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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.
    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.

  • 2. 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.

  • 3.
    Born, Alexandra
    et al.
    Univ Colorado, Dept Biochem & Mol Genet, Anschutz Med Campus,12801 East 17th Ave, Aurora, CO 80045 USA.
    Nichols, Parker J.
    Univ Colorado, Dept Biochem & Mol Genet, Anschutz Med Campus,12801 East 17th Ave, Aurora, CO 80045 USA.
    Henen, Morkos A.
    Univ Colorado, Dept Biochem & Mol Genet, Anschutz Med Campus,12801 East 17th Ave, Aurora, CO 80045 USA;Mansoura Univ, Fac Pharm, Mansoura 35516, Egypt.
    Chi, Celestine N.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Strotz, Dean
    ETH Honggerberg, ETH Zurich, Lab Phys Chem, Zurich, Switzerland.
    Bayer, Peter
    Univ Duisburg Essen, Strukt & Med Biochem, Univ Str 2-5, D-45117 Essen, Germany.
    Tate, Shin-Ichi
    Hiroshima Univ, Dept Math & Life Sci, Hiroshima, Japan.
    Peng, Jeffrey W.
    Univ Notre Dame, Dept Chem & Biochem, Notre Dame, IN 46556 USA;Univ Notre Dame, Dept Phys, Notre Dame, IN 46556 USA.
    Vögeli, Beat
    Univ Colorado, Dept Biochem & Mol Genet, Anschutz Med Campus,12801 East 17th Ave, Aurora, CO 80045 USA;Univ Colorado Denver, Dept Biochem & Mol Genet, Res Ctr 1 South,Room 9103,12801 East 17th Ave, Aurora, CO 80045 USA.
    Backbone and side-chain chemical shift assignments of full-length, apo, human Pin1, a phosphoprotein regulator with interdomain allostery2019In: Biomolecular NMR Assignments, ISSN 1874-2718, E-ISSN 1874-270X, Vol. 13, no 1, p. 85-89Article in journal (Refereed)
    Abstract [en]

    Pin1 is a human peptidyl-prolyl cis-trans isomerase important for the regulation of phosphoproteins that are implicated in many diseases including cancer and Alzheimer's. Further biophysical study of Pin1 will elucidate the importance of the two-domain system to regulate its own activity. Here, we report near-complete backbone and side-chain H-1, C-13 and N-15 NMR chemical shift assignments of full-length, apo Pin1 for the purpose of studying interdomain allostery and dynamics.

  • 4.
    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.

  • 5.
    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.

  • 6.
    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.

  • 7.
    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.

  • 8. 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.

  • 9. 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).

  • 10.
    Jemth, Per
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Karlsson, Elin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Vogeli, Beat
    Univ Colorado Denver, Dept Biochem & Mol Genet, 12801 East 17th Ave, Aurora, CO 80045 USA.
    Guzovsky, Brenda
    Univ Buenos Aires, IQUIBICEN CONICET, FCEyN, Prot Physiol Lab, Intendente Guiraldes 2160,Ciudad Univ,C1428EGA, Buenos Aires, DF, Argentina.
    Andersson, Eva
    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 Pharmacy, Department of Pharmaceutical Biosciences.
    Dogan, Jakob
    Stockholm Univ, Dept Biochem & Biophys, SE-10691 Stockholm, Sweden.
    Guntert, Peter
    Swiss Fed Inst Technol, Lab Phys Chem, ETH Honggerberg, Zurich, Switzerland;Goethe Univ, Ctr Biomol Magnet Resonance, Inst Biophys Chem, D-60438 Frankfurt, Germany;Tokyo Metropolitan Univ, Grad Sch Sci, Tokyo 1920397, Japan.
    Riek, Roland
    Swiss Fed Inst Technol, Lab Phys Chem, ETH Honggerberg, Zurich, Switzerland.
    Chi, Celestine N.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Structure and dynamics conspire in the evolution of affinity between intrinsically disordered proteins2018In: Science Advances, E-ISSN 2375-2548, Vol. 4, no 10, article id eaau4130Article in journal (Refereed)
    Abstract [en]

    In every established species, protein-protein interactions have evolved such that they are fit for purpose. However, the molecular details of the evolution of new protein-protein interactions are poorly understood. We have used nuclear magnetic resonance spectroscopy to investigate the changes in structure and dynamics during the evolution of a protein-protein interaction involving the intrinsically disordered CREBBP (CREB-binding protein) interaction domain (CID) and nuclear coactivator binding domain (NCBD) from the transcriptional coregulators NCOA (nuclear receptor coactivator) and CREBBP/p300, respectively. The most ancient low-affinity "Cambrian-like" [540 to 600 million years (Ma) ago] CID/NCBD complex contained less secondary structure and was more dynamic than the complexes from an evolutionarily younger "Ordovician-Silurian" fish ancestor (ca. 440 Ma ago) and extant human. The most ancient Cambrian-like CID/NCBD complex lacked one helix and several interdomain interactions, resulting in a larger solvent-accessible surface area. Furthermore, the most ancient complex had a high degree of millisecond-to-microsecond dynamics distributed along the entire sequences of both CID and NCBD. These motions were reduced in the Ordovician-Silurian CID/NCBD complex and further redistributed in the extant human CID/NCBD complex. Isothermal calorimetry experiments show that complex formation is enthalpically favorable and that affinity is modulated by a largely unfavorable entropic contribution to binding. Our data demonstrate how changes in structure and motion conspire to shape affinity during the evolution of a protein-protein complex and provide direct evidence for the role of structural, dynamic, and frustrational plasticity in the evolution of interactions between intrinsically disordered proteins.

