MAPPING THE BINDING SITE OF LIGANDS TO PROTEINS USING CHEMICAL EXCHANGE PARAMETERS BY NMR SPECTROSCOPY JANARTHANAN KRISHNAMOORTHY (M.Tech) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY STRUCTURAL BIOLOGY LABS DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2010 Dedicated to The α and the ω, the good Shephard, my teachers, parents and friends Declaration The work in this thesis is based on research carried out by the author at the NMR Structural biology lab, the Department of Biological Sciences, Singapore. This work has been supervised by Associate professor Dr. Henry Mok and co-supervised by Associate professor Dr. Yang Daiwen and submitted after their prior approval. No part of this thesis has been submitted elsewhere for any other degree or qualification and all the work is original unless referenced apropriately in the text. Name: K.Janarthanan Date: 12st November 2010 Copyright © 2010 by K. Janarthanan. “The copyright of this thesis rests with the author. The quotations from it shall be published with the author’s prior consent and information derived from it should be properly acknowledged”. iii Acknowledgements I would like to express my sincere thanks to my advisor Assoc. Prof. Henry Mok, whose guidance throughout these year has instilled a clinical approach and discipline in my research. Discussing with Dr. Henry is always a pleasure as, he always gives new direction and approach in solving tough problems. I admire Dr. Henry’s clarity and simplicity in explaining difficult concepts, which inspires me to emulate in my academic carrier. I extend my gratefulness for the generous assistance offered by Dr. Henry for academic trainings and conferences. I would also like to thank Assc. Prof. Yang Daiwen for his kind co-supervision. Dr. Yang’s critical and pragmatic way of thinking had helped me switch to a different school of thoughts during the course of my research. My sincere thanks to Prof. Kini, along with Prof. Liou Cherng and Prof. Yao sho qin, who inspired me to choose suitable systems to work on, during my priliminary examination. I owe my deepest gratitude to Prof. Bergie Englert and Prof. Choo Ho Hiap, whose lectures on quantum mechanics helped me address my research problems from a theoretical point of view. The wonderful moments spent in those lecture halls are always memberable in my life for their simplified approach which drew not only passion towards science but also the good nature in me. My special thanks to Prof. Anil Kumar for arranging a summer training course at IISC and introducing me to Prof. Ramanathan, Dr. Ragathoma, Dr. Athreya, Dr. Mahesh, Dr. Jeyanthy, Sangeerth and Jarina. My heartfelt thanks to Prof. James Keeler for the generously distributed notes on NMR. Many thanks to Prof. Alex Bain, whose suggestions through emails has helped me complete the final part of this thesis. My word of appreciation to Prof. Ramakrishna and Dr. Naveen iv whose valuable codes and time helped me implement the automation part of analysis in this work. It is my pleasure to thank my colleagues Dr. Fan, Anir, Olga, Siew leong, Yvonne, Rika, Zheng yu, Zou zimming, Karthik, Long dong and others for their wonderful company during my research. Iam grateful to Dr. Sanjay ghosh, Prof. P. V. Sundaram, Dr. Vaidyalingam, Mrs. Susan, Mrs. Vasanty, Mr. Robertson, who helped me in my yesteryears and would be more than happy to see me graduate. I thank my parents and brothers, Ganesh, Sethu and Adisa, who shared their care and concern during those critical times when I was looking for support. Finally, words cannot express the beauty of the intelligent design, whose game called ‘life’, within the confines of laws called ‘nature’, amazes me to wonder and to explore, what life is all about. Thanks to the creative mind who is behind this very existence. v Table of content Declaration iii Acknowledgements iv Summary x List of tables xii List of figures xiii Acronyms xiv Nomenclature xvii Introduction 1.1 Has structural biology delivered what it has promised? . . . . . . . . . . . 1.2 Protein-ligand interaction and drug discovery . . . . . . . . . . . . . . . . . 1.3 Techniques to investigate protein-ligand interaction . . . . . . . . . . . . . 1.4 NMR based methods for protein-ligand interaction . . . . . . . . . . . . . Exchange NOESY experiment . . . . . . . . . . . . . . . . . . . . . . . 1.4.1 vi 1.4.2 Saturation transfer difference experiment . . . . . . . . . . . . . . . . . 1.4.3 waterLOGSY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4.4 HSQC perturbation experiments . . . . . . . . . . . . . . . . . . . . . . 