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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.
    Broo, Kerstin
    et al.
    Uppsala University, Teknisk-naturvetenskapliga vetenskapsområdet, Chemistry, Department of Biochemistry.
    Larsson, Anna-Karin
    Jemth, Per
    Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Mannervik, Bengt
    An ensemble of Theta class glutathione transferases with novel catalytic properties generated by stochastic recombination of fragments of two mammalian enzymes2002In: Journal of Molecular Biology, Vol. 318, p. 59-70Article in journal (Refereed)
  • 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. ETH.
    Bach, Anders
    Stromgaard, Kristian
    Gianni, Stefano
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Ligand binding by PDZ domains2012In: Biofactors, ISSN 0951-6433, E-ISSN 1872-8081, Vol. 38, no 5, p. 338-348Article, review/survey (Refereed)
    Abstract [en]

    The postsynaptic density protein-95/disks large/zonula occludens-1 (PDZ) protein domain family is one of the most common proteinprotein interaction modules in mammalian cells, with paralogs present in several hundred human proteins. PDZ domains are found in most cell types, but neuronal proteins, for example, are particularly rich in these domains. The general function of PDZ domains is to bring proteins together within the appropriate cellular compartment, thereby facilitating scaffolding, signaling, and trafficking events. The many functions of PDZ domains under normal physiological as well as pathological conditions have been reviewed recently. In this review, we focus on the molecular details of how PDZ domains bind their protein ligands and their potential as drug targets in this context.

  • 10.
    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.

  • 11.
    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.

  • 12.
    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.

  • 13.
    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.

  • 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. Di Matteo, Adele
    et al.
    Calosci, Nicoletta
    Gianni, Stefano
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Brunori, Maurizio
    Travaglini-Allocatelle, Carlo
    Structural and functional characterization of CcmG from Pseudomonas aeruginosa, a key component of the bacterial cytochrome c maturation apparatus2010In: Proteins: Structure, Function, and Bioinformatics, ISSN 0887-3585, E-ISSN 1097-0134, ISSN 20544959, Vol. 78, no 10, p. 2213-2221Article in journal (Refereed)
    Abstract [en]

    The cytochrome c maturation process is carried out in the bacterial periplasm, where some specialized thiol-disulfide oxidoreductases work in close synergy for the correct reduction of oxidized apocytochrome before covalent heme attachment. We present a structural and functional characterization of the soluble periplasmic domain of CcmG from the opportunistic pathogen P. aeruginosa (Pa-CcmG), a component of the protein machinery involved in cyt c maturation in gram-negative bacteria. X-ray crystallography reveals that Pa-CcmG is a TRX-like protein; high-resolution crystal structures show that the oxidized and the reduced forms of the enzyme are identical except for the active-site disulfide. The standard redox potential was calculated to be E-0' = -0.213 V at pH 7.0; the pK(a) of the active site thiols were pK(a) = 6.13 +/- 0.05 for the N-terminal Cys74 and pK(a) = 10.5 +/- 0.17 for the C-terminal Cys77. Experiments were carried out to characterize and isolate the mixed disulfide complex between Pa-CcmG and Pa-CcmH (the other redox active component of System I in P. aeruginosa). Our data indicate that the target disulfide of this TRX-like protein is not the intramolecular disulfide of oxidized Pa-CcmH, but the intermolecular disulfide formed between Cys28 of Pa-CcmH and DTNB used for the in vitro experiments. This observation suggests that, in vivo, the physiological substrate of Pa-CcmG may be the mixed-disulfide complex between Pa-CcmH and apo-cyt. Proteins 2010; 78:2213-2221. (C) 2010 Wiley-Liss, Inc.

  • 16.
    Dogan, Jakob
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Gianni, Stefano
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    The binding mechanisms of intrinsically disordered proteins2014In: Physical Chemistry, Chemical Physics - PCCP, ISSN 1463-9076, E-ISSN 1463-9084, Vol. 16, no 14, p. 6323-6331Article in journal (Refereed)
    Abstract [en]

    Intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) of proteins are very common and instrumental for cellular signaling. Recently, a number of studies have investigated the kinetic binding mechanisms of IDPs and IDRs. These results allow us to draw conclusions about the energy landscape for the coupled binding and folding of disordered proteins. The association rate constants of IDPs cover a wide range (10(5)-10(9) M-1 s(-1)) and are largely governed by long-range charge-charge interactions, similarly to interactions between well-folded proteins. Off-rate constants also differ significantly among IDPs (with half-lives of up to several minutes) but are usually around 0.1-1000 s(-1), allowing for rapid dissociation of complexes. Likewise, affinities span from pM to mu M suggesting that the low-affinity high-specificity concept for IDPs is not straightforward. Overall, it appears that binding precedes global folding although secondary structure elements such as helices may form before the protein-protein interaction. Short IDPs bind in apparent two-state reactions whereas larger IDPs often display complex multi-step binding reactions. While the two extreme cases of two-step binding (conformational selection and induced fit) or their combination into a square mechanism is an attractive model in theory, it is too simplistic in practice. Experiment and simulation suggest a more complex energy landscape in which IDPs bind targets through a combination of conformational selection before binding (e. g., secondary structure formation) and induced fit after binding (global folding and formation of short-range intermolecular interactions).

  • 17.
    Dogan, Jakob
    et al.
    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.
    Only kinetics can prove conformational selection2014In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 107, no 8, p. 1997-1998Article in journal (Other academic)
  • 18.
    Dogan, Jakob
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Jonasson, Josefin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Andersson, Eva
    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.
    Binding Rate Constants Reveal Distinct Features of Disordered Protein Domains2015In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 54, no 30, p. 4741-4750Article in journal (Refereed)
    Abstract [en]

    Intrinsically disordered proteins (IDPs) are abundant in the proteome and involved in key cellular functions. However, experimental data about the binding kinetics of IDPs as a function of different environmental conditions are scarce. We have performed an extensive characterization of the ionic strength dependence of the interaction between the molten globular nuclear co-activator binding domain (NCBD) of CREB binding protein and five different protein ligands, including the intrinsically disordered activation domain of p160 transcriptional co-activators (SRC1, TIF2, ACTR), the p53 transactivation domain, and the folded pointed domain (PNT) of transcription factor ETS-2. Direct comparisons of the binding rate constants under identical conditions show that the association rate constant, k(on), for interactions between NCBD and disordered protein domains is high at low salt concentrations (90-350 x 10(6) M-1 s(-1) at 4 degrees C) but is reduced significantly (10-30-fold) with an increasing ionic strength and reaches a plateau around physiological ionic strength. In contrast, the k(on) for the interaction between NCBD and the folded PNT domain is only 7 x 10(6) M-1 s(-1) (4 degrees C and low salt) and displays weak ionic strength dependence, which could reflect a distinctly different association that relies less on electrostatic interactions. Furthermore, the basal rate constant (in the absence of electrostatic interactions) is high for the NCBD interactions, exceeding those typically observed for folded proteins. One likely interpretation is that disordered proteins have a large number of possible collisions leading to a productive on-pathway encounter complex, while folded proteins are more restricted in terms of orientation. Our results highlight the importance of electrostatic interactions in binding involving IDPs and emphasize the significance of including ionic strength as a factor in studies that compare the binding properties of IDPs to those of ordered proteins.

  • 19.
    Dogan, Jakob
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Mu, Xin
    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.
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    The transition state structure for coupled binding and folding of disordered protein domains2013In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 3, p. 2076-Article in journal (Refereed)
    Abstract [en]

    Intrinsically disordered proteins are abundant in the eukaryotic proteome, and they are implicated in a range of different diseases. However, there is a paucity of experimental data on molecular details of the coupled binding and folding of such proteins. Two interacting and relatively well studied disordered protein domains are the activation domain from the p160 transcriptional co-activator ACTR and the nuclear co-activator binding domain (NCBD) of CREB binding protein. We have analyzed the transition state for their coupled binding and folding by protein engineering and kinetic experiments (Phi-value analysis) and found that it involves weak native interactions between the N-terminal helices of ACTR and NCBD, but is otherwise "disordered-like". Most native hydrophobic interactions in the interface between the two domains form later, after the rate-limiting barrier for association. Linear free energy relationships suggest a cooperative formation of native interactions, reminiscent of the nucleation-condensation mechanism in protein folding.

