neuropeptide Y1 receptor and series of eight analogous antagonists. The robustness and accuracy of the method enables univocal interpretation of existing mutagenesis and binding data. We show how these calculations can be used to validate structural models and demonstrate their ability to discriminate against suboptimal ones. Author Summary G-protein coupled receptors constitute a family of drug targets of outstanding interest, with more than 30% of the marketed drugs targeting a GPCR. The combination of site-directed mutagenesis, biochemical experiments and computationally generated 3D structural models has traditionally been used to investigate these receptors. The increasing number of GPCR crystal structures now paves the way for detailed characterization of receptor-ligand interactions and energetics using advanced computer simulations. Here, we present an accurate computational scheme to predict and interpret the effects of alanine scanning experiments, based on molecular dynamics free energy simulations. We apply the technique to antagonist binding to the neuropeptide Y receptor Y1, the structure of which is still unknown. A structural model of a Y1-antagonist complex was derived and used as starting point for computational characterization of the effects on binding of alanine substitutions at thirteen different receptor positions. Further, we used the model and computational scheme to predict the binding of a series of seven antagonist analogs. The results are in excellent agreement with available experimental data and provide validation of both the methodology and structural models of the complexes.

##### National Category
Bioinformatics (Computational Biology) Biochemistry and Molecular Biology
##### Identifiers
urn:nbn:se:uu:diva-212102 (URN)10.1371/journal.pcbi.1003585 (DOI)000336507500014 ()
Available from: 2013-12-05 Created: 2013-12-05 Last updated: 2018-01-11Bibliographically approved
4. Computer Simulations of Structure-Activity Relationships for hERG Channel Blockers
Open this publication in new window or tab >>Computer Simulations of Structure-Activity Relationships for hERG Channel Blockers
2011 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 50, no 27, p. 6146-6156Article in journal (Refereed) Published
##### Abstract [en]

The hERG potassium channel is of major pharmaceutical importance, and its blockade by various compounds, potentially causing serious cardiac side effects, is a major problem in drug development. Despite the large amounts of existing biochemical data on blockade of hERG by drugs and druglike compounds, relatively little is known regarding the structural basis of binding of blockers to the channel. Here, we have used a recently developed homology model of hERG to conduct molecular docking experiments with a series of channel blockers, followed by molecular dynamics simulations of the complexes and evaluation of binding free energies with the linear interaction energy method. The calculations yield a remarkably good agreement with experimental binding affinities and allow for a rationalization of three-dimensional structure-activity relationships in terms of a number of key interactions. Two main interaction regions of the channel are thus identified with implications for further mutagenesis experiments and design of new compounds.

##### National Category
Medical and Health Sciences
##### Identifiers
urn:nbn:se:uu:diva-156474 (URN)10.1021/bi200173n (DOI)000292430600018 ()
Available from: 2011-07-27 Created: 2011-07-25 Last updated: 2017-12-08Bibliographically approved
5. Combining docking, molecular dynamics and the linear interaction energy method to predict binding modes and affinities for non-nucleoside inhibitors to HIV-1 reverse transcriptase
Open this publication in new window or tab >>Combining docking, molecular dynamics and the linear interaction energy method to predict binding modes and affinities for non-nucleoside inhibitors to HIV-1 reverse transcriptase
2008 (English)In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 51, no 9, p. 2648-56Article in journal (Refereed) Published
##### Abstract [en]

Docking, scoring, molecular dynamics (MD), and the linear interaction energy (LIE) method are used here to predict binding modes and affinities for a set of 43 non-nucleoside inhibitors to HIV-1 reverse transcriptase. Starting from a crystallographic structure, the binding modes of 43 inhibitors are predicted using automated docking. The Goldscore scoring function and the LIE method are then used to determine the relative binding free energies for the inhibitors. The Goldscore scoring function does not reproduce the relative binding affinities for the inhibitors, while the standard parametrization of the LIE method reproduces the experimental binding free energies for 39 inhibitors with an R (2) = 0.70 and an unsigned average error of 0.8 kcal/mol. The present calculations provide a validation of the combination of docking, MD, and LIE as a powerful tool in structure-based drug design, and the methodology is easily scalable for attaining a higher throughput of compounds.

##### National Category
Medical and Health Sciences
##### Identifiers
urn:nbn:se:uu:diva-97214 (URN)10.1021/jm7012198 (DOI)000255500000010 ()18410085 (PubMedID)
Available from: 2008-04-29 Created: 2008-04-29 Last updated: 2017-12-14Bibliographically approved
6. Mutagenesis and homology modelling of the Tn21 integron integrase IntI1
Open this publication in new window or tab >>Mutagenesis and homology modelling of the Tn21 integron integrase IntI1
2009 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, no 8, p. 1743-1753Article in journal (Refereed) Published
##### Abstract [en]

Horizontal DNA transfer between bacteria is widespread and a major cause of antibiotic resistance. For logistic reasons, single or combined genes are shuttled between vectors such as plasmids and   bacterial chromosomes. Special elements termed integrons operate in such shuttling and are therefore vital for horizontal gene transfer. Shorter elements carrying genes, cassettes, are integrated in the integrons, or excised from them, by virtue of a recombination site, attC, positioned in the 3' end of each unit. It is a remarkable and   possibly restricting elementary feature of attC that it must be single-stranded while the partner target site, attI, may be double-stranded. The integron integrases belong to the tyrosine recombinase family, and this work reports mutations of the integrase IntI1 from transposon Tn21, chosen within a well-conserved region characteristic of the integron integrases. The mutated proteins were  tested for binding to a bottom strand of an attC substrate, by using an electrophoresis mobility shift assay. To aid in interpreting the   results, a homology model was constructed on the basis of the crystal structure of integron integrase VchIntIA from Vibrio cholerae bound to its cognate attC substrate VCRbs. The local stability and hydrogen bonding network of key domains of the modeled structure were further examined using molecular dynamics simulations. The homology model allowed us to interpret the roles of several amino acid residues, four of which were clearly binding assay responsive upon mutagenesis. Notably, we also observed features indicating that IntI1 may be more prone to base-specific contacts with VCRbs than VchIntIA.

##### National Category
Biological Sciences
##### Identifiers
urn:nbn:se:uu:diva-95309 (URN)10.1021/bi8020235 (DOI)000263697300009 ()
Available from: 2007-01-02 Created: 2007-01-02 Last updated: 2017-12-14Bibliographically approved
• 15. Boutet, Sébastien
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics.
High-resolution protein structure determination by serial femtosecond crystallography2012In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 337, no 6092, p. 362-364Article in journal (Refereed)

Structure determination of proteins and other macromolecules has historically required the growth of high-quality crystals sufficiently large to diffract x-rays efficiently while withstanding radiation damage. We applied serial femtosecond crystallography (SFX) using an x-ray free-electron laser (XFEL) to obtain high-resolution structural information from microcrystals (less than 1 micrometer by 1 micrometer by 3 micrometers) of the well-characterized model protein lysozyme. The agreement with synchrotron data demonstrates the immediate relevance of SFX for analyzing the structure of the large group of difficult-to-crystallize molecules.

• 16.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
Fighting Tuberculosis –: Structural Studies of Three Mycobacterial Proteins2008Doctoral thesis, comprehensive summary (Other academic)

This thesis presents the cloning, purification, crystallization, and structural studies of two unknown proteins from Mycobacterium tuberculosis, and of an aminotransferase from Mycobacterium smegmatis. Structural knowledge of these proteins is of highest interest for structure-based drug design, which is one of the approaches that can be used in order to fight tuberculosis (TB).

The structure of the conserved hypothetical protein Rv0216 was refined to a resolution of 1.9 Å. The structure exhibits a so-called double hotdog-fold, similar to known hydratases. However, only parts of the hydratase active site are conserved in Rv0216, and no function could be assigned to the protein. Several Rv0216-like protein sequences were found in a variety of actino- and proteobacteria, suggesting that these proteins form a new protein family. Furthermore, other hotdog-folded proteins in M. tuberculosis were identified, of which a few are likely to be hydratases or dehydratases involved in the fatty acid metabolism.

