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  • 1. Bach, Anders
    et al.
    Chi, Celestine N
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Olsen, Thomas B
    Pedersen, Søren W
    Røder, Martin U
    Pang, Gar F
    Clausen, Rasmus P
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Strømgaard, Kristian
    Modified peptides as potent inhibitors of the postsynaptic density-95/N-methyl-D-aspartate receptor interaction.2008In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 51, no 20, p. 6450-9Article in journal (Refereed)
    Abstract [en]

    The protein-protein interaction between the NMDA receptor and its intracellular scaffolding protein, PSD-95, is a potential target for treatment of ischemic brain diseases. An undecapeptide corresponding to the C-terminal of the NMDA was used as a template for finding lead candidates for the inhibition of the PSD-95/NMDA receptor interaction. Initially, truncation and alanine scan studies were carried out, which resulted in a pentapeptide with wild-type affinity, as examined in a fluorescence polarization assay. Further examination was performed by systematic substitutions with natural and unnatural amino acids, which disclosed a tripeptide with micromolar affinity and N-methylated tetrapeptides with improved affinities. Molecular modeling studies guided further N-terminal modifications and introduction of a range of N-terminal substitutions dramatically improved affinity. The best compound, N-cyclohexylethyl-ETAV (56), demonstrated up to 19-fold lower K i value ( K i = 0.94 and 0.45 microM against PDZ1 and PDZ2 of PSD-95, respectively) compared to wild-type values, providing the most potent inhibitors of this interaction reported so far. These novel and potent inhibitors provide an important basis for development of small molecule inhibitors of the PSD-95/NMDA receptor interaction.

  • 2. Bach, Anders
    et al.
    Chi, Celestine N.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Pang, Gar F.
    Olsen, Lars
    Kristensen, Anders S.
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Strømgaard, Kristian
    Design and synthesis of highly potent and plasma-stable dimeric inhibitors of the PSD-95-NMDA receptor interaction2009In: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 48, no 51, p. 9685-9689Article in journal (Refereed)
    Abstract [en]

    On the double: Dimerization of monomeric peptide ligands towards the PDZ domains of the protein PSD-95 (postsynaptic density 95) leads to potent inhibitors of protein-protein interactions with stability in blood plasma. Optimization of the length of the polyethylene glycol linker results in unprecedented affinity for inhibitors of the PDZ1-2 domain.

  • 3. Bach, Anders
    et al.
    Clausen, Bettina H.
    Moller, Magda
    Vestergaard, Bente
    Chi, Celestine N.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Round, Adam
    Sorensen, Pernille L.
    Nissen, Klaus B.
    Kastrup, Jette S.
    Gajhede, Michael
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kristensen, Anders S.
    Lundström, Patrik
    Lambertsen, Kate L.
    Stromgaard, Kristian
    A high-affinity, dimeric inhibitor of PSD-95 bivalently interacts with PDZ1-2 and protects against ischemic brain damage2012In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 109, no 9, p. 3317-3322Article in journal (Refereed)
    Abstract [en]

    Inhibition of the ternary protein complex of the synaptic scaffolding protein postsynaptic density protein-95 (PSD-95), neuronal nitric oxide synthase (nNOS), and the N-methyl-D-aspartate (NMDA) receptor is a potential strategy for treating ischemic brain damage, but high-affinity inhibitors are lacking. Here we report the design and synthesis of a novel dimeric inhibitor, Tat-NPEG4(IETDV)(2) (Tat-N-dimer), which binds the tandem PDZ1-2 domain of PSD-95 with an unprecedented high affinity of 4.6 nM, and displays extensive protease-resistance as evaluated in vitro by stability-measurements in human blood plasma. X-ray crystallography, NMR, and small-angle X-ray scattering (SAXS) deduced a true bivalent interaction between dimeric inhibitor and PDZ1-2, and also provided a dynamic model of the conformational changes of PDZ1-2 induced by the dimeric inhibitor. A single intravenous injection of Tat-N-dimer (3 nmol/g) to mice subjected to focal cerebral ischemia reduces infarct volume with 40% and restores motor functions. Thus, Tat-N-dimer is a highly efficacious neuroprotective agent with therapeutic potential in stroke.

  • 4. Bibow, Stefan
    et al.
    Polyhach, Yevhen
    Eichmann, Cédric
    Chi N, Celestine
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. ETH.
    Kowal, Julia
    Albiez, Stefan
    McLeod, Robert A
    Stahlberg, Henning
    Jeschke, Gunnar
    Güntert, Peter
    Riek, Roland
    Solution structure of discoidal high-density lipoprotein particles with a shortened apolipoprotein A-I.2017In: Nature Structural & Molecular Biology, ISSN 1545-9993, E-ISSN 1545-9985, Vol. 24, no 2, p. 187-193Article in journal (Refereed)
    Abstract [en]

    High-density lipoprotein (HDL) particles are cholesterol and lipid transport containers. Mature HDL particles destined for the liver develop through the formation of intermediate discoidal HDL particles, which are the primary acceptors for cholesterol. Here we present the three-dimensional structure of reconstituted discoidal HDL (rdHDL) particles, using a shortened construct of human apolipoprotein A-I, determined from a combination of nuclear magnetic resonance (NMR), electron paramagnetic resonance (EPR) and transmission electron microscopy (TEM) data. The rdHDL particles feature a protein double belt surrounding a lipid bilayer patch in an antiparallel fashion. The integrity of this structure is maintained by up to 28 salt bridges and a zipper-like pattern of cation-π interactions between helices 4 and 6. To accommodate a hydrophobic interior, a gross 'right-to-right' rotation of the helices after lipidation is necessary. The structure reflects the complexity required for a shuttling container to hold a fluid lipid or cholesterol interior at a protein:lipid ratio of 1:50.

