STRUCTURAL ANALYSIS OF CALCIUM-INDUCED CHANGES IN GELSOLIN AND ADSEVERIN NAG SHALINI NATIONAL UNIVERSITY OF SINGAPORE 2010 STRUCTURAL ANALYSIS OF CALCIUM-INDUCED CHANGES IN GELSOLIN AND ADSEVERIN NAG SHALINI (B. Appl. Sc. (1st Class Hons.), NUS) DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2010 Acknowledgements Acknowledgement is due to so many people that it is almost impossible to mention them all here. I have however, made a feeble attempt to thank those without whom this thesis would not exist. I sincerely thank everyone who is mentioned here and everyone who isn’t but has contributed in different and sometimes, intangible ways. Bob, for making me part of your lab when you weren’t sure you wanted to, for reawakening my interest in science and crystallography, for supporting, encouraging and teaching me, for patiently answering my questions, for burning evening and weekend beers (and hours) to help me finish my thesis on time – no thanks can suffice. Dr. Swami, for introducing me to crystallography and giving me the opportunity to start my Ph.D. Prof. Ding for giving me a decent foundation in molecular biology, protein expression and purification. Dr. Pua, for my first opportunity to experience working in a lab. Wang Jing and Liu Pei, for ensuring that I treat ethidium bromide and acrylamide and the like, with the respect that they deserve, and ensuring that I am not an occupational hazard to my colleagues. My collaborators, Les, Toomy, Hui Wang, Bala, for the structures, movies, images, editing and advice that made the publications possible. The BR lab - Alvin, for all the ultra-pure actin and for maintaining the screenmaker – without them no other acknowledgements would have to be; Marten and Albert, for editing, more editing, advisory and query-resolving services, and for the vectors; Wei Lin and Qing, for teaching me so much for collaborating with me; Maria, for your invaluable advice; Khay Chun and David, for all that I’ve learnt from our discussions and everyone else, for helping in so many ways and generally providing good company and lots of fun. My colleagues and friends – Karen and Shee Mei, I could never have done this without your advice and encouragement; Linan, Danny, Kar Lai, Amanda, Vani, Sumana, Soah Yee, for all the impromptu counselling sessions; Bala, Punitha, Sowmya, Akshay, Siddharth, Deepak, Buvana, Dimple, Leslie, Annie, Ana, for keeping me from becoming clinically insane. My family, for always being there for me – both as spring board and safety net. Surya, for being my rudder, sail and anchor, whichever I needed most whenever I needed it – I could never have reached this point without you. i Table of Contents Acknowledgements…………………………………………………………………………. i Table of Contents………………………………………………………………………… . ii Summary…………………………………………………………………………………… vi List of Tables……………………………………………………………………………… . viii List of Figures……………………………………………………………………………… ix Abbreviations……………………………………………………………………………… xi Chapter 1. Introduction……………………………………………………………………. 1.1 Introductory Remarks……………………………………………………………… 1.2 Actin – custom made cellular infrastructure………………………………………… 1.2.1 Actin functions in the cytoplasm……………………………….………… . 1.2.2 Actin functions in the nucleus……………………………… …………… . 1.2.3 In the X-ray beam – the structure of actin……………… ………………… 1.2.3.1 G-actin………………………………………… ………………… 1.2.3.2 F-actin……………………………………………………… …… 1.3 1.4 1.2.4 Actin dynamics………………………………….………………………… 1.2.5 Regulation of actin dynamics – The role of actin binding proteins………… 10 The gelsolin superfamily………………………………………………………….… 11 1.3.1 Overview…………………………………………………………………… 11 1.3.2 An introduction to gelsolin and its mammalian relatives………………… . 14 1.3.3 Cellular functions of gelsolin family proteins……………………………….18 1.3.4 Regulation of gelsolin family proteins……………………………………… 19 Gelsolin……………………………………………………………………………… 21 1.4.1 Key characteristics unique to gelsolin……………………………………… 21 1.4.2 Modular distribution of gelsolin functions………………………………… 22 1.4.3 Molecular gymnast, crystallographer’s bane – conformational antics of gelsolin……………………………………………………………………23 ii 1.4.3.1 Structure of inactive gelsolin………………………………………. 24 1.4.3.2 Structure of the active C-terminal half of gelsolin………………… 24 1.4.3.3 Structure of the active N-terminal half of equine plasma gelsolin….26 1.5 F-actin severing by gelsolin…………………………………………………………. 27 Chapter 2. Materials and Methods……………………………………………………… . 30 2.1 Materials…………………………………………………………………………… 30 2.2 Methods…………………………………………………………………………… . 