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Post-synaptic Density Disc Large Zo-1 (PDZ) Domains: From Folding and Binding to Drug Targeting
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. (Per Jemth)
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Understanding how proteins fold and bind is interesting since these processes are central to most biological activity. Protein folding and protein-protein interaction are by themselves very complex but using a good and robust system to study them could ease some of the hurdles.

In this thesis I have tried to answer some of the fundamental questions of protein folding and binding. I chose to work with PDZ domains, which are protein domains consisting of 90-100 amino acids. They are found in more than 400 human proteins and function mostly as protein-protein interaction units. These proteins are very stable, easy to express and purify and their folding reaction is reversible under most laboratory conditions.

I have characterized the interaction of PSD-95 PDZ3 domain with its putative ligand under different experimental conditions and found out that its binding kinetics is sensitive to salt and pH.  I also demonstrated that the two conserved residues R318 and H372 in PDZ3 are responsible for the salt and pH effect, respectively, on the binding reaction. Moreover, I determined that for PSD 95 PDZ3 coupling of distal residues to peptide binding was better described by a distance relationship and there was a very weak evidence of an allosteric network. Further, I showed that another PDZ domain, SAP97 PDZ2 undergoes conformational change upon ligand binding.

Also, I characterized the binding mechanism of a dimeirc ligand/PDZ1-2 tandem interaction and showed that despite its apparent complexity the binding reaction is best described by a square scheme. Additionally, I determined that for the SAP 97 PDZ/HPV E6 interaction that all three PDZ domains each bind one molecule of the E6 protein and that a set of residues in the PDZ2 of SAP 97 could operate in an unexpected long-range manner during E6 interaction.

Finally, I showed that perhaps all members in the PDZ family could fold via a three state folding mechanism. I characterized the folding mechanism of five different PDZ domains having similar overall fold but different primary structure and the results indicate that all five fold via an intermediate with two transition states. Transition state one is rate limiting at low denaturant concentration and vice versa for transition state two. Comparing and characterizing the structures of the transition states of two PDZ domains using phi value analysis indicated that their early transition states are less similar as compared to their late transition states.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2010. , p. 42
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 573
Keywords [en]
protein-protein interaction, protein folding, and drug design
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:uu:diva-126129ISBN: 978-91-554-7836-0 (print)OAI: oai:DiVA.org:uu-126129DiVA, id: diva2:322083
Public defence
2010-09-03, B22, BMC, Uppsala, 10:15 (English)
Opponent
Supervisors
Available from: 2010-08-16 Created: 2010-06-03 Last updated: 2010-08-25Bibliographically approved
List of papers
1. Two conserved residues govern the salt and pH dependencies of the binding reaction of a PDZ domain
Open this publication in new window or tab >>Two conserved residues govern the salt and pH dependencies of the binding reaction of a PDZ domain
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2006 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 281, no 48, p. 36811-36818Article in journal (Refereed) Published
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.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-22437 (URN)10.1074/jbc.M607883200 (DOI)000242220800039 ()17018532 (PubMedID)
External cooperation:
Available from: 2007-01-17 Created: 2007-01-17 Last updated: 2017-12-07Bibliographically approved
2. Reassessing a sparse energetic network within a single protein domain
Open this publication in new window or tab >>Reassessing a sparse energetic network within a single protein domain
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2008 (English)In: 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) Published
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.

Keywords
allostery, coupling energy, dynamics, energetic network of residues, PDZ domain
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-98242 (URN)10.1073/pnas.0711732105 (DOI)000254772700028 ()18339805 (PubMedID)
Available from: 2009-02-18 Created: 2009-02-18 Last updated: 2017-10-16Bibliographically approved
3. A sequential binding mechanism in a PDZ domain
Open this publication in new window or tab >>A sequential binding mechanism in a PDZ domain
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2009 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, no 30, p. 7089-7097Article in journal (Refereed) Published
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.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-119523 (URN)10.1021/bi900559k (DOI)000268231800002 ()19496620 (PubMedID)
Available from: 2010-02-26 Created: 2010-02-26 Last updated: 2017-10-16Bibliographically approved
4. A conserved folding mechanism for PDZ domains
Open this publication in new window or tab >>A conserved folding mechanism for PDZ domains
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2007 (English)In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 581, no 6, p. 1109-1113Article in journal (Refereed) Published
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.

Keywords
Folding mechanism, Kinetics, PDZ domain, Protein folding
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-15771 (URN)10.1016/j.febslet.2007.02.011 (DOI)000245370600006 ()17316619 (PubMedID)
Available from: 2008-03-05 Created: 2008-03-05 Last updated: 2017-12-08Bibliographically approved
5. Comparison of successive transition states for folding reveals alternative early folding pathways of two homologous proteins
Open this publication in new window or tab >>Comparison of successive transition states for folding reveals alternative early folding pathways of two homologous proteins
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2008 (English)In: 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) Published
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.

Place, publisher, year, edition, pages
The National Academy of Sciences of the USA, 2008
Keywords
energy landscape, kinetics, molecular dynamics, phi analysis, protein folding
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-98177 (URN)10.1073/pnas.0804774105 (DOI)000261706600041 ()19033470 (PubMedID)
Available from: 2009-02-16 Created: 2009-02-16 Last updated: 2017-12-13Bibliographically approved
6. Design and synthesis of highly potent and plasma-stable dimeric inhibitors of the PSD-95-NMDA receptor interaction
Open this publication in new window or tab >>Design and synthesis of highly potent and plasma-stable dimeric inhibitors of the PSD-95-NMDA receptor interaction
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2009 (English)In: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 48, no 51, p. 9685-9689Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
Wiley-VCH, 2009
Keywords
bridging ligands, dimerization, inhibitors, protein-protein interactions, receptors
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-119518 (URN)10.1002/anie.200904741 (DOI)000273093700017 ()19937879 (PubMedID)
Available from: 2010-02-26 Created: 2010-02-26 Last updated: 2017-12-12Bibliographically approved
7. Deciphering the kinetic binding mechanism of dimeric ligands, using a potent plasma-stable dimeric inhibitor of postsynaptic density protein-95 as an example
Open this publication in new window or tab >>Deciphering the kinetic binding mechanism of dimeric ligands, using a potent plasma-stable dimeric inhibitor of postsynaptic density protein-95 as an example
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2010 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 285, no 36, p. 28252-28260Article in journal (Refereed) Published
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.

Keywords
protein-protein interactions, dimeric ligand, PDZ, conformational change, inhibitors
National Category
Other Basic Medicine
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-126083 (URN)10.1074/jbc.M110.124040 (DOI)000281404100068 ()20576616 (PubMedID)
Available from: 2010-06-02 Created: 2010-06-02 Last updated: 2018-01-12Bibliographically approved
8. Conformational change and non-canonical interactions in the complex between human papillomavirus E6 protein and Synapse-Associated Protein 97
Open this publication in new window or tab >>Conformational change and non-canonical interactions in the complex between human papillomavirus E6 protein and Synapse-Associated Protein 97
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(English)In: Article in journal (Refereed) Submitted
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

Keywords
cervical cancer, NMR, kinetics, HPV, SAP97, isothermal calorimetry
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-126084 (URN)
Available from: 2010-06-02 Created: 2010-06-02 Last updated: 2017-10-16Bibliographically approved

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