  • 5.
    Calosci, Nicoletta
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Chi, Celestine N.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Richter, Barbara
    Camilloni, Carlo
    Engström, Åke
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Eklund, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Travaglini-Allocatelli, Carlo
    Gianni, Stefano
    Vendruscolo, Michele
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Comparison of successive transition states for folding reveals alternative early folding pathways of two homologous proteins2008In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 105, no 49, p. 19241-19246Article in journal (Refereed)
    Abstract [en]

    The energy landscape theory provides a general framework for describing protein folding reactions. Because a large number of studies, however, have focused on two-state proteins with single well-defined folding pathways and without detectable intermediates, the extent to which free energy landscapes are shaped up by the native topology at the early stages of the folding process has not been fully characterized experimentally. To this end, we have investigated the folding mechanisms of two homologous three-state proteins, PTP-BL PDZ2 and PSD-95 PDZ3, and compared the early and late transition states on their folding pathways. Through a combination of Phi value analysis and molecular dynamics simulations we obtained atomic-level structures of the transition states of these homologous three-state proteins and found that the late transition states are much more structurally similar than the early ones. Our findings thus reveal that, while the native state topology defines essentially in a unique way the late stages of folding, it leaves significant freedom to the early events, a result that reflects the funneling of the free energy landscape toward the native state.

  • 6.
    Chi, Celestine N.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. ETH, Lab Phys Chem, Zurich, Switzerland.
    Bach, Anders
    Engström, Åke
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Stromgaard, Kristian
    Lundström, Patrik
    Ferguson, Neil
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Biophysical Characterization of the Complex between Human Papillomavirus E6 Protein and Synapse-associated Protein 972011In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 5, p. 3597-3606Article in journal (Refereed)
    Abstract [en]

    The E6 protein of human papillomavirus (HPV) exhibits complex interaction patterns with several host proteins, and their roles in HPV-mediated oncogenesis have proved challenging to study. Here we use several biophysical techniques to explore the binding of E6 to the three PDZ domains of the tumor suppressor protein synapse-associated protein 97 (SAP97). All of the potential binding sites in SAP97 bind E6 with micromolar affinity. The dissociation rate constants govern the different affinities of HPV16 and HPV18 E6 for SAP97. Unexpectedly, binding is not mutually exclusive, and all three PDZ domains can simultaneously bind E6. Intriguingly, this quaternary complex has the same apparent hydrodynamic volume as the unliganded PDZ region, suggesting that a conformational change occurs in the PDZ region upon binding, a conclusion supported by kinetic experiments. Using NMR, we discovered a new mode of interaction between E6 and PDZ: a subset of residues distal to the canonical binding pocket in the PDZ(2) domain exhibited noncanonical interactions with the E6 protein. This is consistent with a larger proportion of the protein surface defining binding specificity, as compared with that reported previously.

  • 7.
    Chi, Celestine N.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Bach, Anders
    University of Copenhagen.
    Engström, Åke
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Strømgaard, Kristian
    University of Copenhagen.
    Lundström, Patrik
    University of Linköping.
    Ferguson, Neil
    4UCD Conway Institute of Biomolecular and Biomedical Research, University College Dublin.
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Conformational change and non-canonical interactions in the complex between human papillomavirus E6 protein and Synapse-Associated Protein 97In: Article in journal (Refereed)
    Abstract [en]

    The E6 protein of human papillomavirus exhibits complex interaction patterns with several host proteins and their roles in HPV mediated oncogensis have proved challenging to study. Here we use several biophysical techniques to explore the binding of E6 to the three PDZ domains of the tumor suppressor protein SAP97. All potential binding sites in SAP97 can bind E6 with low micromolar affinity. Unexpectedly,binding is not mutually exclusive and all three PDZ domains can simultaneously bind E6. Intriguingly, the quaternary complex has the same apparent hydrodynamic volume as the unliganded PDZ region, despite having a doubled mass, suggesting that a conformational change occurs in the PDZ region upon E6 binding. Further, using NMR, we discovered a new mode of interaction between E6 and PDZ, as a subset of residues distal to the binding pocket in the PDZ2 domain was found to exhibit non-canonical interactions with the E6 protein suggesting that a large proportion of the proteins surface defines binding specificity

  • 8.
    Chi, Celestine N.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Bach, Anders
    University of Copenhagen.
    Gottschalk, Marie
    University of Copenhagen.
    Kristensen, S. Anders
    University of Copenhagen.
    Strømgaard, Kristian
    University of Copenhagen.
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Deciphering the kinetic binding mechanism of dimeric ligands, using a potent plasma-stable dimeric inhibitor of postsynaptic density protein-95 as an example2010In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 285, no 36, p. 28252-28260Article in journal (Refereed)
    Abstract [en]

    Dimeric ligands can be potent inhibitors of protein-protein or enzyme-substrate interactions. They have increased affinity and specificity towards their targets due to their ability to bind simultaneously to two binding sites and are therefore very attractive in drug design. However, few studies have addressed the kinetic mechanism of interaction of such bivalent ligands. We have investigated the binding interaction of a recently identified potent plasma-stable dimeric pentapeptide of PDZ1-2 of PSD-95 using protein engineering in combination with fluorescence polarisation, isothermal titration calorimetry and stopped-flow fluorimetry. Our experiments demonstrate that binding occurs via a two-step process, where an initial binding to either one of the two PDZ domains is followed by an intramolecular step, which produces the bidentate complex. We have determined all rate constants involved in the binding reaction and we also find evidence for a conformational transition of the complex. Our data demonstrate the importance of a slow dissociation for a successful dimeric ligand, but also highlight the possibility of optimizing the intramolecular association rate. The results may therefore aid the design of dimeric inhibitors in general.

  • 9.
    Chi, Celestine N.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Elfström, Lisa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Shi, Yao
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Snäll, Tord
    Engström, Åke
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Reassessing a sparse energetic network within a single protein domain2008In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 105, no 12, p. 4679-4684Article in journal (Refereed)
    Abstract [en]

    Understanding the molecular principles that govern allosteric communication is an important goal in protein science. One way allostery could be transmitted is via sparse energetic networks of residues, and one such evolutionary conserved network was identified in the PDZ domain family of proteins by multiple sequence alignment [Lockless SW, Ranganathan R (1999) Science 286:295-299]. We have reassessed the energetic coupling of these residues by double mutant cycles together with ligand binding and stability experiments and found that coupling is not a special property of the coevolved network of residues in PDZ domains. The observed coupling for ligand binding is better explained by a distance relationship, where residues close in space are more likely to couple than distal residues. Our study demonstrates that statistical coupling from sequence analysis is not necessarily a reporter of energetic coupling and allostery.