  • 11.
    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.

  • 12.
    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.

  • 13.
    Nichols, Parker J.
    et al.
    Univ Colorado Denver, Dept Biochem & Mol Genet, Anschutz Med Campus,12801 East 17th Ave, Aurora, CO 80045 USA..
    Born, Alexandra
    Univ Colorado Denver, Dept Biochem & Mol Genet, Anschutz Med Campus,12801 East 17th Ave, Aurora, CO 80045 USA..
    Henen, Morkos A.
    Univ Colorado Denver, Dept Biochem & Mol Genet, Anschutz Med Campus,12801 East 17th Ave, Aurora, CO 80045 USA.;Mansoura Univ, Fac Pharm, Mansoura 35516, Egypt..
    Strotz, Dean
    Swiss Fed Inst Technol, Lab Phys Chem, Vladimir Prelog Weg 2, CH-8093 Zurich, Switzerland..
    Chi, Celestine N.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Güntert, Peter
    Swiss Fed Inst Technol, Lab Phys Chem, Vladimir Prelog Weg 2, CH-8093 Zurich, Switzerland.;Goethe Univ Frankfurt, Inst Biophys Chem, Max Von Laue Str 9, D-60438 Frankfurt, Germany.;Tokyo Metropolitan Univ, Grad Sch Sci, 1-1 Minami Ohsawa, Hachioji, Tokyo 1920397, Japan..
    Vögeli, Beat
    Univ Colorado Denver, Dept Biochem & Mol Genet, Anschutz Med Campus,12801 East 17th Ave, Aurora, CO 80045 USA..
    Extending the Applicability of Exact Nuclear Overhauser Enhancements to Large Proteins and RNA2018In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 19, no 16, p. 1695-1701Article, review/survey (Refereed)
    Abstract [en]

    Distance-dependent nuclear Overhauser enhancements (NOEs) are one of the most popular and important experimental restraints for calculating NMR structures. Despite this, they are mostly employed as semiquantitative upper distance bounds, and this discards the wealth of information that is encoded in the cross-relaxation rate constant. Information that is lost includes exact distances between protons and dynamics that occur on the sub-millisecond timescale. Our recently introduced exact measurement of the NOE (eNOE) requires little additional experimental effort relative to other NMR observables. So far, we have used eNOEs to calculate multistate ensembles of proteins up to approximately 150 residues. Here, we briefly revisit eNOE methodology and present two new directions for the use of eNOEs: applications to large proteins and RNA.

  • 14. Nichols, Parker J.
    et al.
    Born, Alexandra
    Henen, Morkos A.
    Strotz, Dean
    Orts, Julien
    Olsson, Simon
    Güntert, Peter
    Chi, Celestine N.
    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 Advances2017In: Molecules, ISSN 1420-3049, E-ISSN 1420-3049, Vol. 22, no 7, article id 1176Article, review/survey (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.

  • 15.
    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.

  • 16.
    Sundell, Gustav
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Arnold, Roland
    Institute of Cancer and Genomic Sciences, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, UK.
    Ali, Muhammad
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Naksukpaiboon, Piangfan
    Institute of Cancer and Genomic Sciences, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, UK.
    Orts, Julien
    Laboratory of Physical Chemistry, ETH Zürich, Zürich, Switzerland.
    Güntert, Peter
    Laboratory of Physical Chemistry, ETH Zürich, Zürich, Switzerland.
    Chi, Celestine N.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Ivarsson, Ylva
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Proteome‐wide analysis of phospho‐regulated PDZ domain interactions2018In: Molecular Systems Biology, ISSN 1744-4292, E-ISSN 1744-4292, Vol. 14, no 8, article id e8129Article in journal (Refereed)
    Abstract [en]

    A key function of reversible protein phosphorylation is to regulate protein–protein interactions, many of which involve short linear motifs (3–12 amino acids). Motif‐based interactions are difficult to capture because of their often low‐to‐moderate affinities. Here, we describe phosphomimetic proteomic peptide‐phage display, a powerful method for simultaneously finding motif‐based interaction and pinpointing phosphorylation switches. We computationally designed an oligonucleotide library encoding human C‐terminal peptides containing known or predicted Ser/Thr phosphosites and phosphomimetic variants thereof. We incorporated these oligonucleotides into a phage library and screened the PDZ (PSD‐95/Dlg/ZO‐1) domains of Scribble and DLG1 for interactions potentially enabled or disabled by ligand phosphorylation. We identified known and novel binders and characterized selected interactions through microscale thermophoresis, isothermal titration calorimetry, and NMR. We uncover site‐specific phospho‐regulation of PDZ domain interactions, provide a structural framework for how PDZ domains accomplish phosphopeptide binding, and discuss ligand phosphorylation as a switching mechanism of PDZ domain interactions. The approach is readily scalable and can be used to explore the potential phospho‐regulation of motif‐based interactions on a large scale.

  • 17.
    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.

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