32 2.2.1 Cloning and construct preparation………………………………………… 32 2.2.1.1 Vectors used……………………………………………………… . 32 2.2.1.2 Strains and culture conditions……………………………………… 35 2.2.1.3 Cloning of scinlA and scinlB………………………………………. 35 2.2.1.4 Site-directed mutagenesis………………………………………… .35 2.2.2 Protein expression and purification………………………………………….35 2.2.2.1 Gelsolin and calcium-binding mutants, scinderin-like A, scinderin-like B…………………………………………………… 35 2.2.2.2 Gelsolin G2-G3…………………………………………………… 36 2.2.3 Analysis of protein purity………………………………………………… . 37 2.2.4 Dynamic light scattering……………………………………………………. 38 2.2.5 Functional assays…………………………………………………………… 38 2.2.5.1 Actin depolymerization assay……………………………………… 38 2.2.5.2 Actin monomer sequestration assay……………………………… . 40 2.2.5.3 Actin filament nucleation assay……………………………………. 40 2.2.6 Preparation of protein complexes………………………………………… . 41 2.2.7 Crystallization………………………………………………………………. 41 2.2.8 Data collection……………………………………………………………… 42 2.2.9 Structure solution, refinement and analysis………………………………… 42 2.2.9.1 Human G1-G3/actin complex……………………………………… 42 2.2.9.2 Calcium-bound human G3…………………………………………. 43 iii 2.2.9.3 Zebrafish scinderin-like B…………………………………………. 43 Chapter 3. Calcium activation of gelsolin – Part I……………………………………… .45 3.1 Background………………………………………………………………………… 45 3.2 Results………………………………………………………………………………. 48 3.2.1 Å structure of the N-terminal half of human gelsolin in complex with actin……………………………………………………………………. 48 3.2.1.1 Description of human G1-G3/actin structure………………………. 48 3.2.1.2 Comparison with the structure of calcium-bound equine G1-G3/actin………………………………………………………… 51 3.2.1.3 Comparison with the structure of cadmium-bound G2…………… 51 3.2.2 1.2 Å structure of activated G3 in isolation………………………………… 53 3.2.2.1 Preparation and characterization of G2-G3…………………………53 3.2.2.2 Data collection, modeling and refinement of G3………………… . 54 3.2.2.3 The structure of active gelsolin domain in isolation…………… . 54 3.2.2.4 Comparison with active G3 from the G1-G3/actin structure………. 56 3.2.2.5 Comparison with inactive G3……………………………………….57 3.3 Discussion…………………………………………………………………………… 59 Chapter 4. Calcium activation of gelsolin – Part II………………………………………. 66 4.1 Background………………………………………………………………………… 66 4.2 Results………………………………………………………………………………. 68 4.2.1 Preparation of gelsolin calcium-binding site mutants………………………. 68 4.2.2 Actin depolymerization assay – an assay for studying the roles of the type-II calcium-binding sites of gelsolin…………………………………… 68 4.2.3 G2 and G6 calcium-binding sites contribute to gelsolin activity…………… 71 4.2.4 Different domains of gelsolin may have conflicting roles………………… 74 4.2.5 GQMG4G5 has activity comparable to GTL……………………………… 77 4.2.6 Six-site mutants can deploymerize actin to the same extent as activated GFL…………………………………………………………………………. 79 4.2.7 The six-site gelsolin mutants sequester monomers but not nucleate filaments…………………………………………………………………… 79 iv 4.3 Discussion…………………………………………………………………………… 82 Chapter 5. Adseverin and scinderin-like B offer insights into the structure, function and regulation of the gelsolin family……………………………… . 89 5.1 The adseverin C-terminal half imitates the structure and actin binding mechanism of gelsolin……………………………………………………………… 89 5.1.1 Background – Adseverin and gelsolin: homologous sequences, variable function and regulation…………………………………………………… . 89 5.1.2 Results……………………………………………………………………… 92 5.1.2.1 Actin-binding by adseverin mirrors actin-binding by gelsolin…… 92 5.1.2.2 Adseverin A5-A6 is unable to nucleate actin filaments……………. 92 5.1.2.3 The N-terminal half confers calcium regulation on adseverin…… . 94 5.2 The structure of calcium-free scinderin-like B……………………………………… 95 5.2.1 Background – Divergent sequences, novel functions – scinderin-like genes from zebrafish……………………………………………………… . 95 5.2.2 Results……………………………………………………………………… 96 5.2.2.1 Sequence comparison of scinlA and scinlB with adseverin and gelsolin………………………………………………………… 96 5.2.2.2 Protein preparation and crystallization…………………………… .96 5.2.2.3 Actin-related functions of zebrafish scinderin-like genes………… 96 5.2.2.4 Structural data for scinlB………………………………………… . 99 5.2.2.5 Structure of inactive scinlB………………………………………… 101 5.2.2.6 Comparative study of the scinlB domains…………………………. 105 5.2.2.7 Comparative analysis of individual domains of scinlB and gelsolin…………………………………………………………… . 105 5.2.2.8 Towards the structure of scinlB/actin………………………………109 5.3 Discussion……………………………………………….………………………… . 109 Chapter 6. Perspectives and Conclusions…………………………………………………. 119 6.1 Matters arising – insights and questions………………………………………… . 