The structure of Rv0130 exhibits a single hotdog-fold and contains a highly conserved R-hydratase motif. Rv0130 was shown to hydrate fatty acid coenzyme A derivatives with a length of six to eight carbons. The Rv0130 active site is situated in a long tunnel, formed by a kink in the central hotdog-helix, which indicate that it can utilize long fatty acid chains as well. A number of previously predicted hotdog-folded proteins also feature a similar tunnel.

The structure of branched chain aminotransferase (BCAT) of M. smegmatis was determined in the apo-form and in complex with an aminooxy inhibitor. Mycobacterial BCAT is very similar to the human BCAT, apart for one important difference in the active site. Gly243 is a threonine in the human BCAT, a difference that offers specificity in inhibition and substrate recognition of these proteins. The aminooxy compound and MES were found to inhibit the mycobacterial BCAT activities. The aminooxy compound inhibits by blocking the substrate-pocket. A second inhibitor-binding site was identified through the binding of a MES molecule. Therefore, both the MES-binding site and the substrate-pocket of M. smegmatis BCAT are suggested to be potential sites for the development of new inhibitors against tuberculosis.

1. Rv0216, a conserved hypothetical protein from Mycobacterium tuberculosis that is essential for bacterial
Open this publication in new window or tab >>Rv0216, a conserved hypothetical protein from Mycobacterium tuberculosis that is essential for bacterial
##### Identifiers
urn:nbn:se:uu:diva-97709 (URN)
Available from: 2008-11-06 Created: 2008-11-06 Last updated: 2010-01-13Bibliographically approved
2. Structure and function of Rv0130, a conserved hypothetical protein from Mycobacterium tuberculosis
Open this publication in new window or tab >>Structure and function of Rv0130, a conserved hypothetical protein from Mycobacterium tuberculosis
2006 (English)In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 15, no 10, p. 2300-2309Article in journal (Refereed) Published
##### Abstract [en]

A large fraction of the Mycobacterium tuberculosis genome codes for proteins of unknown function. We here report the structure of one of these proteins, Rv0130, solved to a resolution of 1.8 angstrom. The Rv0130 monomer features a single hotdog fold composed of a highly curved beta-sheet on top of a long and a short alpha-helix. Two monomers in turn pack to form a double-hotdog-folded homodimer, similar to a large group of enzymes that use thiol esters as substrates. Rv0130 was found to contain a highly conserved R-specific hydratase motif buried deeply between the two monomers. Our biochemical studies show that the protein is able to hydrate a short trans-2-enoyl-coenzyme A moiety with a k(cat) of 1.1 x 10(2) sec(-1). The importance of the side chains of D40 and H45 for hydratase activity is demonstrated by site-directed mutagenesis. In contrast to many hotdog-folded proteins, a proline residue distorts the central helix of Rv0130. This distortion allows the creation of a long, curved tunnel, similar to the substrate-binding channels of long-chain eukaryotic hydratase 2 enzymes.

##### Keywords
Rv0130, Mycobacterium tuberculosis, hydratase, hotdog fold, crystal structure
##### National Category
Biological Sciences
##### Identifiers
urn:nbn:se:uu:diva-94234 (URN)10.1110/ps.062309306 (DOI)000240851300008 ()16963641 (PubMedID)
Available from: 2006-04-12 Created: 2006-04-12 Last updated: 2017-12-14Bibliographically approved
3. Structural analysis of
Open this publication in new window or tab >>Structural analysis of
##### Identifiers
urn:nbn:se:uu:diva-97711 (URN)
Available from: 2008-11-06 Created: 2008-11-06 Last updated: 2010-01-13Bibliographically approved
• 17.
Lawrence Berkeley Natl Lab, Mol Biophys & Integrated Bioimaging Div, Berkeley, CA 94720 USA.
Lawrence Berkeley Natl Lab, Mol Biophys & Integrated Bioimaging Div, Berkeley, CA 94720 USA. Lawrence Berkeley Natl Lab, Mol Biophys & Integrated Bioimaging Div, Berkeley, CA 94720 USA. Lawrence Berkeley Natl Lab, Mol Biophys & Integrated Bioimaging Div, Berkeley, CA 94720 USA. Lawrence Berkeley Natl Lab, Mol Biophys & Integrated Bioimaging Div, Berkeley, CA 94720 USA. Humboldt Univ, Inst Biol, D-10099 Berlin, Germany. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics. Umea Univ, Inst Kemi, Kemiskt Biol Centrum, S-90187 Umea, Sweden. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics. Humboldt Univ, Inst Biol, D-10099 Berlin, Germany. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics. Umea Univ, Inst Kemi, Kemiskt Biol Centrum, S-90187 Umea, Sweden. Lawrence Berkeley Natl Lab, Mol Biophys & Integrated Bioimaging Div, Berkeley, CA 94720 USA. Lawrence Berkeley Natl Lab, Mol Biophys & Integrated Bioimaging Div, Berkeley, CA 94720 USA. Lawrence Berkeley Natl Lab, Mol Biophys & Integrated Bioimaging Div, Berkeley, CA 94720 USA.
Structural isomers of the S-2 state in photosystem II: do they exist at room temperature and are they important for function?2019In: Physiologia Plantarum: An International Journal for Plant Biology, ISSN 0031-9317, E-ISSN 1399-3054, Vol. 166, no 1, p. 60-72Article in journal (Refereed)

In nature, an oxo-bridged Mn4CaO5 cluster embedded in photosystem II (PSII), a membrane-bound multi-subunit pigment protein complex, catalyzes the water oxidation reaction that is driven by light-induced charge separations in the reaction center of PSII. The Mn4CaO5 cluster accumulates four oxidizing equivalents to enable the four-electron four-proton catalysis of two water molecules to one dioxygen molecule and cycles through five intermediate S-states, S-0-S-4 in the Kok cycle. One important question related to the catalytic mechanism of the oxygen-evolving complex (OEC) that remains is, whether structural isomers are present in some of the intermediate S-states and if such equilibria are essential for the mechanism of the O-O bond formation. Here we compare results from electron paramagnetic resonance (EPR) and X-ray absorption spectroscopy (XAS) obtained at cryogenic temperatures for the S-2 state of PSII with structural data collected of the S-1, S-2 and S-3 states by serial crystallography at neutral pH (approximate to 6.5) using an X-ray free electron laser at room temperature. While the cryogenic data show the presence of at least two structural forms of the S-2 state, the room temperature crystallography data can be well-described by just one S-2 structure. We discuss the deviating results and outline experimental strategies for clarifying this mechanistically important question.

• 18.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Structural and Biochemical Studies of Antibiotic Resistance and Ribosomal Frameshifting2013Doctoral thesis, comprehensive summary (Other academic)

Protein synthesis, translation, performed by the ribosome, is a fundamental process of life and one of the main targets of antibacterial drugs. This thesis provides structural and biochemical understanding of three aspects of bacterial translation.

Elongation factor G (EF-G) is the target for the antibiotic fusidic acid (FA). FA binds to EF-G only on the ribosome after GTP hydrolysis and prevents EF-G dissociation from the ribosome. Point mutations in EF-G can lead to FA resistance but are often accompanied by a fitness cost in terms of slower growth of the bacteria. Secondary mutations can compensate for this fitness cost while resistance is maintained. Here we present the crystal structure of the clinical FA drug target, Staphylococcus aureus EF-G, together with the mapping and analysis of all known FA-resistance mutations in EF-G. We also present crystal structures of the FA-resistant mutant F88L, the FA-hypersensitive mutant M16I and the FA-resistant but fitness-compensated double mutant F88L/M16I. Analysis of mutant structures together with biochemical data allowed us to propose that fitness loss and compensation are caused by effects on the conformational dynamics of EF-G on the ribosome.

Aminoglycosides are another group of antibiotics that target the decoding region of the 30S ribosomal subunit. Resistance to aminoglycosides can be acquired by inactivation of the drugs via enzymatic modification. Here, we present the first crystal structure an aminoglycoside 3’’ adenyltransferase, AadA from Salmonella enterica. AadA displays two domains and unlike related structures most likely functions as a monomer.

Frameshifts are deviations the standard three-base reading frame of translation. -1 frameshifting can be caused by normal tRNASer3 at GCA alanine codons and tRNAThr3 at CCA/CCG proline codons. This process has been proposed to involve doublet decoding using non-standard codon-anticodon interactions. In our study, we showed by equilibrium binding that these tRNAs bind with low micromolar Kd to the frameshift codons. Our results support the doublet-decoding model and show that non-standard anticodon loop structures need to be adopted for the frameshifts to happen.

