CRYSTAL STRUCTURE AND REGULATORY MECHANISM OF CALPAIN Insights into Calcium-Dependent Proteol y sis CHRISTOPHER MARK HOSFIELD A thesis submitted to the Department of Biochemistry in conformity with the requirements for the degree of Doctor of Philosophy Queen's University Kingston?Ontario, Canada Apnl, 2001 copyright Q Christopher Mark Hosfield, 2001 National Library Bibliothèque nationale l*l of Canada du Canada Acquisitions and Acquisitions et Bibliographie SeMces selvices bibliographiques The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive pennethnt à la National Library of Canada to Bibliothéque nationale du Canada de reproduce, loan, distriiute or seil reproduire, prêter, distribuer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/nùn, de reproduction sur papier ou sur format électronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. The ubiquitous calpains (p- and m-calpain) are heterodimeric enzymes that combine cysteine protease activity togetha with ca2+-binding EF-hand motifs in one molecule. This featrire is unique to the calpains and has linked them to signaling pathways stimulated by ca2'. Excessive activation of calpains due to deregdation of intracellular ca2+homeostasis is implicated in neurodegeneration (as in Alzheimer's and Parkinson's diseases) and ischemic tissue damage (following heart attack or stroke). Consequently, calpains are considered important therapeutic targets and much interest exists to develop effective calpain-specific inhibitors. The enzymatic activity of calpain is tightly regulated by several factors including heterodimer formation, limited autolysis, phospholipid membranes, activator proteins and calpastatin. Even regulation through ca2', the only absolute requirement for activity, is complicated by the fact that calpain requires significantly more ca2* for activation in vino than is available in vivo. The precise rnechanism of calpain activation by ca2' is a fundamental biochemical question that has remained poorly understood, largely owing to the lack of available structural information. To better undentand the functional replation of this intriguing enzyme, we have det mined the three-dimensional structure of rat m-calpain by X-ray aystallography. m-Calpain was crystallized in the absence of ca2+in two crystd foms and a selenomethionine-derivative was used to determine the structure using the method of multiwavelength anomalous dispersion. Refined to 2.6 A resolution, this structure is the first reported for any calpain heterodimer. Structural analysis reveais that the calpain heterodimer is an elongated molecule with several discrete domains. The cysteine protease cornponent (domains 1 and II) and the EF-hand domains (IV and VI) are situated at opposite poles of the enzyme. linked covalently through an extended linker and p- sandwich domain similar to a C2-motif (domain-III), as well as non-covalently by a unique, pro-segment-like N-terminal a-helix. This inactive form of calpain exhibits a novel mechanisrn of cysteine protease zymogen-inactivation: protease domains 1 and II are held apart and consequently the catalytic triad and substrate-binding cleft are not formed. The a-helical pro-segment does not occupy a pre-forrned active site, as in other cysteine proteases, but instead inhiiits its assernbly by senring as a conformational restraint on protease domain-1, anchoring it to the reglatory subunit (domain-VI). It is likely, therefore, that auto1ytic removal of the pro-segment reduces the ca2'-requirement of calpain by removing this remaint A set of highly conserveci inter-domain salt-bridges between lysine residues in protease domain-11 and acidic residues in the Crlike domain III serve as a conformational restraint on protease dornain-II. Disruption of these key interactions through site-directed mutagenesis has verified their inhiiitory role, and was shown to significantly reduce the [ca2'] required for activation in vitro. The finding of a Cl-like domain in the m-calpain structure has provided a potentiaiiy novel insight, suggestiDg that the EF-hand domains are not the sole replators of the ~a~+-res~onse. Indeed, the mutagenesis studies have indicated the afhity of m-calpain for ca2+ is influenced by domain III, which is far-removed fiom the EF-han& Although a structure of an activated, caN-bound enzyme has not yet been determined, it is clear that ca2+- binding faditates calpain activation through conformational changes that re-orient the protease domains and allow the formation of the substrate-binding clefl