119 6.2 Future Work…………………………………………………………………………. 125 References…………………………………………………………………………………. 128 Appendices………………………………………………………………………………… 144 v Summary The actin cytoskeleton is a dynamic machine that constantly undergoes rearrangements to perform diverse cellular functions, which range from powering movement to providing mechanical strength and shape. Actin function is regulated by numerous actin-binding proteins (ABPs) that modulate its polymerization kinetics and interactions with other cellular components. The gelsolin superfamily of calcium-dependent ABPs, are multifunctional regulators of actin dynamics that participate in numerous cellular processes. Calcium binding, to the six conserved type-II sites, activates gelsolin by inducing large structural rearrangements that expose the actin-binding sites. Gelsolin can then bind monomeric actin or sever, cap and nucleate filaments. However, the mechanisms through which calcium binding is translated into large domain movements, and the contribution of the individual type-II sites, remain unclear. This thesis describes the structures of the actin-bound N-terminal half (G1-G3) and active domain-3 (G3) of human cytoplasmic gelsolin. These data, in combination with the functional characterization of calcium-binding site mutants of gelsolin, provide insights into gelsolin activation. Release of the C-terminal tail-latch partially activates gelsolin, by allowing it to dynamically shift between open and closed conformations, and appears to be one of the early steps of activation. Cooperative binding of calcium to domains (G2) and (G6) mediates tail-latch release by inducing localized structural changes, possibly resulting in clashes that push these two domains apart. Cooperative binding of calcium to G2, G4 and G6 or G2, G5 and G6, appear to activate the C-terminal half of gelsolin (G4-G6) by springing the G4-G6 latch in addition to the tail-latch. The role of the gelsolin mutations, studied here, has relevance for familial amyloidosis of the Finnish-type (FAF). The G2 mutation causing FAF was observed to be activating at low calcium concentrations and had a destabilizing effect at high calcium concentrations. Thus, mutant gelsolin possibly gets trapped in intermediate conformations where the furin cleavage site is exposed and gets cleaved into the deposit-forming fragments. vi The functional characterization studies of adseverin, presented here, suggest that the C-terminal half of human adseverin (A4-A6) is indistinguishable from that of G4-G6, placing the major actin-binding site of A4-A6 within A4. A5-A6 lacks any ability to nucleate actin polymerization. However, while the isolated G1-G3 has calcium-independent activity, the adseverin N-terminal half (A1-A3) must bind calcium to function, thus explaining why adseverin is inactive in the absence of calcium, despite the lack of the C-terminal tail-latch. It appears that calcium-independent activity of isolated G1-G3 may be attributed to conformational changes induced by the binding of actin to G2 and the straightening of the G3 helix and AB loops. Finally, the structure of calcium-free full-length zebrafish scinderin-like B (scinlB) was elucidated and observed to be broadly similar to the structure human gelsolin. However, despite being calcium-free, the structure of scinlB appears to be in an early activation stage, possibly because it crystallized at a semi-activating pH. This structure suggests that calciumand pH-dependent activation may share some common mechanisms, and also offers an avenue for elucidating pH-mediated activation of gelsolin family proteins. Hence, this study has elucidated some of the mechanisms involved in calciumactivation of gelsolin-family members, delineated the roles of the G2, G4, G5 and G6 type-II calcium-binding sites in activation, highlighted the functional similarities and differences between gelsolin and adseverin, and provided avenues for further exploration of the roles and functions of the other gelsolin calcium-binding sites as well as the mechanisms of pH activation. vii List of Tables Table 1.1 Some human ABPs with an example of their functions……………… 12 Table 2.1 Reagents used in this study…………………………………………… 30 Table 2.2 Oligonucleotides/primers used in this study…………………………… 30 Table 3.1 Statistics for G1-G3/actin………………………………………………. 49 Table 3.2 Data collection and refinement statistics for calcium-bound G3…… 55 Table 4.1 Gelsolin mutants used in this study……………………………………. 69 Table 5.1 Data collection and refinement statistics for scinderin-like B (ScinlB) …. 