1.4.5 Isotope edited or filtered experiments . . . . . . . . . . . . . . . . . . . 1.4.6 CPMG experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 1.5 Proteins involved in cancer: an excellent target system for drug design . . 10 1.5.1 Mechanism of Apoptosis . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 1.5.2 Structure aided drug design for cancer treatment . . . . . . . . . . . . . 14 1.6 Using NMR to understand the dynamic protein-ligand interactions . . . . 16 Material and Methods 17 2.1 Protein sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 2.2 ITC titration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 2.3 15 N HSQC titration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 2.4 J-Surface mapping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 2.5 Molecular docking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 Analysing fast chemical exchange systems vii 21 3.1 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 3.2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 3.3 Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 3.3.1 Mechanisms of protein-ligand interaction . . . . . . . . . . . . . . . . . 25 3.3.2 Correction for free ligand concentration . . . . . . . . . . . . . . . . . . 27 3.3.3 Automation using genetic algorithm . . . . . . . . . . . . . . . . . . . . 28 3.3.4 Mapping the binding site of BH3I-1 onto hBclXL . . . . . . . . . . . . . 29 3.A Automated data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 3.B Deriving complex models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 3.C Calculating [L] from [LT ] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 Analysing all chemical exchange systems 45 4.1 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 4.2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 4.3 Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 4.3.1 Automation using genetic algorithm . . . . . . . . . . . . . . . . . . . . 51 4.3.2 Analysis of fast exchange titration (hBclXL and BH3I-1 ) . . . . . . . . . 55 4.3.3 Analysis of the slow exchange titration (mMCL-1 and NOXA-B ) . . . . 58 viii . . . . . . . . . . 4.3.4 Mechanism of interaction of mMCL-1 and NOXA-B 4.3.5 Comparison of optimized parameters of fast and slow exchange 62 regimes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 4.4 Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68 4.A Appendix: Theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70 4.A.1 Mechanisms based ligand correction and calculation of population . . . 72 4.A.2 Mechanism dependent setting up of the Liouville, relaxation 4.A.3 and kinetic matrix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74 Data preperation for line shape analysis . . . . . . . . . . . . . . . . . . 75 Conclusion and future direction 76 5.1 Quantum mechanical approach to study protein-ligand interactions . . . 78 5.2 In-silico drug design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78 Bibliography 95 ix Summary Mapping the binding site of ligands to proteins using chemical exchange parameters by NMR Spectroscopy NMR spectroscopy, along with X-ray crystallography, have advanced to such an extent that structure aided drug design is no longer just a concept on paper. With a myriad of techniques in array, NMR spectroscopy is routinely used to screen ligands, locate binding site and design site specific molecules. Almost all NMR experiments takes advantage of the fundamental behaviour of nuclei namely, the chemical exchange and relaxation phenomena, which can explain the dynamic nature of macromolecules (proteins) and their complexes (protein + ligand) in solution. Classical Bloch-McConnell equations are commonly used to study mechanisms ranging from simple to complex interactions in a quantitative way. We adopted the same approach, to understand the relationship between NMR derived kinetic parameters and the underlying interaction mechanism for protein-ligand systems. Using hBclXL (protein) and BH3I-1 (ligand) as a standard system for the fast exchange regime (weak binding case), we have shown that the rate of change within a population from the free to bound state, can differentiate the binding site residues from the non binding site residues. 