  • 10.
    Chi, Celestine N.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Engström, Åke
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Gianni, Stefano
    Larsson, Mårten
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Two conserved residues govern the salt and pH dependencies of the binding reaction of a PDZ domain2006In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 281, no 48, p. 36811-36818Article in journal (Refereed)
    Abstract [en]

    PDZ domains are protein-protein interaction modules found in hundreds of human proteins. Their binding reactions are sensitive to variations in salt and pH but the basis of the respective dependence has not been clear. We investigated the binding reaction between PSD-95 PDZ3 and a peptide corresponding to a native ligand with protein engineering in conjunction with stopped-flow and equilibrium fluorimetry and found that the two conserved residues Arg-318 and His-372 were responsible for the salt and pH dependencies, respectively. The basis of the salt-dependent variation of the affinity was explored by mutating all charged residues in and around the peptide-binding pocket. Arg-318 was found to be crucial, as mutation to alanine obliterated the effect of chloride on the binding constants. The direct interaction of chloride with Arg-318 was demonstrated by time-resolved urea denaturation experiments, where the Arg-318 --> Ala mutant was less stabilized by addition of chloride as compared with wild-type PDZ3. We also demonstrated that protonation of His-372 was responsible for the increase of the equilibrium dissociation constant at low pH. Both chloride concentration and pH (during ischemia) vary in the postsynaptic density, where PSD-95 is present, and the physiological buffer conditions may thus modulate the interaction between PSD-95 and its ligands through binding of chloride and protons to the "molecular switches" Arg-318 and His-372, respectively.

  • 11.
    Chi, Celestine N.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Gianni, Stefano
    Calosci, Nicoletta
    Travaglini-Allocatelli, Carlo
    Engström, Åke
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    A conserved folding mechanism for PDZ domains2007In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 581, no 6, p. 1109-1113Article in journal (Refereed)
    Abstract [en]

    An important question in protein folding is whether the folding mechanism is sequence dependent and conserved for homologous proteins. In this work we compared the kinetic folding mechanism of five postsynaptic density protein-95, disc-large tumor suppressor protein, zonula occludens-1 (PDZ) domains, sharing similar topology but having different primary structures. Investigation of the different proteins under various experimental conditions revealed that the folding kinetics of each member of the PDZ family can be described by a model with two transition states separated by an intermediate. Moreover, the positions of the two transition states along the reaction coordinate (as given by their βT-values) are fairly constant for the five PDZ domains.

  • 12.
    Chi, Celestine N.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. ETH.
    Haq, S. Raza
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Rinaldo, S.
    Dogan, Jakob
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Cutruzzolà, F.
    Engström, Åke
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Gianni, S.
    Lundström, P.
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Interactions outside the boundaries of the canonical binding groove of a pdz domain influence ligand binding2012In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 51, no 44, p. 8971-8979Article in journal (Refereed)
    Abstract [en]

    The postsynaptic density protein-95/discs large/zonula occludens-1 (PDZ) domain is a protein-protein interaction module with a shallow binding groove where protein ligands bind. However, interactions that are not part of this canonical binding groove are likely to modulate peptide binding. We have investigated such interactions beyond the binding groove for PDZ3 from PSD-95 and a peptide derived from the C-terminus of the natural ligand CRIPT. We found via nuclear magnetic resonance experiments that up to eight residues of the peptide ligand interact with the PDZ domain, showing that the interaction surface extends far outside of the binding groove as defined by the crystal structure. PDZ3 contains an extra structural element, a C-terminal helix (α3), which is known to affect affinity. Deletion of this helix resulted in the loss of several intermolecular nuclear Overhauser enhancements from peptide residues outside of the binding pocket, suggesting that α3 forms part of the extra binding surface in wild-type PDZ3. Site-directed mutagenesis, isothermal titration calorimetry, and fluorescence intensity experiments confirmed the importance of both α3 and the N-terminal part of the peptide for the affinity. Our data suggest a general mechanism in which different binding surfaces outside of the PDZ binding groove could provide sites for specific interactions.

  • 13.
    Chi, Celestine N.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Strotz, Dean
    Riek, Roland
    Vögeli, Beat Rolf
    NOE-derived methyl distances from a 360 kDa proteasome complex2018In: Chemistry - A European Journal, ISSN 0947-6539, E-ISSN 1521-3765, Vol. 24, no 9, p. 2270-2276Article in journal (Refereed)
    Abstract [en]

    Nuclear magnetic resonance spectroscopy is the prime tool to probe structure and dynamics of biomolecules at atomic resolution. A serious challenge for that method is the size limit imposed on molecules to be studied. Standard studies are typically restricted to ca. 30-40 kDa. More recent developments lead to spin relaxation measurements in methyl groups in single proteins or protein complexes as large as a mega-Dalton, which directly allow the extraction of angular information or experiments with paramagnetic samples. However, these probes are all of indirect nature in contrast to the most intuitive and easy-to-interpret structural/dynamics restraint, the internuclear distance, which can be measured by nuclear Overhauser enhancement (NOE). Here, we demonstrate time-averaged distance measurements on the 360 kDa half proteasome from Thermoplasma acidophilium. The approach is based on exact quantification of the NOE (eNOE). Our findings open up an avenue for such measurements on very large molecules. These restraints will help in a detailed determination of conformational changes upon perturbation such as ligand binding.