and a functional catalytic triad ACKNOWLEDGEMENTS Above dl, 1 would like to thank my supervisor Dr. Zongchao Jia for allowing me the opportunity to study such an interestkg topic in an exciting and extremely important field of biochernistry. Most important to me were the excellent guidance and support I received fÏom Dr. Jia throughout this entire project. His open-mindedness allowed me to investigate many independent questions, and he always made himself available to hear my fmdings and concerns. On a persona1 note, 1 enjoyed many hurnorous and non- scientific discussions with Dr. Jia and his entire family. in particular 1 enjoyed the practical conversations we had about crystallography, science, careers, and life during our many late nights while away at the synchrotron. A special thank you is also reserved for my brother David, who was a Ph.D candidate in the laboratory of Dr. John Tainer at The Scripps Research Institute in San Diego over the same time period. In the initial stages of my research, 1 benefited greatly fiorn his help, as he was second only to Dr. Jia as far as offering me advice to overcome the problerns encountered in this project. Many members of Dr. Aa's laboratory contributed to my success as a graduate snident at Queen's. Dr. Qilu Ye, Daniel Lim, Ante Tocilj, Eeva Leinala, Brent Wathen, Steffen Graether and Dr. Gour Pal have dl provided me with help; be it teaching me the hdamentals of crystallization or cornputer skills, or by assisting me on several trips to the synchrotron. Special recognition should also be given to Yih-Cherng Liou and Leslie Magtanong, students who also assisted with data collection at the synchrotron. Additionally, I must thank undergraduate students Alex Mok for assistance with molecular modeling and Michael Sung for technical help with protein purification and mutagenesis. The help fiom our pnmary collaborators Dr. John Elce and Dr. Peter Davies and their laboratones is also gratefully acknowledged. Both Dr. Elce and Dr. Davies provided me with insights into the world of biochemistry that are not typically found in the X-ray lab. 1 must also thank Dr. Elce for his patience and his uncanny ability for instilling me with motivation. The excellent technical support of Carol Hegadom and Sherry Gauthier is also recognized as a significant contribution to my project. A very special thank you is saved for Tudor Moldoveanu, a doctoral student in Dr. Davies' laboratory. Tudor provided me with both unparalleled fiiendship and academic challenges throughout the entire duration of this thesis. Tudor sees the world of science much differently than 1, which made for an endless nurnber of conversations that ended with very insightful conclusions. It is very appropriate that our combined efforts will go hand-in-hand in contributing a new and unforeseen understanding to the field of calpain research. It goes without saying that my most heart-felt thank you is reserved for my brothers David and Scott, and in particular my parents Robert and Dome. It is arnazing how much I have continued to lem from than, in spite of the fact they have no idea what a protein is. Finally, 1 would like to thank the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Canadian Institutes of Health Research (CIHR) for providing me with scholarships. Grants hm the CIHR, the Protein Engineering Network of Centers of ExceIlence, and Warner-Lambert Canada fundeci this research. LIST OF ABBREVIATIONS APS Advanced Photon Source BNL Brookhaven National Laboratory CCD charge coupled device CHESS Comell High Energy Synchrotron Source DEAE diethylamine ethyl OMS0 dimethyl sulfoxide D?T dithiothreitol EDTA ethylene-diamine tetraacetic acid FPLC fast protein liquid chromatography IPTG iso-propyl P-thiogalactoside keV kilo electron volts LB Luria Bertani medium MAD multiwavelength anomalous dispersion MALDI matrix-assisted laser desorption ionization MES 2-morpholinoethanesulfonicacid MIR multiple isomorphous replacement MIRAS multiple isomorphous replacement with anomalous scattering MOI multiplicity of infection MPD 2-methyl-2,4-pent anediol NSLS National Synchrotron Light Source PAGE polyacrylamide gel electrophoresis PEG pol yethy lene glycol PMSF phenyiniethyl sulfonyl fluoride r.m.s.d, root mean square deviation SDS sodium dodecylsulfate ssDNA single-stranded DNA SSRL Stanford Synchrotron Radiation Laboratory TCA trichloroacetic acid UV ultraviolet TABLE OF CONTENTS ACKNOWEDGEMENTS ...........................................................................................