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Procaspase-8 Fig 1.5: Mechanism of apoptosis 1.5.1 Mechanism of Apoptosis In the intrinsic pathway, stress factors like cytokine deprivation trigger the release of cytochromec from mitochondria[49] The released cytochrome-c is sequestered by a factor called Apaf-1 (Apoptosis activation factor) present in the cytoplasm This factor oligomerises to form a complex named as ‘apoptosome’ The apoptosome has peptidase... identification of the binding pocket for these molecules is the subsequent stage which greatly assists in shaping these molecules to lead molecules Once the scaffold of the lead structure has been decided upon, a diverse set of molecules are then synthesized to imporve on the selectivity and affinity towards the target protein[15, 16] The promising candidates are taken to the next stage to characterize their toxicity... preventing the cell from responding to normal apoptotic regulation[59, 60, 61] Drugs like gossypol inhibit Bcl-2 oligomerization by binding to their hydrophobic groove, which sensitizes the cells to apoptosis SMACs (Second mitochondria derived activators of caspase), are natural apoptotic proteins released from mitochondria IAP’s (Inhibitors of apoptosis) bind to SMACs and prevent apoptosis Inhibitors of IAP’s... enhance the understanding of the mechanism of interaction but also help us to improve a weakly interacting molecule in to a highly specific therapeutic molecule In the first stage of drug design, a large library of ligands are screened against the target protein and molecules showing characteristic selectivity towards the target protein are chosen for further study into the nature of the interaction[14] The. .. the progression of apoptosis The pro-apoptotic proteins are classified into two types, namely, ‘Bax like’ proteins and ‘BH3 only proteins The ‘Bax like’ proteins contain three BH domains namely, BH1 through BH3, but lack the BH4 domain additionally present in the pro-survival proteins Bax, Bak and Bok are members of this group The second class of proteins, the ‘BH3 only proteins are the minimalists,... only the BH3 domain[55] Bim, Bid, Bmf, Noxa and Puma are members of this group Both of these classes induce apoptosis either directly or indirectly, e.g Bax and Bak can directly interact with the mitochondrial membrane and cause release of cytochrome-c , which in turn activates the apoptosome Whereas, ‘BH3 only proteins indirectly induce apoptosis by binding to the hydrophobic groove of pro-survival proteins. . .exchange system (tight binding case), was analysed and showed that there are regime dependent limitations on using kinetic parameters to interpret binding processes xi List of tables 3.1 Thermodynamic parameters obtained from ITC experiment by fitting the data to sequential three site binding model 30 3.2 Parameters determined by fitting of chemical shifts to model equations... dominate the spectrum, whereas the most informative intermolecular NOEs are seen only through scrupulous optimization of the mixing time in the experiment[28, 29] As the NOE pattern of the bound form would be different from that of the free form of the ligand, the conformation of the protein-ligand complex can be calculated using exchange NOE as the experimental constrain 5 CHAPTER 1 Introduction NMR based... perturbation of chemical shifts, which are represented as arrows in the right side figure The 15 N HSQC spectrum of a protein will contain all the amide and amine protons present in the peptide linkages and side chains The same experiment, when recorded with the addition of ligand, the chemical shifts of the amide protons will shift due to conformational changes caused by ligand interactions[34] The perturbed... procaspase 9 to its active form caspase 9 Upon activation, caspase 9 catalyses the cleavage of caspase 3, which irreversibly leads the cell to its death, by activating further caspases downstream The members of the Bcl-2 family are a set of proteins which regulate (stimulate/inhibit) the activity of the apoptosome and caspases at various levels[50] Bcl-2 family proteins are broadly classified into pro-survival . MAPPING THE BINDING SITE OF LIGANDS TO PROTEINS USING CHEMICAL EXCHANGE PARAMETERS BY NMR SPECTROSCOPY JANARTHANAN KRISHNAMOORTHY (M.Tech) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY STRUCTURAL. 95 ix Summary Mapping the binding site of ligands to proteins using chemical exchange parameters by NMR Spectroscopy NMR spectroscopy, along with X-ray crystallography, have advanced to such an extent that structure. characteristic selectivity towards the target protein are chosen for further study into the nature of the interaction[14]. The identification of the binding pocket for these molecules is the subsequent stage