  • 20.
    Dogan, Jakob
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Schmidt, Tanja
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Mu, Xin
    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.
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Fast Association and Slow Transitions in the Interaction between Two Intrinsically Disordered Protein Domains2012In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 287, no 41, p. 34316-34324Article in journal (Refereed)
    Abstract [en]

    Proteins that contain long disordered regions are prevalent in the proteome and frequently associated with diseases. However, the mechanisms by which such intrinsically disordered proteins (IDPs) recognize their targets are not well understood. Here, we report the first experimental investigation of the interaction kinetics of the nuclear co-activator binding domain of CREB-binding protein and the activation domain from the p160 transcriptional co-activator for thyroid hormone and retinoid receptors. Both protein domains are intrinsically disordered in the free state and synergistically fold upon binding each other. Using the stopped-flow technique, we found that the binding reaction is fast, with an association rate constant of 3 x 10(7) M-1 s(-1) at 277 K. Mutation of a conserved buried intermolecular salt bridge showed that electrostatics govern the rapid association. Furthermore, upon mutation of the salt bridge or at high salt concentration, an additional kinetic phase was detected (similar to 20 and similar to 40 s(-1), respectively, at 277 K), suggesting that the salt bridge may steer formation of the productive bimolecular complex in an intramolecular step. Finally, we directly measured slow kinetics for the IDP domains (similar to 1 s(-1) at 277 K) related to conformational transitions upon binding. Together, the experiments demonstrate that the interaction involves several steps and accumulation of intermediate states. Our data are consistent with an induced fit mechanism, in agreement with previous simulations. We propose that the slow transitions may be a consequence of the multipartner interactions of IDPs.

  • 21.
    Eildal, Jonas N. N.
    et al.
    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.
    Ye, Fei
    Zhang, Mingjie
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Stromgaard, Kristian
    Rigidified Clicked Dimeric Ligands for Studying the Dynamics of the PDZ1-2 Supramodule of PSD-952015In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 16, no 1, p. 64-69Article in journal (Refereed)
    Abstract [en]

    PSD-95 is a scaffolding protein of the MAGUK protein family, and engages in several vital protein-protein interactions in the brain with its PDZ domains. It has been suggested that PSD-95 is composed of two supramodules, one of which is the PDZ1-2 tandem domain. Here we have developed rigidified high-affinity dimeric ligands that target the PDZ1-2 supramodule, and established the biophysical parameters of the dynamic PDZ1-2/ligand interactions. By employing ITC, protein NMR, and stopped-flow kinetics this study provides a detailed insight into the overall conformational energetics of the interaction between dimeric ligands and tandem PDZ domains. Our findings expand our understanding of the dynamics of PSD-95 with potential relevance to its biological role in interacting with multivalent receptor complexes and development of novel drugs.

  • 22.
    Eildal, Jonas N. N.
    et al.
    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.
    Balle, Thomas
    Stuhr-Hansen, Nicolai
    Padrah, Shahrokh
    Gianni, Stefano
    Stromgaard, Kristian
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Probing the Role of Backbone Hydrogen Bonds in Protein-Peptide Interactions by Amide-to-Ester Mutations2013In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 135, no 35, p. 12998-13007Article in journal (Refereed)
    Abstract [en]

    One of the most frequent protein-protein interaction modules in mammalian cells is the postsynaptic density 95/discs large/zonula occludens 1 (PDZ) domain, involved in scaffolding and signaling and emerging as an important drug target for several diseases. Like many other protein-protein interactions, those of the PDZ domain family involve formation of intermolecular hydrogen bonds: C-termini or internal linear motifs of proteins bind as beta-strands to form an extended antiparallel beta-sheet with the PDZ domain. Whereas extensive work has focused on the importance of the, amino acid side chains of the protein ligand, the role of the backbone hydrogen bonds in the binding reaction is not known. Using amide-to-ester substitutions to perturb the backbone hydrogen-bonding pattern, we have systematically probed putative backbone hydrogen bonds between four different PDZ domains and peptides corresponding to natural protein ligands. Amide-to-ester mutations of the three C-terminal amides of the peptide ligand severely affected the affinity with the PDZ domain, demonstrating that hydrogen bonds contribute significantly to ligand binding (apparent changes in binding energy, Delta Delta G = 1.3 to >3.8 kcal mol(-1)). This decrease in affinity was mainly due to an increase in the dissociation rate constant, but a significant decrease in the association rate constant was found for some amide-to-ester mutations Suggesting that native hydrogen bonds have begun to form in the transition state of the binding reaction. This study provides a general framework for studying the role of backbone hydrogen bonds in protein-peptide interactions and for the first time specifically addresses these for PDZ domain-peptide interactions.

  • 23.
    Eklund, Sandra
    et al.