These findings provide new insights in antibiotic resistance and reading-frame maintenance and will contribute to a better understanding of the translation elongation process.

1. Staphylococcus aureus elongation factor G - structure and analysis of a target for fusidic acid
Open this publication in new window or tab >>Staphylococcus aureus elongation factor G - structure and analysis of a target for fusidic acid
2010 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 277, no 18, p. 3789-3803Article in journal (Refereed) Published
##### Abstract [en]

Fusidic acid (FA) is a bacteriostatic antibiotic that locks elongation factor G (EF-G) on the ribosome in a post-translocational state. It is used clinically against Gram-positive bacteria such as pathogenic strains of Staphylococcus aureus, but no structural information has been available for EF-G from these species. We have solved the apo crystal structure of EF-G from S. aureus to 1.9 A resolution. This structure shows a dramatically different overall conformation from previous structures of EF-G, although the individual domains are highly similar. Between the different structures of free or ribosome-bound EF-G, domains III-V move relative to domains I-II, resulting in a displacement of the tip of domain IV relative to domain G. In S. aureus EF-G, this displacement is about 25 A relative to structures of Thermus thermophilus EF-G in a direction perpendicular to that in previous observations. Part of the switch I region (residues 46-56) is ordered in a helix, and has a distinct conformation as compared with structures of EF-Tu in the GDP and GTP states. Also, the switch II region shows a new conformation, which, as in other structures of free EF-G, is incompatible with FA binding. We have analysed and discussed all known fusA-based fusidic acid resistance mutations in the light of the new structure of EF-G from S. aureus, and a recent structure of T. thermophilus EF-G in complex with the 70S ribosome with fusidic acid [Gao YG et al. (2009) Science326, 694-699]. The mutations can be classified as affecting FA binding, EF-G-ribosome interactions, EF-G conformation, and EF-G stability.

##### Keywords
antibiotic resistance, crystallography, elongation factor G (EF-G), fusidic acid, switch region
##### National Category
Biological Sciences
##### Identifiers
urn:nbn:se:uu:diva-134901 (URN)10.1111/j.1742-4658.2010.07780.x (DOI)000281555600016 ()
Available from: 2010-12-02 Created: 2010-12-02 Last updated: 2017-12-12Bibliographically approved
2. Mechanism of Elongation Factor-G-mediated Fusidic Acid Resistance and Fitness Compensation in Staphylococcus aureus
Open this publication in new window or tab >>Mechanism of Elongation Factor-G-mediated Fusidic Acid Resistance and Fitness Compensation in Staphylococcus aureus
2012 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 36, p. 30257-30267Article in journal (Refereed) Published
##### Abstract [en]

Antibiotic resistance in bacteria is often associated with fitness loss, which is compensated by secondary mutations. Fusidic acid (FA), an antibiotic used against pathogenic bacteria Staphylococcus aureus, locks elongation factor-G (EF-G) to the ribosome after GTP hydrolysis. To clarify the mechanism of fitness loss and compensation in relation to FA resistance, we have characterized three S. aureus EF-G mutants with fast kinetics and crystal structures. Our results show that a significantly slower tRNA translocation and ribosome recycling, plus increased peptidyl-tRNA drop-off, are the causes for fitness defects of the primary FA-resistant mutant F88L. The double mutant F88L/M16I is three to four times faster than F88L in both reactions and showed no tRNA drop-off, explaining its fitness compensatory phenotype. The M16I mutation alone showed hypersensitivity to FA, higher activity, and somewhat increased affinity to GTP. The crystal structures demonstrate that Phe-88 in switch II is a key residue for FA locking and also for triggering interdomain movements in EF-G essential for its function, explaining functional deficiencies in F88L. The mutation M16I loosens the hydrophobic core in the G domain and affects domain I to domain II contact, resulting in improved activity both in the wild-type and F88L background. Thus, FA-resistant EF-G mutations causing fitness loss and compensation operate by affecting the conformational dynamics of EF-G on the ribosome.

##### Keywords
Antibiotic Resistance, GTPase, Staphylococcus aureus, Translation, Translation Elongation Factors
Cell Biology
##### Identifiers
urn:nbn:se:uu:diva-182775 (URN)10.1074/jbc.M112.378521 (DOI)000308579800019 ()
Available from: 2012-10-17 Created: 2012-10-15 Last updated: 2017-12-07Bibliographically approved
3. Crystal structure of AadA at 2.5 Å resolution - an aminoglycoside 3" adenyltransferase
Open this publication in new window or tab >>Crystal structure of AadA at 2.5 Å resolution - an aminoglycoside 3" adenyltransferase
##### National Category
Structural Biology
##### Research subject
Biology with specialization in Structural Biology
##### Identifiers
urn:nbn:se:uu:diva-205128 (URN)
Available from: 2013-08-14 Created: 2013-08-14 Last updated: 2016-11-30
4. tRNASer and tRNAThr induce -1 frameshifting using alternative anticodon-loop structures
Open this publication in new window or tab >>tRNASer and tRNAThr induce -1 frameshifting using alternative anticodon-loop structures
##### National Category
Biochemistry and Molecular Biology
Biology
##### Identifiers
urn:nbn:se:uu:diva-205130 (URN)
Available from: 2013-08-14 Created: 2013-08-14 Last updated: 2014-01-22
• 19.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
• 20.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
Structure of AadA from Salmonella enterica: a monomeric aminoglycoside (3'')(9) adenyltransferase2015In: Acta Crystallographica Section D: Biological Crystallography, ISSN 0907-4449, E-ISSN 1399-0047, Vol. 71, p. 2267-2277Article in journal (Refereed)

Aminoglycoside resistance is commonly conferred by enzymatic modification of drugs by aminoglycoside-modifying enzymes such as aminoglycoside nucleo\-tidyltransferases (ANTs). Here, the first crystal structure of an ANT(3$^\prime$$^\prime$)(9) adenyltransferase, AadA from Salmonella enterica, is presented. AadA catalyses the magnesium-dependent transfer of adenosine monophosphate from ATP to the two chemically dissimilar drugs streptomycin and spectinomycin. The structure was solved using selenium SAD phasing and refined to 2.5Å resolution. AadA consists of a nucleotidyltransferase domain and an α-helical bundle domain. AadA crystallizes as a monomer and is a monomer in solution as confirmed by small-angle X-ray scattering, in contrast to structurally similar homodimeric adenylating enzymes such as kanamycin nucleotidyltransferase. Isothermal titration calorimetry experiments show that ATP binding has to occur before binding of the aminoglycoside substrate, and structure analysis suggests that ATP binding repositions the two domains for aminoglycoside binding in the interdomain cleft. Candidate residues for ligand binding and catalysis were subjected to site-directed mutagenesis. In vivo resistance and in vitro binding assays support the role of Glu87 as the catalytic base in adenylation, while Arg192 and Lys205 are shown to be critical for ATP binding.

• 21.
Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Institute of Molecular and Cell Biology, A*STAR, Singapore. epartment of Chemistry and Centre for Blood Research, Life Sciences Institute, The University of British Columbia, Canada. Institute of Molecular and Cell Biology, A*STAR, Singapore.
The crystal structure of the C-terminus of adseverin: Implications for actin bindingManuscript (Other (popular science, discussion, etc.))

• 22. Claesson, Magnus
Karolinska Institutet.
Crystal structure of the glycosyltransferase SnogD from the biosynthetic pathway of nogalamycin in Streptomyces nogalater2012In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 279, no 17, p. 3251-3263Article in journal (Refereed)

The glycosyltransferase SnogD from Streptomyces nogalater transfers a nogalamine moiety to the metabolic intermediate 3',4'-demethoxynogalose-1-hydroxynogalamycinone during the final steps of biosynthesis of the aromatic polyketide nogalamycin. The crystal structure of recombinant SnogD, as an apo-enzyme and with a bound nucleotide, 2-deoxyuridine-5'-diphosphate, was determined to 2.6 Å resolution. Reductive methylation of SnogD was crucial for reproducible preparation of diffraction quality crystals due to creation of an additional intermolecular salt bridge between methylated lysine residue Lys384 and Glu374* from an adjacent molecule in the crystal lattice. SnogD is a dimer both in solution and in the crystal, and the enzyme subunit displays a fold characteristic of the GT-B family of glycosyltransferases. Binding of the nucleotide is associated with rearrangement of two active-site loops. Site-directed mutagenesis shows that two active-site histidine residues, His25 and His301, are critical for the glycosyltransferase activities of SnogD both in vivo and in vitro. The crystal structures and the functional data are consistent with a role for His301 in binding of the diphosphate group of the sugar donor substrate, and a function of His25 as a catalytic base in the glycosyl transfer reaction.