  • 14.
    Chi N, Celestine
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. ETH.
    Bach, Anders
    Engström, Åke
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Wang, Huiqun
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Strømgaard, Kristian
    Gianni, Stefano
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    A sequential binding mechanism in a PDZ domain2009In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, no 30, p. 7089-7097Article in journal (Refereed)
    Abstract [en]

    Conformational selection and induced fit are two well-known mechanisms of allosteric protein-ligand interaction. Some proteins, like ubiquitin, have recently been found to exist in multiple conformations at equilibrium, suggesting that the conformational selection may be a general mechanism of interaction, in particular for single-domain proteins. Here, we found that the PDZ2 domain of SAP97 binds its ligand via a sequential (induced fit) mechanism. We performed binding experiments using SAP97 PDZ2 and peptide ligands and observed biphasic kinetics with the stopped-flow technique, indicating that ligand binding involves at least a two-step process. By using an ultrarapid continuous-flow mixer, we then detected a hyperbolic dependence of binding rate constants on peptide concentration, corroborating the two-step binding mechanism. Furthermore, we found a similar dependence of the rate constants on both PDZ and peptide concentration, demonstrating that the PDZ2-peptide interaction involves a precomplex, which then undergoes a conformational change, and thereby follows an induced fit mechanism.

  • 15.
    Chi N, Celestine
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. ETH.
    Strotz, Dean
    Riek, Roland
    Vögeli, Beat
    Extending the eNOE data set of large proteins by evaluation of NOEs with unresolved diagonals.2015In: Journal of Biomolecular NMR, ISSN 0925-2738, E-ISSN 1573-5001, Vol. 62, no 1, p. 63-9Article in journal (Refereed)
    Abstract [en]

    The representation of a protein's spatial sampling at atomic resolution is fundamental for understanding its function. NMR has been established as the best-suited technique toward this goal for small proteins. However, the accessible information content rapidly deteriorates with increasing protein size. We have recently demonstrated that for small proteins distance restraints with an accuracy smaller than 0.1 Å can be obtained by replacing traditional semi-quantitative Nuclear Overhauser Effects (NOEs) with exact NOEs (eNOE). The high quality of the data allowed us to calculate structural ensembles of the small model protein GB3 consisting of multiple rather than a single state. The analysis has been limited to small proteins because NOEs of spins with unresolved diagonal peaks cannot be used. Here we propose a simple approach to translate such NOEs into correct upper distance restraints, which opens access to larger biomolecules. We demonstrate that for 16 kDa cyclophilin A the collection of such restraints extends the original 1254 eNOEs to 3471.

  • 16.
    Chi N, Celestine
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. ETH.
    Vögeli, Beat
    Bibow, Stefan
    Strotz, Dean
    Orts, Julien
    Güntert, Peter
    Riek, Roland
    A Structural Ensemble for the Enzyme Cyclophilin Reveals an Orchestrated Mode of Action at Atomic Resolution.2015In: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 54, no 40, p. 11657-61Article in journal (Refereed)
    Abstract [en]

    For enzyme activity, an exact structural and motional orchestration of the active site and its surroundings is believed to be key. In order to reveal such possible phenomena at atomic resolution on the basis of experimental evidence, an experimental restraint driven two-state ensemble of the prototypical enzyme cyclophilin was determined by using a recently introduced exact NOE approach. The ensemble description reveals the presence of an open and a closed state of cyclophilin, which is indicative of large-scale correlated motion. In the open state, the catalytic site is preorganized for catalysis, thus suggesting the mechanism of action to be conformational sampling, while the ligand-binding loop appears to act through an induced fit mechanism. This finding is supported by affinity measurements of a cyclophilin designed to be more open. Overall, more than 60-70 % of the side-chain conformations of cyclophilin appear to be correlated.

  • 17. Daskalov, Asen
    et al.
    Gantner, Matthias
    Wälti, Marielle Aulikki
    Schmidlin, Thierry
    Chi N, Celestine
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. ETH.
    Wasmer, Christian
    Schütz, Anne
    Ceschin, Johanna
    Clavé, Corinne
    Cescau, Sandra
    Meier, Beat
    Riek, Roland
    Saupe, Sven J
    Contribution of specific residues of the β-solenoid fold to HET-s prion function, amyloid structure and stability.2014In: PLoS Pathogens, ISSN 1553-7366, E-ISSN 1553-7374, Vol. 10, no 6, article id e1004158Article in journal (Refereed)
    Abstract [en]

    The [Het-s] prion of the fungus Podospora anserina represents a good model system for studying the structure-function relationship in amyloid proteins because a high resolution solid-state NMR structure of the amyloid prion form of the HET-s prion forming domain (PFD) is available. The HET-s PFD adopts a specific β-solenoid fold with two rungs of β-strands delimiting a triangular hydrophobic core. A C-terminal loop folds back onto the rigid core region and forms a more dynamic semi-hydrophobic pocket extending the hydrophobic core. Herein, an alanine scanning mutagenesis of the HET-s PFD was conducted. Different structural elements identified in the prion fold such as the triangular hydrophobic core, the salt bridges, the asparagines ladders and the C-terminal loop were altered and the effect of these mutations on prion function, fibril structure and stability was assayed. Prion activity and structure were found to be very robust; only a few key mutations were able to corrupt structure and function. While some mutations strongly destabilize the fold, many substitutions in fact increase stability of the fold. This increase in structural stability did not influence prion formation propensity in vivo. However, if an Ala replacement did alter the structure of the core or did influence the shape of the denaturation curve, the corresponding variant showed a decreased prion efficacy. It is also the finding that in addition to the structural elements of the rigid core region, the aromatic residues in the C-terminal semi-hydrophobic pocket are critical for prion propagation. Mutations in the latter region either positively or negatively affected prion formation. We thus identify a region that modulates prion formation although it is not part of the rigid cross-β core, an observation that might be relevant to other amyloid models.