101 Table 5.2 Structural variations between scinlB and gelsolin domains………………107 viii Chellaiah, M.A. (2006). 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(1999) Protein Crystallization Techniques, Strategies, and Tips. Chapter 9, 79-90 Zhai, L., Zhao, P., Panebra, A., Guerrerio, A.L., and Khurana, S. (2001). Tyrosine phosphorylation of villin regulates the organization of the actin cytoskeleton. J Biol Chem. 276, 36163-36167. 142 Zhang, Y., Vorobiev, S.M., Gibson, B.G., Hao, B., Sidhu, G.S., Mishra, V.S., Yarmola, E.G., Bubb, M.R., Almo, S.C., and Southwick, F.S. (2006). A CapG gain-of-function mutant reveals critical structural and functional determinants for actin filament severing. EMBO J. 25, 4458-4467. 143 Appendix A Paper I Ca2+ binding by domain plays a critical role in the activation and stabilization of gelsolin Authors Shalini Nag1; Qing Ma1; Hui Wang1; Sakesit Chumnarnsilpa; Wei Lin Lee; Mårten Larsson; Balakrishnan Kannan; Maria Hernandez-Valladares; Leslie D. Burtnick and Robert C. Robinson S.N., Q.M., and H.W. contributed equally to this work. Abstract Gelsolin consists of six homologous domains (G1–G6), each containing a conserved Cabinding site. Occupation of a subset of these sites enables gelsolin to sever and cap actin filaments in a Ca-dependent manner. Here, we present the structures of Ca-free human gelsolin and of Ca-bound human G1–G3 in a complex with actin. These structures closely resemble those determined previously for equine gelsolin. However, the G2 Ca-binding site is occupied in the human G1–G3/actin structure, whereas it is vacant in the equine version. Indepth comparison of the Ca-free and Ca-activated, actin-bound human gelsolin structures suggests G2 and G6 to be cooperative in binding Ca2+ and responsible for opening the G2–G6 latch to expose the F-actin-binding site on G2. Mutational analysis of the G2 and G6 Cabinding sites demonstrates their interdependence in maintaining the compact structure in the absence of calcium. Examination of Ca binding by G2 in human G1–G3/actin reveals that the Ca2+ locks the G2–G3 interface. Thermal denaturation studies of G2–G3 indicate that Ca binding stabilizes this fragment, driving it into the active conformation. The G2 Ca-binding site is mutated in gelsolin from familial amyloidosis (Finnish-type) patients. This disease initially proceeds through protease cleavage of G2, ultimately to produce a fragment that forms amyloid fibrils. The data presented here support a mechanism whereby the loss of Ca binding by G2 prolongs the lifetime of partially activated, intermediate conformations in which the protease cleavage site is exposed. 144 Appendix B Paper II The crystal structure of the C-terminus of adseverin reveals the actin-binding interface Authors Chumnarnsilpa Sakesit; Lee Wei Lin; Nag Shalini; Kannan Balakrishnan; Larsson Marten; Burtnick Leslie D. and Robinson Robert C. Abstract Adseverin is a member of the calcium-regulated gelsolin superfamily of actin severing and capping proteins. Adseverin comprises homologous domains (A1-A6), which share 60% identity with the domains from gelsolin (G1-G6). Adseverin is truncated in comparison to gelsolin, lacking the C-terminal extension that masks the F-actin binding site in calcium-free gelsolin. Biochemical assays have indicated differences in the interaction of the C-terminal halves of adseverin and gelsolin with actin. Gelsolin contacts actin through a major site on G4 and a minor site on G6, whereas adseverin uses a site on A5. Here, we present the X-ray structure of the activated C-terminal half of adseverin (A4-A6). This structure is highly similar to that of the activated form of the C-terminal half of gelsolin (G4-G6), both in arrangement of domains and in the bound calcium ions. Comparative analysis of the actinbinding surfaces observed in the G4-G6/actin structure suggests that adseverin in this conformation will also be able to interact with actin through A4 and A6, whereas the A5 surface is obscured. A single residue mutation in A4-A6 located at the predicted A4/actin interface completely abrogates actin sequestration. A model of calcium-free adseverin, constructed from the structure of gelsolin, predicts that in the absence of a gelsolin-like Cterminal extension the interaction between A2 and A6 provides the steric inhibition to prevent interaction with F-actin. We propose that calcium binding to the N terminus of adseverin dominates the activation process to expose the F-actin binding site on A2. 145 [...]