• 23.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
University of Oxford. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
Lipids Shape the Electron Acceptor-Binding Site of the Peripheral Membrane Protein Dihydroorotate Dehydrogenase2018In: Cell Chemical Biology, ISSN 2451-9456, E-ISSN 2451-9448, Vol. 25, no 3, p. 309-317Article in journal (Refereed)

The interactions between proteins and biological membranes are important for drug development, but remain notoriously refractory to structural investigation. We combine non-denaturing mass spectrometry (MS) with molecular dynamics (MD) simulations to unravel the connections among co-factor, lipid, and inhibitor binding in the peripheral membrane protein dihydroorotate dehydrogenase (DHODH), a key anticancer target. Interrogation of intact DHODH complexes by MS reveals that phospholipids bind via their charged head groups at a limited number of sites, while binding of the inhibitor brequinar involves simultaneous association with detergent molecules. MD simulations show that lipids support flexible segments in the membrane-binding domain and position the inhibitor and electron acceptor-binding site away from the membrane surface, similar to the electron acceptor-binding site in respiratory chain complex I. By complementing MS with MD simulations, we demonstrate how a peripheral membrane protein uses lipids to modulate its structure in a similar manner as integral membrane proteins.

• 24.
Karolinska Institutet.
Crystallization and X-ray diffraction analysis of dihydropyrimidinase from Saccharomyces kluyveri2005In: Acta Crystallographica. Section F: Structural Biology and Crystallization Communications, ISSN 1744-3091, E-ISSN 1744-3091, Vol. 61, no Pt 4, p. 359-362Article in journal (Refereed)

Dihydropyrimidinase (EC 3.5.2.2) catalyzes the second step in the reductive pathway of pyrimidine degradation, the hydrolysis of 5,6-dihydrouracil and 5,6-dihydrothymine to the corresponding N-carbamylated beta-amino acids. Crystals of the recombinant enzyme from the yeast Saccharomyces kluyveri diffracting to 2.6 A at a synchrotron-radiation source have been obtained by the hanging-drop vapour-diffusion method. They belong to space group P2(1) (unit-cell parameters a = 91.0, b = 73.0, c = 161.4 A, beta = 91.4 degrees), with one homotetramer per asymmetric unit.

• 25.
Karolinska Institutet.
Crystallization and preliminary X-ray analysis of beta-alanine synthase from the yeast Saccharomyces kluyveri2003In: Acta Crystallographica Section D: Biological Crystallography, ISSN 0907-4449, E-ISSN 1399-0047, Vol. 59, no Pt 7, p. 1267-1269Article in journal (Refereed)

In eukaryotes and some bacteria, the third step of reductive pyrimidine catabolism is catalyzed by beta-alanine synthase (EC 3.5.1.6). Crystals of the recombinant enzyme from the yeast Saccharomyces kluyveri were obtained using sodium citrate as a precipitant. The crystals belong to space group P2(1) (unit-cell parameters a = 117.2, b = 77.1, c = 225.5 A, beta = 95.0 degrees ) and contain four homodimers per asymmetric unit. Data were collected to 2.7 A resolution. Introduction of heavy atoms into the crystal lattice induced a different set of unit-cell parameters (a = 61.0, b = 77.9, c = 110.1 A, beta = 97.2 degrees ) in the same space group P2(1), with only one homodimer per asymmetric unit.

• 26.
Martin-Luther-Universität Halle-Wittenberg.
High resolution crystal structure of pyruvate decarboxylase from Zymomonas mobilis: Implications for substrate activation in pyruvate decarboxylases1998In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 273, no 32, p. 20196-20204Article in journal (Refereed)

The crystal structure of tetrameric pyruvate decarboxylase from Zymomonas mobilis has been determined at 1.9 A resolution and refined to a crystallographic R-factor of 16.2% and Rfree of 19.7%. The subunit consists of three domains, all of the alpha/beta type. Two of the subunits form a tight dimer with an extensive interface area. The thiamin diphosphate binding site is located at the subunit-subunit interface, and the cofactor, bound in the V conformation, interacts with residues from the N-terminal domain of one subunit and the C-terminal domain of the second subunit. The 2-fold symmetry generates the second thiamin diphosphate binding site in the dimer. Two of the dimers form a tightly packed tetramer with pseudo 222 symmetry. The interface area between the dimers is much larger in pyruvate decarboxylase from Z. mobilis than in the yeast enzyme, and structural differences in these parts result in a completely different packing of the subunits in the two enzymes. In contrast to other pyruvate decarboxylases, the enzyme from Z. mobilis is not subject to allosteric activation by the substrate. The tight packing of the dimers in the tetramer prevents large rearrangements in the quaternary structure as seen in the yeast enzyme and locks the enzyme in an activated conformation. The architecture of the cofactor binding site and the active site is similar in the two enzymes. However, the x-ray analysis reveals subtle but significant structural differences in the active site that might be responsible for variations in the biochemical properties in these enzymes.

• 27.
Karolinska Institutet.
Crystal structure of an arthritogenic anticollagen immune complex2011In: Arthritis and Rheumatism, ISSN 0004-3591, E-ISSN 1529-0131, Vol. 63, no 12, p. 3740-3748Article in journal (Refereed)

OBJECTIVE: In rheumatoid arthritis, joint inflammation and cartilage destruction are mediated by autoantibodies directed to various self antigens. Type II collagen (CII)-specific antibodies are likely to play a role in this process and have been shown to induce experimental arthritis in susceptible animals. The purpose of this study was to reveal how arthritogenic autoantibodies recognize native CII in its triple-helical conformation.

METHODS: Site-directed mutagenesis and crystallographic studies were performed to reveal crucial contact points between the CII antibody and the triple-helical CII peptide.

RESULTS: The crystal structure of a pathogenic autoantibody bound to a major triple-helical epitope present on CII was determined, allowing a first and detailed description of the interactions within an arthritogenic complex that is frequently occurring in both mice and humans with autoimmune arthritis. The crystal structure emphasizes the role of arginine residues located in a commonly recognized motif on CII and reveals that germline-encoded elements are involved in the interaction with the epitope.

CONCLUSION: The crystal structure of an arthritogenic antibody binding a triple-helical epitope on CII indicates a crucial role of germline-encoded and arginine residues as the target structures.

• 28.
Karolinska Institutet.
Crystallization and preliminary X-ray study of pig liver dihydropyrimidine dehydrogenase2001In: Acta Crystallographica Section D: Biological Crystallography, ISSN 0907-4449, E-ISSN 1399-0047, Vol. 57, no Pt 1, p. 153-155Article in journal (Refereed)

Dihydropyrimidine dehydrogenase catalyzes the first and rate-limiting reaction in pyrimidine catabolism. The enzyme contains one FMN, one FAD and four Fe-S clusters per subunit of 1025 amino acids as prosthetic groups. It is also the major determinant of bioavailability and toxicity of 5-fluorouracil, a chemotherapeutic agent widely used in the treatment of solid tumors. Crystals of this enzyme diffracting to at least 2.5 A have been obtained by the hanging-drop vapour-diffusion method and belong to space group P2(1) (unit-cell parameters a = 82.0, b = 159.3, c = 163.6 A, beta = 96.1 degrees ), with two homodimers per asymmetric unit.