  • 18. Gianni, Stefano
    et al.
    Engström, Åke
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Larsson, Mårten
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Calosci, Nicoletta
    Malatesta, Francesco
    Eklund, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Chi N, Celestine
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. ETH.
    Travaglini-Allocatelli, Carlo
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    The kinetics of PDZ domain-ligand interactions and implications for the binding mechanism2005In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 280, no 41, p. 34805-34812Article in journal (Refereed)
    Abstract [en]

    PDZ domains are protein adapter modules present in a few hundred human proteins. They play important roles in scaffolding and signal transduction. PDZ domains usually bind to the C termini of their target proteins. To assess the binding mechanism of this interaction we have performed the first in-solution kinetic study for PDZ domains and peptides corresponding to target ligands. Both PDZ3 from postsynaptic density protein 95 and PDZ2 from protein tyrosine phosphatase L1 bind their respective target peptides through an apparent A + B --> A.B mechanism without rate-limiting conformational changes. But a mutant with a fluorescent probe (Trp) outside of the binding pocket suggests that slight changes in the structure take place upon binding in protein tyrosine phosphatase-L1 PDZ2. For PDZ3 from postsynaptic density protein 95 the pH dependence of the binding reaction is consistent with a one-step mechanism with one titratable group. The salt dependence of the interaction shows that the formation of electrostatic interactions is rate-limiting for the association reaction but not for dissociation of the complex.

  • 19. Gianni, Stefano
    et al.
    Haq, Syed Raza Ul
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Montemiglio, Linda C.
    Jürgens, Maike C.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Engström, Åke
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Chi, Celestine N.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Brunori, Maurizio
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Sequence-specific Long Range networks in PSD-95/Discs Large/ZO-1 (PDZ) Domains Tune Their Binding Selectivity2011In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 31, p. 27167-27175Article in journal (Refereed)
    Abstract [en]

    Protein-protein interactions mediated by modular protein domains are critical for cell scaffolding, differentiation, signaling, and ultimately, evolution. Given the vast number of ligands competing for binding to a limited number of domain families, it is often puzzling how specificity can be achieved. Selectivity may be modulated by intradomain allostery, whereby a remote residue is energetically connected to the functional binding site via side chain or backbone interactions. Whereas several energetic pathways, which could mediate intradomain allostery, have been predicted in modular protein domains, there is a paucity of experimental data to validate their existence and roles. Here, we have identified such functional energetic networks in one of the most common protein-protein interaction modules, the PDZ domain. We used double mutant cycles involving site-directed mutagenesis of both the PDZ domain and the peptide ligand, in conjunction with kinetics to capture the fine energetic details of the networks involved in peptide recognition. We performed the analysis on two homologous PDZ-ligand complexes and found that the energetically coupled residues differ for these two complexes. This result demonstrates that amino acid sequence rather than topology dictates the allosteric pathways. Furthermore, our data support a mechanism whereby the whole domain and not only the binding pocket is optimized for a specific ligand. Such cross-talk between binding sites and remote residues may be used to fine tune target selectivity.

  • 20. Haq, S. Raza
    et al.
    Chi, Celestine N.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Bach, Anders
    Dogan, Jakob
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Engström, Åke
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Hultqvist, Greta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Karlsson, O. Andreas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Lundstrom, Patrik
    Montemiglio, Linda C.
    Stromgaard, Kristian
    Gianni, Stefano
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Side-Chain Interactions Form Late and Cooperatively in the Binding Reaction between Disordered Peptides and PDZ Domains2012In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 134, no 1, p. 599-605Article in journal (Refereed)
    Abstract [en]

    Intrinsically disordered proteins are very common and mediate numerous protein-protein and protein-DNA interactions. While it is clear that these interactions are instrumental for the life of the mammalian cell, there is a paucity of data regarding their molecular binding mechanisms. Here we have used short peptides as a model system for intrinsically disordered proteins. Linear free energy relationships based on rate and equilibrium constants for the binding of these peptides to ordered target proteins, PDZ domains, demonstrate that native side-chain interactions form mainly after the rate-limiting barrier for binding and in a cooperative fashion. This finding suggests that these disordered peptides first form a weak encounter complex with non-native interactions. The data do not support the recent notion that the affinities of intrinsically disordered proteins toward their targets are generally governed by their association rate constants. Instead, we observed the opposite for peptide-PDZ interactions, namely, that changes in K-d correlate with changes in k(off).

  • 21.
    Haq, Syed Raza
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Jürgens, Maike C.
    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.
    Chi N, Celestine
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. ETH.
    Koh, Cha San
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Elfström, Lisa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Selmer, Maria
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Gianni, Stefano
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    The plastic energy landscape of protein folding: a triangular folding mechanism with an equilibrium intermediate for a small protein domain2010In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 285, no 23, p. 18051-18059Article in journal (Refereed)
    Abstract [en]

    Protein domains usually fold without or with only transiently populated intermediates, possibly to avoid misfolding, which could result in amyloidogenic disease. Whether observed intermediates are productive and obligatory species on the folding reaction pathway or dispensable by-products is a matter of debate. Here, we solved the crystal structure of a small protein domain, SAP97 PDZ2 I342W C378A, and determined its folding pathway. The presence of a folding intermediate was demonstrated both by single and double-mixing kinetic experiments using urea-induced (un) folding as well as ligand-induced folding. This protein domain was found to fold via a triangular scheme, where the folding intermediate could be either on-or off-pathway, depending on the experimental conditions. Furthermore, we found that the intermediate was present at equilibrium, which is rarely seen in folding reactions of small protein domains. The folding mechanism observed here illustrates the roughness and plasticity of the protein folding energy landscape, where several routes may be employed to reach the native state. The results also reconcile the folding mechanisms of topological variants within the PDZ domain family.

  • 22.
    Hultqvist, Greta
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Haq, Raza
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Punekar, Avinash
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Chi, Celestine
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. ETH.
    Engström, Åke
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Bach, Anders
    Strømgaard, Kristian
    University of Copenhagen.
    Selmer, Maria
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Gianni, Stefano
    Sapienza Università di Roma.
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Energetic pathway sampling in a protein interaction domain2013In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 21, p. 1193-1202Article in journal (Other academic)
    Abstract [en]

    The affinity and specificity of protein-ligand interactions are influenced by energeticcrosstalk within the protein domain. However, the molecular details of such intradomain allostery are still unclear. Here, we have experimentally detected and computationally predicted interactionpathways in the postsynaptic density 95/discs large/zonula occludens 1 (PDZ)-peptide ligand model system using wild-type and circularly permuted PDZ proteins. The circular permutant introduced small perturbations in the tertiary structure and a concomitant rewiring of allosteric pathways, allowing us to describe how subtle changes may reshape energetic signaling. The results were analyzed in the context of other members of the PDZ family, which were found to contain distinct interaction pathways for different peptide ligands. The data reveal a fascinating scenario whereby several energetic pathways are sampled within one single domain and distinct pathways are activated by specific protein ligands. 