... actin filaments Twinfilin inhibits actin nucleotide exchange Actin/Bundling/Crosslinking Proteins α-actinin connects and organizes actin filaments EPLIN stabilizes and bundles actin filaments Espin forms parallel actin bundles Fascin bundles actin filaments Filamin orthogonally cross-links actin filaments Fimbrin/plastin bundles actin filaments 12 Chapter 1 Introduction Figure 1.6 Roles of actin binding... wild-type gelsolin, residues 25-741 GFL wild-type full-length gelsolin, residues 25-755 A1-A3 adseverin N-terminal half A4-A6 adseverin C-terminal half A4-A6M adseverin C-terminal half carrying a Phe455Asp mutation A1 A2 A3 adseverin domains 1 through 6 A4 A5 A6 scinlA scinderin-like A scinlB scinderin-like B SB1-SB3 scinlB N-terminal half SB4-SB6 scinlB C-terminal half SB1 SB2 SB3 scinlB domains 1 through... domains with corresponding inactive and active gelsolin domains………………………………………108 Figure 5.11 Towards the structure of scinlA/actin and scinlB/actin complexes……… 110 Figure 5.12 Model of inactive calcium- free scinlB created by homology modeling of SWISS-MODEL…………………………………………… 112 Figure 5.13 Cooperative activation by domains 2 and 6 of scinlB and gelsolin …….114 Figure 5.14 Actin severing by scinlB…………………………………………………... study, gelsolin is used as the reference point for comparing the sequences, domain architectures and functions of related proteins Gelsolin has six homologous core domains and eight calcium- binding sites that fall into two classes (Burtnick et al., 1997; Kwiatkowski et al., 1986) The structure of gelsolin, the calcium- binding sites and the effects of calcium binding on the structure and function of the... released into the plasma during injury, from clogging the microvasculature (Vasconcellos and Lind, 1993) Also distinctive within the family, is the physiological role of the isolated N-terminal half of gelsolin During apoptosis, caspase-3 cleaves gelsolin between domains 3 and 4, yielding a calcium- independent, threedomain, actin-severing protein (Kothakota et al., 1997) Intriguingly, the calciumindependence... Formins G-Actin Binding Proteins CAP/Srv2 recycles actin monomers Profilin prevents actin monomer addition at pointed ends DnaseI sequesters G-actin Thymosin β4 sequesters G-actin Verprolin/WIP binds actin monomers, WASP F-Actin Binding Proteins Ena/VASP family MIM antagonizes CapZ capping at filament ends, recruits profilin binds both G- and F-actin, may regulate nucleation-promoting factors Pointed... Capping Proteins Tropomodulin/Tmod caps pointed ends, binds tropomyosin Barbed End Capping Proteins AIP1 promotes depolymerization by ADF/cofilin CapG caps barbed ends (calcium- dependent) capping protein heterodimer that caps barbed ends Eps8 caps barbed ends, involved in Rac signalling Actin Depolymerizing/Severing Proteins ADF/cofilin promotes actin filament assembly and disassembly Gelsolin/ villin... presence of five gelsolin- like domains (corresponding to gelsolin s domains 2-6), and a villin-like headpiece in the C-terminal third while the Nterminal two-thirds comprise a unique domain containing four nuclear localization signals Detailed domain analysis reveals that contrary to the predictions, the gelsolin- like C-terminal half binds F-actin weakly, the villin-like headpiece lacks F-actin binding... Profilin-actin binds to formin and adds actin to the barbed end of the filament (C) Nucleation by arp2/3 complex Nucleation-promoting factors such as WASp bind an actin monomer and arp2/3 complex The barbed end of the daughter filament grows from arp2/3 complex (D) Reactions of actin filaments Capping proteins bind to and block barbed ends; cofilin and gelsolin sever filaments; cross-linking proteins... binding proteins in actin dynamics (A) Actin monomer binding proteins Thymosin-β4 blocks all assembly reactions; profilin promotes nucleotide exchange and inhibits pointed-end elongation and nucleation; cofilin inhibits nucleotide exchange and promotes nucleation (B) Nucleation and elongation by formins Formins initiate polymerization from free actin monomers and remain associated with the growing barbed . STRUCTURAL ANALYSIS OF CALCIUM- INDUCED CHANGES IN GELSOLIN AND ADSEVERIN NAG SHALINI NATIONAL UNIVERSITY OF SINGAPORE 2010 STRUCTURAL. cytoplasmic gelsolin. These data, in combination with the functional characterization of calcium- binding site mutants of gelsolin, provide insights into gelsolin activation. Release of the C-terminal. 5. Adseverin and scinderin-like B offer insights into the structure, function and regulation of the gelsolin family……………………………… 89 5.1 The adseverin C-terminal half imitates the structure and