• 29.
Karolinska Institutet.
Crystal structure of the productive ternary complex of dihydropyrimidine dehydrogenase with NADPH and 5-iodouracil: Implications for mechanism of inhibition and electron transfer2002In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 277, no 15, p. 13155-13166Article in journal (Refereed)

Dihydroprymidine dehydrogenase catalyzes the first and rate-limiting step in pyrimidine degradation by converting pyrimidines to the corresponding 5,6- dihydro compounds. The three-dimensional structures of a binary complex with the inhibitor 5-iodouracil and two ternary complexes with NADPH and the inhibitors 5-iodouracil and uracil-4-acetic acid were determined by x-ray crystallography. In the ternary complexes, NADPH is bound in a catalytically competent fashion, with the nicotinamide ring in a position suitable for hydride transfer to FAD. The structures provide a complete picture of the electron transfer chain from NADPH to the substrate, 5-iodouracil, spanning a distance of 56 A and involving FAD, four [Fe-S] clusters, and FMN as cofactors. The crystallographic analysis further reveals that pyrimidine binding triggers a conformational change of a flexible active-site loop in the alpha/beta-barrel domain, resulting in placement of a catalytically crucial cysteine close to the bound substrate. Loop closure requires physiological pH, which is also necessary for correct binding of NADPH. Binding of the voluminous competitive inhibitor uracil-4-acetic acid prevents loop closure due to steric hindrance. The three-dimensional structure of the ternary complex enzyme-NADPH-5-iodouracil supports the proposal that this compound acts as a mechanism-based inhibitor, covalently modifying the active-site residue Cys-671, resulting in S-(hexahydro-2,4-dioxo-5-pyrimidinyl)cysteine.

• 30.
Karolinska Institutet.
Crystal structure of dihydropyrimidine dehydrogenase, a major determinant of the pharmacokinetics of the anti-cancer drug 5-fluorouracil2001In: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 20, no 4, p. 650-660Article in journal (Refereed)

Dihydropyrimidine dehydrogenase catalyzes the first step in pyrimidine degradation: the NADPH-dependent reduction of uracil and thymine to the corresponding 5,6-dihydropyrimidines. Its controlled inhibition has become an adjunct target for cancer therapy, since the enzyme is also responsible for the rapid breakdown of the chemotherapeutic drug 5-fluorouracil. The crystal structure of the homodimeric pig liver enzyme (2x 111 kDa) determined at 1.9 A resolution reveals a highly modular subunit organization, consisting of five domains with different folds. Dihydropyrimidine dehydrogenase contains two FAD, two FMN and eight [4Fe-4S] clusters, arranged in two electron transfer chains that pass the dimer interface twice. Two of the Fe-S clusters show a hitherto unobserved coordination involving a glutamine residue. The ternary complex of an inactive mutant of the enzyme with bound NADPH and 5-fluorouracil reveals the architecture of the substrate-binding sites and residues responsible for recognition and binding of the drug.

• 31.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab. Univ Oxford, Chem Res Lab, 12 Mansfield Rd, Oxford OX1 3TA, England.;Univ Oxford, Phys & Theoret Chem Lab, S Parks Rd, Oxford OX1 3QZ, England..
Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics. Uppsala University, Science for Life Laboratory, SciLifeLab. Univ Sheffield, Dept Chem, Sheffield S3 7HF, S Yorkshire, England.. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
The Competing Mechanisms of Phosphate Monoester Dianion Hydrolysis2016In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 138, no 33, p. 10664-10673Article in journal (Refereed)

Despite the numerous experimental and theoretical studies on phosphate monoester hydrolysis, significant questions remain concerning the mechanistic details of these biologically critical reactions. In the present work we construct a linear free energy relationship for phosphate monoester hydrolysis to explore the effect of modulating leaving group plc on the competition between solvent- and substrate-assisted pathways for the hydrolysis of these compounds. Through detailed comparative electronic-structure studies of methyl phosphate and a series of substituted aryl phosphate monoesters, we demonstrate that the preferred mechanism is dependent on the nature of the leaving group. For good leaving groups, a strong preference is observed for a more dissociative solvent-assisted pathway. However, the energy difference between the two pathways gradually reduces as the leaving group pK(a) increases and creates mechanistic ambiguity for reactions involving relatively poor alkoxy leaving groups. Our calculations show that the transition-state structures vary smoothly across the range of pK(a)s studied and that the pathways remain discrete mechanistic alternatives. Therefore, while not impossible, a biological catalyst would have to surmount a significantly higher activation barrier to facilitate a substrate-assisted pathway than for the solvent-assisted pathway when phosphate is bonded to good leaving groups. For poor leaving groups, this intrinsic preference disappears.

• 32.
Aix Marseille Univ, Inst Paoli Calmettes, INSERM, CRCM,U1068,CNRS UMR7258, F-13009 Marseille, France; Katholieke Univ Leuven, Dept Human Genet, ON1 Herestr 49,Box 602, B-3000 Leuven, Belgium.
Katholieke Univ Leuven, Dept Human Genet, ON1 Herestr 49,Box 602, B-3000 Leuven, Belgium;Katholieke Univ Leuven, Dept Mol Cellular & Mol Med, VIB, VIB Switch Lab, B-3000 Leuven, Belgium. Vrije Univ Brussel, Struct Biol Brussels, Dept Biotechnol DBIT, Pl Laan 2, B-1050 Brussels, Belgium; VIB, Mol Recognit Unit, Struct Biol Res Ctr, Pl Laan 2, B-1050 Brussels, Belgium; Univ Libre Bruxelles, Biol Struct & Biophys, CP300,Rue Prof Jeener & Brachet 12, B-6041 Gosselies, Belgium. Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry. Katholieke Univ Leuven, Dept Human Genet, ON1 Herestr 49,Box 602, B-3000 Leuven, Belgium. Katholieke Univ Leuven, Dept Human Genet, ON1 Herestr 49,Box 602, B-3000 Leuven, Belgium. Aix Marseille Univ, Inst Paoli Calmettes, INSERM, CRCM,U1068,CNRS UMR7258, F-13009 Marseille, France; Katholieke Univ Leuven, Dept Human Genet, ON1 Herestr 49,Box 602, B-3000 Leuven, Belgium. Vrije Univ Brussel, Struct Biol Brussels, Dept Biotechnol DBIT, Pl Laan 2, B-1050 Brussels, Belgium; VIB, Mol Recognit Unit, Struct Biol Res Ctr, Pl Laan 2, B-1050 Brussels, Belgium. Katholieke Univ Leuven, Dept Mol Cellular & Mol Med, VIB, VIB Switch Lab, B-3000 Leuven, Belgium. Katholieke Univ Leuven, Dept Mol Cellular & Mol Med, VIB, VIB Switch Lab, B-3000 Leuven, Belgium. Aix Marseille Univ, Inst Paoli Calmettes, INSERM, CRCM,U1068,CNRS UMR7258, F-13009 Marseille, France; Katholieke Univ Leuven, Dept Human Genet, ON1 Herestr 49,Box 602, B-3000 Leuven, Belgium.
Frizzled 7 and PIP2 binding by syntenin PDZ2 domain supports Frizzled 7 trafficking and signalling2016In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 7, article id 12101Article in journal (Refereed)

PDZ domain-containing proteins work as intracellular scaffolds to control spatio-temporal aspects of cell signalling. This function is supported by the ability of their PDZ domains to bind other proteins such as receptors, but also phosphoinositide lipids important for membrane trafficking. Here we report a crystal structure of the syntenin PDZ tandem in complex with the carboxy-terminal fragment of Frizzled 7 and phosphatidylinositol 4,5-bisphosphate (PIP2). The crystal structure reveals a tripartite interaction formed via the second PDZ domain of syntenin. Biophysical and biochemical experiments establish co-operative binding of the tripartite complex and identify residues crucial for membrane PIP2-specific recognition. Experiments with cells support the importance of the syntenin-PIP2 interaction for plasma membrane targeting of Frizzled 7 and c-jun phosphorylation. This study contributes to our understanding of the biology of PDZ proteins as key players in membrane compartmentalization and dynamics.

• 33.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology.
Purification, functional characterization and crystallization of the PerR peroxide sensor from Saccharopolyspora erythraea2019Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis

This report summarizes the work on the cloning, expression, and purification of PerR, a metal sensing regulator from Saccharopolyspora erythraea and the subsequent characterization using small angle X-ray scattering and other biochemical methods. The report aims to provide an insight into prokaryotic metal homeostasis, provide a better understanding of how PerR works and provide valuable information for the continued work on the crystallization of PerR.