  • 23.
    Hultqvist, Greta
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Pedersen, Søren
    University of Copenhagen.
    Strømgaard, Kristian
    University of Copenhagen.
    Chi, Celestine
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. ETH.
    Gianni, Stefano
    Sapienza Università di Roma.
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    An expanded view of the protein folding landscape of PDZ domains2012In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 421, no 3, p. 550-553Article in journal (Refereed)
    Abstract [en]

    Most protein domains fold in an apparently co-operative and two-state manner with only the native and denatured states significantly populated at any experimental condition. However, the protein folding energy landscape is often rugged and different transition states may be rate limiting for the folding reaction under different conditions, as seen for the PDZ protein domain family. We have here analyzed the folding kinetics of two PDZ domains and found that a previously undetected third transition state is rate limiting under conditions that stabilize the native state relative to the denatured state. In light of these results, we have re-analyzed previous folding data on PDZ domains and present a unified folding mechanism with three distinct transition states separated by two high-energy intermediates. Our data show that sequence composition tunes the relative stabilities of folding transition states within the PDZ family, while the overall mechanism is determined by topology. This model captures the kinetic folding mechanism of all PDZ domains studied to date.

  • 24.
    Hultqvist, Greta
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Punekar, Avinash
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Morrone, Angela
    Sapienza Università di Roma.
    Chi, Celestine
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. ETH.
    Engström, Åke
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Selmer, Maria
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Gianni, Stefano
    Sapienza Università di Roma.
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Tolerance of Protein Folding to a Circular Permutation in a PDZ Domain2012In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 7, no 11, p. e50055-Article in journal (Refereed)
    Abstract [en]

    Circular permutation is a common molecular mechanism for evolution of proteins. However, such re-arrangement of secondary structure connectivity may interfere with the folding mechanism causing accumulation of folding intermediates, which in turn can lead to misfolding. We solved the crystal structure and investigated the folding pathway of a circularly permuted variant of a PDZ domain, SAP97 PDZ2. Our data illustrate how well circular permutation may work as a mechanism for molecular evolution. The circular permutant retains the overall structure and function of the native protein domain. Further, unlike most examples in the literature, this circular permutant displays a folding mechanism that is virtually identical to that of the wild type. This observation contrasts with previous data on the circularly permuted PDZ2 domain from PTP-BL, for which the folding pathway was remarkably affected by the same mutation in sequence connectivity. The different effects of this circular permutation in two homologous proteins show the strong influence of sequence as compared to topology. Circular permutation, when peripheral to the major folding nucleus, may have little effect on folding pathways and could explain why, despite the dramatic change in primary structure, it is frequently tolerated by different protein folds.

  • 25.
    Hultqvist, Greta
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Åberg, Emma
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Camilloni, Carlo
    Univ Cambridge, Dept Chem, Cambridge, England.;Tech Univ Munich, Dept Chem, Munich, Germany.;Tech Univ Munich, Inst Adv Study, Munich, Germany..
    Sundell, Gustav
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Andersson, Eva
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Dogan, Jakob
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Stockholm Univ, Dept Biochem & Biophys, Stockholm, Sweden..
    Chi, Celestine N.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. ETH, Lab Phys Chem, Zurich, Switzerland.
    Vendruscolo, Michele
    Univ Cambridge, Dept Chem, Cambridge, England.
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala Univ, Dept Med Biochem & Microbiol, Uppsala, Sweden..
    Emergence and evolution of an interaction between intrinsically disordered proteins2017In: eLIFE, E-ISSN 2050-084X, Vol. 6, article id e16059Article in journal (Refereed)
    Abstract [en]

    Protein-protein interactions involving intrinsically disordered proteins are important for cellular function and common in all organisms. However, it is not clear how such interactions emerge and evolve on a molecular level. We performed phylogenetic reconstruction, resurrection and biophysical characterization of two interacting disordered protein domains, CID and NCBD. CID appeared after the divergence of protostomes and deuterostomes 450-600 million years ago, while NCBD was present in the protostome/deuterostome ancestor. The most ancient CID/NCBD formed a relatively weak complex (K(d similar to)5 mu M). At the time of the first vertebrate-specific whole genome duplication, the affinity had increased (K-d\similar to 200 nM) and was maintained in further speciation. Experiments together with molecular modeling using NMR chemical shifts suggest that new interactions involving intrinsically disordered proteins may evolve via a low-affinity complex which is optimized by modulating direct interactions as well as dynamics, while tolerating several potentially disruptive mutations.

  • 26.
    Karlsson, O. Andreas
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Chi, Celestine N.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. ETH, Lab Phys Chem, Zurich, Switzerland.
    Engström, Åke
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    The Transition State of Coupled Folding and Binding for a Flexible beta-Finger2012In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 417, no 3, p. 253-261Article in journal (Refereed)
    Abstract [en]

    Flexible and fully disordered protein regions that fold upon binding mediate numerous protein protein interactions. However, little is known about their mechanism of interaction. One such coupled folding and binding occurs when a flexible region of neuronal nitric oxide synthase adopts a beta-finger structure upon binding to its protein ligand, a PDZ [PSD-95 (postsynaptic density protein-95)/Discs large/ZO-1] domain from PSD-95. We have analyzed this binding reaction by protein engineering combined with kinetic experiments. Mutational destabilization of the beta-finger changed mainly the dissociation rate constant of the proteins and, to a lesser extent, the association rate constant. Thus, mutation affected late events in the coupled folding and binding reaction. Our results therefore suggest that the native binding interactions of the beta-finger are not present in the rate-limiting transition state for binding but form on the downhill side in a cooperative manner. However, by mutation, we could destabilize the beta-finger further and change the rate-limiting step such that an initial conformational change becomes rate limiting. This switch in rate-limiting step shows that multistep binding mechanisms are likely to be found among flexible and intrinsically disordered regions of proteins.

