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Interactions of Native Peptides and Small Molecules with the Pdz Domains of Psd-95 and Sap97

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Interactions of Native Peptides and Small Molecules with the Pdz Domains of Psd-95 and Sap97 INTERACTIONS OF NATIVE PEPTIDES AND SMALL MOLECULES WITH THE PDZ DOMAINS OF PSD-95 AND SAP97 Thesis submitted in accordance with the requirements of the University of Liverpool for the degree of Doctor in Philosophy by LIAM ANTHONY DORR July 2013 Abstract ABSTRACT A PDZ domain is a small, ~ 90 amino acid residue region of a protein that acts as a protein-protein interaction module. There are currently 267 known PDZ domain- containing proteins in the human genome, with the predominant function of a PDZ domain being the recognition and binding of C-terminal motifs in partner proteins. Examples of well-studied multi PDZ domain-containing proteins are the postsynaptic density-95 protein (PSD-95) and the synapse-associated protein 97 (SAP97); different PSD-95 and SAP97 PDZ domain-mediated interactions have been implicated in a variety of pathological conditions. The interaction of the PSD-95 PDZ domains with the 5-hydroxytryptamine receptor 2a (5-HT2a) & 2c (5-HT2c) variants is known to be important in inducing hyperalgesia in neuropathic pain; the PDZ-mediated interaction of SAP97 with the human papillomavirus type 18 (HPV18) E6 protein is an important event in a p53-independent pathway of cervical carcinogenesis. As PDZ domains have been shown to bind small organic molecules and that the majority of free energy contributions of the PDZ domain interaction interface to binding, are due to a select few ‘hotspot’ regions; the development of novel, reversible small molecule inhibitors of the PSD-95 and SAP97 PDZ domains was deemed a viable research target. This was the overriding aim of the research programme detailed in this thesis and encompassed biophysical techniques such as: protein production, NMR spectroscopy, isothermal titration calorimetry (ITC), restraint-driven docking and structure determination methodologies. The PDZ-mediated interactions of 5-HT2a and 5-HT2c with the PSD-95 and SAP97 PDZ1 & PDZ2 were confirmed using NMR spectroscopy and then, quantified by ITC. The structure of the physiologically relevant complex formed between PSD-95 i Abstract PDZ1 and 5-HT2c was determined using NMR solution state spectroscopic determination methods; this is the first example of a PSD-95 PDZ – 5-HT2x complex structure. A systematic investigation into the interaction between the SAP97 PDZ2 domain and the HPV18 E6 protein was carried out by ITC; this process involved binding experiments utilising commercially acquired peptides of varying lengths and sequences, containing natural and non-natural amino acids. A variety of small organic molecules that bind to the PSD-95 PDZ1 domain have been developed by an NMR screening process. The PSD-95 PDZ1 binding affinity of two of the small molecules was determined by NMR spectroscopy and their inhibitory effect on the native PSD-95 PDZ1 – 5-HT2c interaction was quantified using ITC. The structure of the individual PSD-95 PDZ1 – small molecule complexes formed were determined by NMR restraint-driven docking, using the high-ambiguity driven docking (HADDOCK) programme; this is the first instance of HADDOCK being utilised to determine a PDZ domain – small molecule complex structure. The research detailed in this thesis alludes to the possibility that PSD-95 and SAP97 PDZ domain-mediated interaction inhibition by a small molecule compound may not be a feasible research aim and that future PDZ domain inhibition research may need to be focus on other inhibitor structures i.e. β-strand peptidomimetics. ii Table of Contents Abstract ........................................................................................................................ i Table of Contents ...................................................................................................... iii Publications .............................................................................................................. viii Acknowledgements .................................................................................................... ix Abbreviations ............................................................................................................. x 1. Introduction ............................................................................................................ 1 1.1 Protein-Protein Interactions ................................................................................ 2 1.2 Synapses ............................................................................................................. 4 1.3 Postsynaptic Density Membrane-Associated Guanylate Kinases ...................... 5 1.4 PDZ Domains ..................................................................................................... 6 1.5 PDZ Proteins: PSD-95 and SAP97 ..................................................................... 9 1.5.1 PDZ Domain Comparison ........................................................................... 9 1.5.2 Postsynaptic Density-95 Protein (PSD-95) ............................................... 10 1.5.1 General Overview ............................................................................... 10 1.5.2 PSD-95 PDZ Domain-Mediated Interactions ..................................... 11 1.5.3 Synapse-Associated Protein 97 (SAP97)................................................... 14 1.5.1 General Overview ............................................................................... 14 1.5.2 SAP97 PDZ Domain-Mediated Interactions ...................................... 14 1.5.4 Roles of PSD-95 and SAP97 in LTD, LTP and Synaptic Plasticity ......... 17 1.5.5 Summary .................................................................................................... 21 1.6 Previous PDZ Domain Inhibition Research ..................................................... 21 1.6.1 Peptides ...................................................................................................... 21 1.6.2 Peptidomimetics ........................................................................................ 26 1.6.3 Small Molecule Inhibitors ......................................................................... 30 1.6.3.1 Rational Design ............................................................................... 30 1.6.3.2 Virtual Library Screening/NMR Spectroscopy ............................... 32 1.6.3.3 High-Throughput Screening ............................................................ 34 1.6.3.4 Re-Purposing of Existing Drugs ...................................................... 35 1.6.4 Summary of Previous PDZ Domain Inhibition Research.......................... 36 1.7 Conclusion ........................................................................................................ 37 1.8 Research Aims .................................................................................................. 38 2. Introduction to Biophysical Techniques ............................................................ 41 2.1 General ............................................................................................................. 42 2.2 Protein Nuclear Magnetic Resonance (NMR) Spectroscopy ........................... 42 2.2.1 Introduction to NMR Spectroscopy ........................................................... 42 2.2.2 Classical NMR Spectroscopy .................................................................... 42 2.2.3 Experimental Aspects of NMR Spectroscopy ........................................... 52 iii Table of Contents 2.2.4 Applications of NMR Spectroscopy .......................................................... 64 2.2.4.1 Protein Structure Determination ...................................................... 64 2.2.4.2 Characterisation of Intermolecular Interactions .............................. 64 2.3 Isothermal Titration Calorimetry (ITC) ............................................................ 65 2.3.1 Introduction to ITC .................................................................................... 65 2.3.2 Advantages of ITC ..................................................................................... 66 2.3.3 Instrumentation and Experimental Overview ............................................ 67 2.3.4 Analysis of Experimental Data .................................................................. 69 2.3.5 Applications of ITC ................................................................................... 71 3. Materials and Methods ........................................................................................ 72 3.1 Materials ........................................................................................................... 73 3.1.1 Water.......................................................................................................... 73 3.1.2 General Solvents ........................................................................................ 73 3.1.3 General Reagents ....................................................................................... 73 3.1.4 General Equipment .................................................................................... 73 3.1.5 General Experimental Considerations ....................................................... 74 3.1.6 Expression Vectors .................................................................................... 74 3.1.7 Bacterial Strains ......................................................................................... 74 3.1.8 Proteases ...................................................................................................
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