• 34.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
HisA mutants with minor structural differences display major functional deviations2016Independent thesis Advanced level (degree of Master (Two Years)), 40 credits / 60 HE creditsStudent thesis

Even though enzymes tend to specialize on one reaction during evolution, enzyme promiscuity is an abundant phenomenon. The subject of this thesis is the Salmonella enterica N’-[(5’-phosphosoribosyl)-formimino]-5-aminoimidazole-4 carboxamide-ribonucleotide (ProFAR) isomerase (SeHisA), a (βα)8-barrel enzyme from the histidine biosynthesis that catalyzes one reaction on one substrate in one organism. In Actinobacteria HisA has evolved to a bifunctional enzyme called phosphoribosyl isomerase A (PriA): it is capable of catalyzing the reaction normally done by the N’-(5’-phosphoribosyl) anthranilate (PRA) isomerase (TrpF) as well. The functional plasticity of PriA is possible due to the common reaction mechanism of HisA and TrpF, called Amadori rearrangement. The Amadori rearrangement is an acid-base catalyzed isomerization reaction where the aminoaldose (ProFAR or PRA) is converted into the corresponding ketose (PRFAR or CdRP). A SeHisA mutant with a glutamine to arginine mutation in position 18 (Q18R) shows a detectable TrpF activity, whereas another mutant with a duplication of residues from 13 to 15 (dup13-15) loses its HisA activity and gains TrpF activity.

My first aim was to improve the TrpF activity of the Q18R mutant. A G79S mutation was introduced inspired by PriA. Proteins were purified and crystallized. In order to gain complex structures with either the TrpF reaction product analogue reduced CdRP (rCdRP) or ProFAR, co-crystallization and soaking were done. ProFAR is not commercially available and had to be synthetized and purified. In vitro TrpF activities of the Q18R and Q18R/G79S mutants were measured. My second aim was to compare the Q18R mutant with the dup13-15 mutant, since there is very little structural difference between them, but they show high difference in catalytic activity. Mutants, which would bridge the functional gap between them, were designed and by using lambda red recombineering were introduced into a Salmonella typhimurium genome. In vivo growth rate was measured and relative fitness was calculated for each mutant in respect to their HisA and TrpF activity.

HisA mutants Q18R and Q18R/G79S showed very poor TrpF activity in in vitro assays. No dissociation constants could be measured for either of the mutants using microscale thermophoresis, and a very low kcat/KM value (~2 s-1M-1) with a high error rate could be determined for Q18R/G79S. Complex structures of Q18R and Q18R/G79S mutants with ProFAR were solved at a 2.47 Å and a 1.78 Å, respectively, from soaked crystals. No structure with rCdRP was obtained. Growth rate measurements in comparison with a strain with wild type HisA and TrpF, gave striking results pointing out the important role of the residue in the position 16 when three residues are inserted after the arginine in position 18. A leucine in position 16 yielded wild type HisA activity (94%) and poor TrpF activity (0-5%), with a valine, no HisA activity (0%) and a moderate TrpF activity (38-46%) were found compared to the wild type. These results pointed out how small the barrier between a specialist, a promiscuous and a bifunctional enzyme can be. SeHisA, being a specialized enzyme can easily be modified in order to gain TrpF function, and as I have showed in the present study, a single methyl group (the difference between a leucine and a valine) can turn the activity of an enzyme inside out.

• 35.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
Tunnels and Grooves: Structure-Function Studies in Two Disparate Enzymes2009Doctoral thesis, comprehensive summary (Other academic)

This thesis describes structural and binding studies in enzymes from two different  organisms: ribonucleotide reductase from Mycobacterium tuberculosis (RNR) and lipase A from Candida antarctica (CalA).

RNR is viable as a target for new drugs against the causative agent of tuberculosis. The biologically active form of RNR is a heterotetramer with an α2β2 substructure. Here we show that an N-acetylated heptapeptide based on the C-terminal sequence of the smaller RNR subunit can disrupt the formation of the holoenzyme sufficiently to inhibit its function. An N-terminal truncation, an alanine scan and a novel statistical molecular design approach based on the heptapeptide Ac-Glu-Asp-Asp-Asp-Trp-Asp-Phe-OH were applied. A full-length acetylated heptapeptide was necessary for inhibition, and Trp5 and Phe7 were also essential. Exchanging the acetyl for the N-terminal Fmoc protective-group increased the binding potency ten-fold. Based on this, several truncated and N-protected peptides were evaluated in a competitive fluorescence polarization assay. The single-amino acid Fmoc-Trp inhibits the RNR holoenzyme formation with a dissociation constant of 12µM, making it an attractive candidate for further development of non-peptidic inhibitors

Lipases are enzymes with major biotechnological applications. We report the x-ray structure of CalA, the first member of a novel family of lipases. The fold includes a well-defined lid as well as a classical α/β hydrolase domain. The structure is that of the closed/inactive state of the enzyme, but loop movements near Phe431 will provide virtually unlimited access to solvent for the alcohol moiety of an ester substrate. The structure thus provides a basis for understanding the enzyme's preference for acyl moieties with long, straight tails, and for its highly promiscuous acceptance of widely different alcohol and amine moieties. An unconventional oxyanion hole is observed in the present structure, although the situation may change during interfacial activation.

1. Design, synthesis and evaluation of peptide inhibitors of Mycobacterium tuberculosis ribonucleotide reductase
Open this publication in new window or tab >>Design, synthesis and evaluation of peptide inhibitors of Mycobacterium tuberculosis ribonucleotide reductase
2007 (English)In: Journal of Peptide Science, ISSN 1075-2617, E-ISSN 1099-1387, Vol. 13, no 12, p. 822-832Article in journal (Refereed) Published
##### Abstract [en]

Mycobacterium tuberculosis ribonucleotide reductase (RNR) is a potential target for new antitubercular drugs. Herein we describe the synthesis and evaluation of peptide inhibitors of RNR derived from the C-terminus of the small subunit of M. tuberculosis RNR. An N-terminal truncation, an alanine scan and a novel statistical molecular design (SMD) approach based on the heptapeptide Ac-Glu-Asp-Asp-Asp-Trp-Asp-Phe-OH were applied in this study. The alanine scan showed that TrP5 and Phe7 were important for inhibitory potency. A quantitative structure relationship (QSAR) model was developed based on the synthesized peptides which showed that a negative charge in positions 2, 3, and 6 is beneficial for inhibitory potency. Finally, in position 5 the model coefficients indicate that there is room for a larger side chain., as compared to Trp5.

##### Keywords
mycobacterium tuberculosis, ribonucleotide reductase, peptide inhibitors, alanine scan, statistical molecular design, structure activity relationships, FHDoE
##### National Category
Pharmaceutical Sciences
##### Identifiers
urn:nbn:se:uu:diva-14261 (URN)10.1002/psc.906 (DOI)000252000600007 ()17918768 (PubMedID)
Available from: 2008-05-29 Created: 2008-05-29 Last updated: 2018-01-12Bibliographically approved
2. Identification of small peptides mimicking the R2 C-terminus of Mycobacterium tuberculosis ribonucleotide reductase
Open this publication in new window or tab >>Identification of small peptides mimicking the R2 C-terminus of Mycobacterium tuberculosis ribonucleotide reductase
2010 (English)In: Journal of Peptide Science, ISSN 1075-2617, E-ISSN 1099-1387, Vol. 16, no 3, p. 159-164Article in journal (Refereed) Published
##### Abstract [en]

Ribonucleotide reductase (RNR) is a viable target for new drugs against the causative agent of tuberculosis, Mycobacterium tuberculosis. Previous work has shown that an N-acetylated heptapeptide based on the C-terminal sequence of the smaller RNR subunit can disrupt the formation of the holoenzyme sufficiently to inhibit its function. Here the synthesis and binding affinity, evaluated by competitive fluorescence polarization, of several truncated and N-protected peptides are described. The protected single-amino acid Fmoc-Trp shows binding affinity comparable to the N-acetylated heptapeptide, making it an attractive candidate for further development of non-peptidic RNR inhibitors.