  • 27.
    Karlsson, O. Andreas
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Ramirez, Juan
    Öberg, Daniel
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Malmqvist, Tony
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Engström, Åke
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Friberg, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Chi, Celestine N.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Trave, Gilles
    Nilsson, Mikael T. I.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Design of a PDZbody, a bivalent binder of the E6 protein from human papillomavirus2015In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 5, article id 9382Article in journal (Refereed)
    Abstract [en]

    Chronic infection by high risk human papillomavirus (HPV) strains may lead to cancer. Expression of the two viral oncoproteins E6 and E7 is largely responsible for immortalization of infected cells. The HPV E6 is a small (approximately 150 residues) two domain protein that interacts with a number of cellular proteins including the ubiquitin ligase E6-associated protein (E6AP) and several PDZ-domain containing proteins. Our aim was to design a high-affinity binder for HPV E6 by linking two of its cellular targets. First, we improved the affinity of the second PDZ domain from SAP97 for the C-terminus of HPV E6 from the high-risk strain HPV18 using phage display. Second, we added a helix from E6AP to the N-terminus of the optimized PDZ variant, creating a chimeric bivalent binder, denoted PDZbody. Full-length HPV E6 proteins are difficult to express and purify. Nevertheless, we could measure the affinity of the PDZbody for E6 from another high-risk strain, HPV16 (K-d = 65 nM). Finally, the PDZbody was used to co-immunoprecipitate E6 protein from HPV18-immortalized HeLa cells, confirming the interaction between PDZbody and HPV18 E6 in a cellular context.

  • 28. Nichols, Parker J
    et al.
    Born, Alexandra
    Henen, Morkos A
    Strotz, Dean
    Orts, Julien
    Olsson, Simon
    Güntert, Peter
    Chi N, Celestine
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. ETH.
    Vögeli, Beat
    The Exact Nuclear Overhauser Enhancement: Recent Advances.2017In: Molecules, ISSN 1420-3049, E-ISSN 1420-3049, Vol. 22, no 7, article id E1176Article in journal (Refereed)
    Abstract [en]

    Although often depicted as rigid structures, proteins are highly dynamic systems, whose motions are essential to their functions. Despite this, it is difficult to investigate protein dynamics due to the rapid timescale at which they sample their conformational space, leading most NMR-determined structures to represent only an averaged snapshot of the dynamic picture. While NMR relaxation measurements can help to determine local dynamics, it is difficult to detect translational or concerted motion, and only recently have significant advances been made to make it possible to acquire a more holistic representation of the dynamics and structural landscapes of proteins. Here, we briefly revisit our most recent progress in the theory and use of exact nuclear Overhauser enhancements (eNOEs) for the calculation of structural ensembles that describe their conformational space. New developments are primarily targeted at increasing the number and improving the quality of extracted eNOE distance restraints, such that the multi-state structure calculation can be applied to proteins of higher molecular weights. We then review the implications of the exact NOE to the protein dynamics and function of cyclophilin A and the WW domain of Pin1, and finally discuss our current research and future directions.

  • 29.
    Strotz, Dean
    et al.
    ETH Honggerberg, Zurich, Switzerland.
    Orts, Julien
    ETH Honggerberg, Zurich, Switzerland.
    Chi, Celestine N.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Riek, Roland
    ETH Honggerberg, Zurich, Switzerland.
    Vögeli, Beat
    Univ Colorado Denver, Aurora, USA.
    eNORA2 Exact NOE Analysis Program2017In: Journal of Chemical Theory and Computation, ISSN 1549-9618, E-ISSN 1549-9626, Vol. 13, no 9, p. 4336-4346Article in journal (Refereed)
    Abstract [en]

    We have recently developed an NMR protocol to extract exact distances between nuclei in proteins from an exact interpretation of NOESY buildup intensities (eNOEs). This enabled us to calculate multistate structural ensembles that exhibit realistic spatial sampling and long-range correlations. Our initial studies were laborious and required a deep understanding of the underlying spin dynamics. Here, we present a MatLab package that integrates all data processing steps required to convert intensities of assigned peaks in NOESY series into upper and lower distance limits for structure calculation. Those steps include organization of the data in object format, extraction of autorelaxation and cross-relaxation rate constants by fitting of diagonal peak decays and cross peak buildups, validation of the data, correction for spin diffusion, graphical display of the results, and generation of distance limits in CYANA compatible format. The analysis may be carried out using a full relaxation matrix or a simplified "divide and conquer" approach that allows for partial deuteration of protons. As the program does not require expertise beyond that of standard resonance assignment/structure calculation, it is suitable for experts and nonexperts alike.

  • 30.
    Sundell, Gustav
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Vögeli, Beat
    Department of Biochemistry and Molecular Genetics, University of Colorado at Denver, 12801 East 17th Avenue, Aurora, Colorado 80045, United States.
    Ivarsson, Ylva
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Chi, Celestine N.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    The Sign of Nuclear Magnetic Resonance Chemical Shift Difference as a Determinant of the Origin of Binding Selectivity: Elucidation of the Position Dependence of Phosphorylation in Ligands Binding to Scribble PDZ12018In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 57, no 1, p. 66-71Article in journal (Refereed)
    Abstract [en]

    The use of nuclear magnetic resonance chemical shift perturbation to monitor changes taking place around the binding site of a ligand-protein interaction is a routine and widely applied methodology in the field of protein biochemistry. Shifts are often acquired by titrating various concentrations of ligand to a fixed concentration of the receptor and may serve the purpose, among others, of determining affinity constants, locating binding surfaces, or differentiating between binding mechanisms. Shifts are quantified by the so-called combined chemical shift difference. Although the directionality of shift changes is often used for detailed analysis of specific cases, the approach has not been adapted in standard chemical shift monitoring. This is surprising as it would not require additional effort. Here, we demonstrate the importance of the sign of the chemical shift difference induced by ligand-protein interaction. We analyze the sign of the 15N/1H shift changes of the PDZ1 domain of Scribble upon interaction with two pairs of phosphorylated and unphosphorylated peptides. We find that detailed differences in the molecular basis of this PDZ-ligand interaction can be obtained from our analysis to which the classical method of combined chemical shift perturbation analysis is insensitive. In addition, we find a correlation between affinity and millisecond motions. Application of the methodology to Cyclophilin a, a cis-trans isomerase, reveals molecular details of peptide recognition. We consider our directionality vector chemical shift analysis as a method of choice when distinguishing the molecular origin of binding specificities of a class of similar ligands, which is often done in drug discovery.

  • 31.
    Toto, Angelo
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Univ Rome, Fdn Cenci Bolognetti, Ist Pasteur, Dipartimento Sci Biochim A Rossi Fanelli Sapienza, I-00185 Rome, Italy.;Univ Rome, CNR, Ist Biol & Patol Mol, I-00185 Rome, Italy..
    Pedersen, Soren W.
    Univ Copenhagen, Ctr Biopharmaceut, Dept Drug Design & Pharmacol, Univ Pk 2, DK-2100 Copenhagen, Denmark..
    Karlsson, O. Andreas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Moran, Griffin E.
    Univ Copenhagen, Ctr Biopharmaceut, Dept Drug Design & Pharmacol, Univ Pk 2, DK-2100 Copenhagen, Denmark..
    Andersson, Eva
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Chi, Celestine N.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Stromgaard, Kristian
    Univ Copenhagen, Ctr Biopharmaceut, Dept Drug Design & Pharmacol, Univ Pk 2, DK-2100 Copenhagen, Denmark..
    Gianni, Stefano
    Univ Rome, Fdn Cenci Bolognetti, Ist Pasteur, Dipartimento Sci Biochim A Rossi Fanelli Sapienza, I-00185 Rome, Italy.;Univ Rome, CNR, Ist Biol & Patol Mol, I-00185 Rome, Italy.;Univ Cambridge, Dept Chem, Lensfield Rd, Cambridge CB2 1EW, England..
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Ligand binding to the PDZ domains of postsynaptic density protein 952016In: Protein Engineering Design & Selection, ISSN 1741-0126, E-ISSN 1741-0134, Vol. 29, no 5, p. 169-175Article in journal (Refereed)
    Abstract [en]

    Cellular scaffolding and signalling is generally governed by multidomain proteins, where each domain has a particular function. Postsynaptic density protein 95 (PSD-95) is involved in synapse formation and is a typical example of such a multidomain protein. Protein-protein interactions of PSD-95 are well studied and include the following three protein ligands: (i) N-methyl-d-aspartate-type ionotropic glutamate receptor subunit GluN2B, (ii) neuronal nitric oxide synthase and (iii) cysteine-rich protein (CRIPT), all of which bind to one or more of the three PDZ domains in PSD-95. While interactions for individual PDZ domains of PSD-95 have been well studied, less is known about the influence of neighbouring domains on the function of the respective individual domain. We therefore performed a systematic study on the ligand-binding kinetics of PSD-95 using constructs of different size for PSD-95 and its ligands. Regarding the canonical peptide-binding pocket and relatively short peptides (up to 15-mer), the PDZ domains in PSD-95 by and large work as individual binding modules. However, in agreement with previous studies, residues outside of the canonical binding pocket modulate the affinity of the ligands. In particular, the dissociation of the 101 amino acid CRIPT from PSD-95 is slowed down at least 10-fold for full-length PSD-95 when compared with the individual PDZ3 domain.

  • 32. Vögeli, Beat
    et al.
    Bibow, Stefan
    Chi N, Celestine
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. ETH.
    Enzyme Selectivity Fine-Tuned through Dynamic Control of a Loop.2016In: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 55, no 9, p. 3096-100Article in journal (Refereed)
    Abstract [en]

    Allostery has been revealed as an essential property of all proteins. For enzymes, shifting of the structural equilibrium distribution at one site can have substantial impacts on protein dynamics and selectivity. Promising sites of remotely shifting such a distribution by changing the dynamics would be at flexible loops because relatively large changes may be achieved with minimal modification of the protein. A ligand-selective change of binding affinity to the active site of cyclophilin is presented involving tuning of the dynamics of a highly flexible loop. Binding affinity is increased upon substitution of double Gly to Ala at the hinge regions of the loop. Quenching of the motional amplitudes of the loop slightly rearranges the active site. In particular, key residues for binding Phe60 and His126 adopt a more fixed orientation in the bound protein. Our system may serve as a model system for studying the effects of various time scales of loop motion on protein function tuned by mutations.

  • 33. Vögeli, Beat
    et al.
    Orts, Julien
    Strotz, Dean
    Chi N, Celestine
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. ETH.
    Minges, Martina
    Wälti, Marielle Aulikki
    Güntert, Peter
    Riek, Roland
    Towards a true protein movie: a perspective on the potential impact of the ensemble-based structure determination using exact NOEs.2014In: Journal of magnetic resonance, ISSN 1090-7807, E-ISSN 1096-0856, Vol. 241, p. 53-9, article id S1090-7807(13)00307-8Article in journal (Refereed)
    Abstract [en]

    Confined by the Boltzmann distribution of the energies of the states, a multitude of structural states are inherent to biomolecules. For a detailed understanding of a protein's function, its entire structural landscape at atomic resolution and insight into the interconversion between all the structural states (i.e. dynamics) are required. Whereas dedicated trickery with NMR relaxation provides aspects of local dynamics, and 3D structure determination by NMR is well established, only recently have several attempts been made to formulate a more comprehensive description of the dynamics and the structural landscape of a protein. Here, a perspective is given on the use of exact NOEs (eNOEs) for the elucidation of structural ensembles of a protein describing the covered conformational space.

1 - 33 of 33
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