##### Keywords
Fluorescence polarization, Mycobacterium tuberculosis, Peptide inhibitors, Ribonucleotide reductase
##### National Category
Pharmaceutical Sciences
##### Identifiers
urn:nbn:se:uu:diva-112344 (URN)10.1002/psc.1214 (DOI)000275448300007 ()20127854 (PubMedID)
Available from: 2010-01-26 Created: 2010-01-13 Last updated: 2018-01-12Bibliographically approved
3. X-ray structure of Candida antarctica lipase A shows a novel lid structure and a likely mode of interfacial activation
Open this publication in new window or tab >>X-ray structure of Candida antarctica lipase A shows a novel lid structure and a likely mode of interfacial activation
2008 (English)In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 376, no 1, p. 109-119Article in journal (Refereed) Published
##### Abstract [en]

In nature, lipases (EC 3.1.1.3) catalyze the hydrolysis of triglycerides to form glycerol and fatty acids. Under the appropriate conditions, the reaction is reversible, and so biotechnological applications commonly make use of their capacity for esterification as well as for hydrolysis of a wide variety of compounds. In the present paper, we report the X-ray structure of lipase A from Candida antarctica, solved by single isomorphous replacement with anomalous scattering, and refined to 2.2-A resolution. The structure is the first from a novel family of lipases. Contrary to previous predictions, the fold includes a well-defined lid as well as a classic alpha/beta hydrolase domain. The catalytic triad is identified as Ser184, Asp334 and His366, which follow the sequential order considered to be characteristic of lipases; the serine lies within a typical nucleophilic elbow. Computer docking studies, as well as comparisons to related structures, place the carboxylate group of a fatty acid product near the serine nucleophile, with the long lipid tail closely following the path through the lid that is marked by a fortuitously bound molecule of polyethylene glycol. For an ester substrate to bind in an equivalent fashion, loop movements near Phe431 will be required, suggesting the primary focus of the conformational changes required for interfacial activation. Such movements will provide virtually unlimited access to solvent for the alcohol moiety of an ester substrate. The structure thus provides a basis for understanding the enzyme's preference for acyl moieties with long, straight tails, and for its highly promiscuous acceptance of widely different alcohol and amine moieties. An unconventional oxyanion hole is observed in the present structure, although the situation may change during interfacial activation.

##### Keywords
lipase, interfacial activation, hydrolase, X-ray structure, substrate specificity
##### National Category
Biochemistry and Molecular Biology
##### Identifiers
urn:nbn:se:uu:diva-14221 (URN)10.1016/j.jmb.2007.10.079 (DOI)000253181500011 ()18155238 (PubMedID)
Available from: 2008-01-29 Created: 2008-01-29 Last updated: 2017-12-11Bibliographically approved
• 36. Fenn, Sebastian
Crystal Structure of an Anti-Ang2 CrossFab Demonstrates Complete Structural and Functional Integrity of the Variable Domain2013In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 8, no 4Article in journal (Refereed)

Bispecific antibodies are considered as a promising class of future biotherapeutic molecules. They comprise binding specificities for two different antigens, which may provide additive or synergistic modes of action. There is a wide variety of design alternatives for such bispecific antibodies, including the "CrossMab" format. CrossMabs contain a domain crossover in one of the antigen-binding (Fab) parts, together with the "knobs-and-holes" approach, to enforce the correct assembly of four different polypeptide chains into an IgG-like bispecific antibody. We determined the crystal structure of a hAng-2-binding Fab in its crossed and uncrossed form and show that CH1-CL-domain crossover does not induce significant perturbations of the structure and has no detectable influence on target binding.

• 37.
Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Uppsala University, Science for Life Laboratory, SciLifeLab. Center of Biotechnology, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil . Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology. Univ Konstanz, Dept Chem, Univ Str 10, D-78457 Constance, Germany. Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
Influence of Na+ and Mg2+ ions on RNA structures studied with molecular dynamics simulations2018In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 46, no 10, p. 4872-4882Article in journal (Refereed)

The structure of ribonucleic acid (RNA) polymers is strongly dependent on the presence of, in particular Mg2+ cations to stabilize structural features. Only in high-resolution X-ray crystallography structures can ions be identified reliably. Here, we perform molecular dynamics simulations of 24 RNA structures with varying ion concentrations. Twelve of the structures were helical and the others complex folded. The aim of the study is to predict ion positions but also to evaluate the impact of different types of ions (Na+ or Mg2+) and the ionic strength on structural stability and variations of RNA. As a general conclusion Mg2+ is found to conserve the experimental structure better than Na+ and, where experimental ion positions are available, they can be reproduced with reasonable accuracy. If a large surplus of ions is present the added electrostatic screening makes prediction of binding-sites less reproducible. Distinct differences in ion-binding between helical and complex folded structures are found. The strength of binding (Delta G(+) for breaking RNA atom-ion interactions) is found to differ between roughly 10 and 26 kJ/mol for the different RNA atoms. Differences in stability between helical and complex folded structures and of the influence of metal ions on either are discussed.

• 38.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology.
Old Lineages in a New Ecosystem: Diversification of Arcellinid Amoebae (Amoebozoa) and Peatland Mosses2014In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 4, p. e95238-Article in journal (Refereed)

Arcellinid testate amoebae (Amoebozoa) form a group of free-living microbial eukaryotes with one of the oldest fossil records known, yet several aspects of their evolutionary history remain poorly understood. Arcellinids occur in a range of terrestrial, freshwater and even brackish habitats; however, many arcellinid morphospecies such as Hyalosphenia papilio are particularly abundant in Sphagnum-dominated peatlands, a relatively new ecosystem that appeared during the diversification of Sphagnum species in the Miocene (5-20 Myr ago). Here, we reconstruct divergence times in arcellinid testate amoebae after selecting several fossils for clock calibrations and then infer whether or not arcellinids followed a pattern of diversification that parallels the pattern described for Sphagnum. We found that the diversification of core arcellinids occurred during the Phanerozoic, which is congruent with most arcellinid fossils but not with the oldest known amoebozoan fossil (i.e. at ca. 662 or ca. 750 Myr). Overall, Sphagnum and the Hyalospheniidae exhibit different patterns of diversification. However, an extensive molecular phylogenetic analysis of distinct clades within H. papilio species complex demonstrated a correlation between the recent diversification of H. papilio, the recent diversification of Sphagnum mosses, and the establishment of peatlands.

• 39. Flores, Samuel
Turning limited experimental information into 3D models of RNA2010In: RNA: A publication of the RNA Society, ISSN 1355-8382, E-ISSN 1469-9001, Vol. 16, no 9, p. 1769-1778Article in journal (Refereed)
• 40. Flores, Samuel
Stanford University.
Structural insights into pre-translocation ribosome motions2011In: Proceedings of the Pacific Symposium on BiocomputingArticle in journal (Refereed)
• 41.
Stanford University.
Stanford University.
Methods for building and refining 3D models of RNA2012In: "RNA 3D Structure Analysis and Prediction / [ed] Neocles Leontis and Eric Westhof, Springer London, 2012, 1Chapter in book (Other academic)
• 42. Frank, Matthias
Linac Coherent Light Source, 2575 Sand Hill Road, Menlo Park, CA 94025, USA.
Femtosecond X-ray diffraction from two-dimensional protein crystals2014In: IUCrJ, ISSN 2052-2525, Vol. 1, no 2, p. 95-100Article in journal (Refereed)

X-ray diffraction patterns from two-dimensional (2-D) protein crystals obtained using femtosecond X-ray pulses from an X-ray free-electron laser (XFEL) are presented. To date, it has not been possible to acquire transmission X-ray diffraction patterns from individual 2-D protein crystals due to radiation damage. However, the intense and ultrafast pulses generated by an XFEL permit a new method of collecting diffraction data before the sample is destroyed. Utilizing a diffract-before-destroy approach at the Linac Coherent Light Source, Bragg diffraction was acquired to better than 8.5 Å resolution for two different 2-D protein crystal samples each less than 10 nm thick and maintained at room temperature. These proof-of-principle results show promise for structural analysis of both soluble and membrane proteins arranged as 2-D crystals without requiring cryogenic conditions or the formation of three-dimensional crystals.

• 43.
University Bordeaux, INSERM, CNRS, Laboratoire Acides Nucléiques Régulations Naturelle et Artificielle (ARNA, U1212, UMR5320), IECB, Pessac, France.
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
Fundamentals of ion mobility spectrometry2018In: Current opinion in chemical biology, ISSN 1367-5931, E-ISSN 1879-0402, Vol. 42, p. 51-59, article id S1367-5931(17)30122-9Article, review/survey (Refereed)

Fundamental questions in ion mobility spectrometry have practical implications for analytical applications in general, and omics in particular, in three respects. (1) Understanding how ion mobility and collision cross section values depend on the collision gas, on the electric field and on temperature is crucial to ascertain their transferability across instrumental platforms. (2) Predicting collision cross section values for new analytes is necessary to exploit the full potential of ion mobility in discovery workflows. (3) Finally, understanding the fate of ion structures in the gas phase is essential to infer meaningful information on solution structures based on gas-phase ion mobility measurements. We review here the most recent advances in ion mobility fundamentals, relevant to these three aspects.

• 44. Galli, Lorenzo
Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Molecular and condensed matter physics. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular biophysics. Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Molecular and condensed matter physics.
Towards phasing using high X-ray intensity2015In: IUCrJ, ISSN 0972-6918, E-ISSN 2052-2525, Vol. 2, p. 627-634Article in journal (Refereed)

X-ray free-electron lasers (XFELs) show great promise for macromolecular structure determination from sub-micrometre-sized crystals, using the emerging method of serial femtosecond crystallography. The extreme brightness of the XFEL radiation can multiply ionize most, if not all, atoms in a protein, causing their scattering factors to change during the pulse, with a preferential ‘bleaching’ of heavy atoms. This paper investigates the effects of electronic damage on experimental data collected from a Gd derivative of lysozyme microcrystals at different X-ray intensities, and the degree of ionization of Gd atoms is quantified from phased difference Fourier maps. A pattern sorting scheme is proposed to maximize the ionization contrast and the way in which the local electronic damage can be used for a new experimental phasing method is discussed.

• 45.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Biology. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
Inhibition of translation termination by small molecules targeting ribosomal release factors2019In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, article id 15424Article in journal (Refereed)

The bacterial ribosome is an important drug target for antibiotics that can inhibit different stages of protein synthesis. Among the various classes of compounds that impair translation there are, however, no known small-molecule inhibitors that specifically target ribosomal release factors (RFs). The class I RFs are essential for correct termination of translation and they differ considerably between bacteria and eukaryotes, making them potential targets for inhibiting bacterial protein synthesis. We carried out virtual screening of a large compound library against 3D structures of free and ribosome-bound RFs in order to search for small molecules that could potentially inhibit termination by binding to the RFs. Here, we report identification of two such compounds which are found both to bind free RFs in solution and to inhibit peptide release on the ribosome, without affecting peptide bond formation.

• 46. Gorbalenya, Alexander E.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology. Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
Practical application of bioinformatics by the multidisciplinary VIZIER consortium2010In: Antiviral Research, ISSN 0166-3542, E-ISSN 1872-9096, Vol. 87, no 2, p. 95-110Article, review/survey (Refereed)

This review focuses on bioinformatics technologies employed by the EU-sponsored multidisciplinary VIZIER consortium (Comparative Structural Genomics of Viral Enzymes Involved in Replication, FP6 Project: 2004-511960, active from 1 November 2004 to 30 April 2009), to achieve its goals. From the management of the information flow of the project, to bioinformatics-mediated selection of RNA viruses and prediction of protein targets, to the analysis of 3D protein structures and antiviral compounds, these technologies provided a communication framework and integrated solutions for steady and timely advancement of the project. RNA viruses form a large class of major pathogens that affect humans and domestic animals. Such RNA viruses as HIV, Influenza virus and Hepatitis C virus are of prime medical concern today, but the identities of viruses that will threaten human population tomorrow are far from certain. To contain outbreaks of common or newly emerging infections, prototype drugs against viruses representing the Virus Universe must be developed. This concept was championed by the VIZIER project which brought together experts in diverse fields to produce a concerted and sustained effort for identifying and validating targets for antivirus therapy in dozens of RNA virus lineages.

• 47.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Biology Education Centre.
Crystallisation using cross-seeding2017Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
• 48.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology. Stockholm Univ, Dept Biochem & Biophys, S-10691 Stockholm, Sweden.
Karolinska Inst, Sci Life Lab, Dept Oncol Pathol, Canc Prote Mass Spectrometry, Box 1031, S-17121 Solna, Sweden. Stockholm Univ, Dept Biochem & Biophys, S-10691 Stockholm, Sweden. Stockholm Univ, Dept Biochem & Biophys, S-10691 Stockholm, Sweden.
Ether cross-link formation in the R2-like ligand-binding oxidase2018In: Journal of Biological Inorganic Chemistry, ISSN 0949-8257, E-ISSN 1432-1327, Vol. 23, no 6, p. 879-886Article in journal (Refereed)

R2-like ligand-binding oxidases contain a dinuclear metal cofactor which can consist either of two iron ions or one manganese and one iron ion, but the heterodinuclear Mn/Fe cofactor is the preferred assembly in the presence of Mn-II and Fe-II in vitro. We have previously shown that both types of cofactor are capable of catalyzing formation of a tyrosine-valine ether cross-link in the protein scaffold. Here we demonstrate that Mn/Fe centers catalyze cross-link formation more efficiently than Fe/Fe centers, indicating that the heterodinuclear cofactor is the biologically relevant one. We further explore the chemical potential of the Mn/Fe cofactor by introducing mutations at the cross-linking valine residue. We find that cross-link formation is possible also to the tertiary beta-carbon in an isoleucine, but not to the secondary beta-carbon or tertiary gamma-carbon in a leucine, nor to the primary beta-carbon of an alanine. These results illustrate that the reactivity of the cofactor is highly specific and directed.

• 49. Griese, Julia J.
Structure and DNA-binding activity of the Pyrococcus furiosus SMC protein hinge domain2011In: Proteins: Structure, Function, and Bioinformatics, ISSN 0887-3585, E-ISSN 1097-0134, Vol. 79, no 2, p. 558-568Article in journal (Refereed)

Structural Maintenance of Chromosomes (SMC) proteins are essential for a wide range of processes including chromosome structure and dynamics, gene regulation, and DNA repair. While bacteria and archaea have one SMC protein that forms a homodimer, eukaryotes possess three distinct SMC complexes, consisting of heterodimeric pairs of six different SMC proteins. SMC holocomplexes additionally contain several specific regulatory subunits. The bacterial SMC complex is required for chromosome condensation and segregation. In eukaryotes, this function is carried out by the condensin (SMC2-SMC4) complex. SMC proteins consist of N-terminal and C-terminal domains that fold back onto each other to create an ATPase "head" domain, connected to a central "hinge" domain via a long coiled-coil region. The hinge domain mediates dimerization of SMC proteins and binds DNA. This activity implicates a direct involvement of the hinge domain in the action of SMC proteins on DNA. We studied the SMC hinge domain from the thermophilic archaeon Pyrococcus furiosus. Its crystal structure shows that the SMC hinge domain fold is largely conserved between archaea and bacteria as well as eukarya. Like the eukaryotic condensin hinge domain, the P. furiosus SMC hinge domain preferentially binds single-stranded DNA (ssDNA), but its affinity for DNA is weaker than that of its eukaryotic counterpart, and point mutations reveal that its DNA-binding surface is more confined. The ssDNA-binding activity of its hinge domain might play a role in the DNA-loading process of the prokaryotic SMC complex during replication.

• 50.
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Biology. Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden.
Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden.
Location-specific quantification of protein-bound metal ions by X-ray anomalous dispersion: Q-XAD2019In: Acta Crystallographica Section D: Structural Biology, ISSN 2059-7983, Vol. 75, no 8, p. 764-771Article in journal (Refereed)

Here, a method is described which exploits X-ray anomalous dispersion (XAD) to quantify mixtures of metal ions in the binding sites of proteins and can be applied to metalloprotein crystals of average quality. This method has successfully been used to study site-specific metal binding in a protein from the R2-like ligand-binding oxidase family which assembles a hetero­dinuclear Mn/Fe cofactor. While previously only the relative contents of Fe and Mn in each metal-binding site have been assessed, here it is shown that the method can be extended to quantify the relative occupancies of at least three different transition metals, enabling complex competition experiments. The number of different metal ions that can be quantified is only limited by the number of high-quality anomalous data sets that can be obtained from one crystal, as one data set has to be collected for each transition-metal ion that is present (or is suspected to be present) in the protein, ideally at the absorption edge of each metal. A detailed description of the method, Q-XAD, is provided.

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