Structural analysis of calcium induced changes in gelsolin and adseverin

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). Regulation of podosomes by integrin alphavbeta3 and Rho GTPasefacilitated phosphoinositide signaling. Eur J Cell Biol. 85, 311-317. Chen, C.D., Huff, M.E., Matteson, J., Page, L., Phillips, R., Kelly, J.W., and Balch, W.E. (2001). Furin initiates gelsolin familial amyloidosis in the Golgi through a defect in Ca(2+) stabilization. EMBO J. 20, 6277-6287. Chen, K.S., Gunaratne, P.H., Hoheisel, J.D., Young, I.G., Miklos, G.L., Greenberg, F., Shaffer, L.G., Campbell, H.D., and Lupski, J.R. (1995). The human homologue of the Drosophila melanogaster flightless-I gene (flil) maps within the Smith-Magenis microdeletion critical region in 17p11.2. Am J Hum Genet. 56, 175-182. Chen, Y., Takizawa, N., Crowley, J.L., Oh, S.W., Gatto, C.L., Kambara, T., Sato, O., Li, X.D., Ikebe, M., and Luna, E.J. (2003). F-actin and myosin II binding domains in supervilin. J Biol Chem. 278, 46094-46106. Choe, H., Burtnick, L.D., Mejillano, M., Yin, H.L., Robinson, R.C., and Choe, S. (2002). The Calcium activation of gelsolin: Insights from the A Structure of the G4-G6/Actin Complex. Journal of Molecular Biology. 324, 691-702. Chumnarnsilpa, S., Lee, W.L., Nag, S., Kannan, B., Larsson, M., Burtnick, L.D., and Robinson, R.C. (2009). The crystal structure of the C-terminus of adseverin reveals the actinbinding interface. Proc Natl Acad Sci U S A. 106, 13719-13724. Chumnarnsilpa, S., Loonchanta, A., Xue, B., Choe, H., Urosev, D., Wang, H., Lindberg, U., Burtnick, L.D., and Robinson, R.C. (2006). Calcium Ion Exchange in Crystalline Gelsolin. Journal of Molecular Biology. 357, 773-782. Claudianos, C., and Campbell, H.D. (1995). The novel flightless-I gene brings together two gene families, actin-binding proteins related to gelsolin and leucine-rich-repeat proteins involved in Ras signal transduction. Mol Biol Evol. 12, 405-414. Collaborative Computational Project, Number (1994). The CCP4 suite: Programs for protein crystallography. Acta Crystallographica Section D-Biological Crystallography. 50, 760-763. Cooper, J.A., Buhle, E.L., Jr., Walker, S.B., Tsong, T.Y., and Pollard, T.D. (1983a). Kinetic evidence for a monomer activation step in actin polymerization. Biochemistry. 22, 21932202. Cooper, J.A., Walker, S.B., and Pollard, T.D. (1983b). Pyrene actin: documentation of the validity of a sensitive assay for actin polymerization. J Muscle Res Cell Motil. 4, 253-262. Cossart, P. (2004). Bacterial invasion: a new strategy to dominate cytoskeleton plasticity. Dev Cell. 6, 314-315. Dabrowska, R., Hinssen, H., Galazkiewicz, B., and Nowak, E. (1996). Modulation of gelsolin-induced actin-filament severing by caldesmon and tropomyosin and the effect of these proteins on the actin activation of myosin Mg(2+)-ATPase activity. Biochem J. 315 ( Pt 3), 753-759. De Corte, V., Gettemans, J., and Vandekerckhove, J. (1997). Phosphatidylinositol 4,5bisphosphate specifically stimulates PP60(c-src) catalyzed phosphorylation of gelsolin and related actin-binding proteins. FEBS Lett. 401, 191-196. 130 De Corte, V., Van Impe, K., Bruyneel, E., Boucherie, C., Mareel, M., Vandekerckhove, J., and Gettemans, J. (2004). Increased importin-beta-dependent nuclear import of the actin modulating protein CapG promotes cell invasion. J Cell Sci. 117, 5283-5292. de la Chapelle, A., Tolvanen, R., Boysen, G., Santavy, J., Bleeker-Wagemakers, L., Maury, C.P., and Kere, J. (1992). Gelsolin-derived familial amyloidosis caused by asparagine or tyrosine substitution for aspartic acid at residue 187. Nat Genet. 2, 157-160. Disanza, A., Steffen, A., Hertzog, M., Frittoli, E., Rottner, K., and Scita, G. (2005). Actin polymerization machinery: the finish line of signaling networks, the starting point of cellular movement. Cell Mol Life Sci. 62, 955-970. Ditsch, A., and Wegner, A. (1995). Two low-affinity Ca(2+)-binding sites of gelsolin that regulate association with actin. Eur J Biochem. 229, 512-516. Doering, D.S., and Matsudaira, P. (1996). Cysteine scanning mutagenesis at 40 of 76 positions in villin headpiece maps the F-actin binding site and structural features of the domain. Biochemistry. 35, 12677-12685. Doi, Y., Hashimoto, T., Yamaguchi, H., and Vertut-Doi, A. (1991). Modification of gelsolin with 4-fluoro-7-nitrobenz-2-oxa-1,3-diazole. Eur J Biochem. 199, 277-283. Dolman, N.J., and Tepikin, A.V. (2006). Calcium gradients and the Golgi. Cell Calcium. 40, 505-512. dos Remedios, C.G., Chhabra, D., Kekic, M., Dedova, I.V., Tsubakihara, M., Berry, D.A., and Nosworthy, N.J. (2003). Actin binding proteins: regulation of cytoskeletal microfilaments. Physiol Rev. 83, 433-473. El Sayegh, T.Y., Kapus, A., and McCulloch, C.A. (2007). Beyond the epithelium: cadherin function in fibrous connective tissues. FEBS Lett. 581, 167-174. Emsley, P., Cowtan, K. (2004). Coot: model-building tools for molecular graphics. Acta Crystallographica Section D-Biological Crystallography 60: 2126-2132 Part 12 Sp. Iss. Faul, C., Asanuma, K., Yanagida-Asanuma, E., Kim, K., and Mundel, P. (2007). Actin up: regulation of podocyte structure and function by components of the actin cytoskeleton. Trends Cell Biol. 17, 428-437. Feng, L., Mejillano, M., Yin, H.L., Chen, J., and Prestwich, G.D. (2001). Full-contact domain labeling: identification of a novel phosphoinositide binding site on gelsolin that requires the complete protein. Biochemistry. 40, 904-913. Frieden, C., and Goddette, D.W. (1983). Polymerization of actin and actin-like systems: evaluation of the time course of polymerization in relation to the mechanism. Biochemistry. 22, 5836-5843. Furukawa, K., Fu, W., Li, Y., Witke, W., Kwiatkowski, D.J., and Mattson, M.P. (1997). The actin-severing protein gelsolin modulates calcium channel and NMDA receptor activities and vulnerability to excitotoxicity in hippocampal neurons. J Neurosci. 17, 8178-8186. Gaszner, B., Nyitrai, M., Hartvig, N., Koszegi, T., Somogyi, B., and Belagyi, J. (1999). Replacement of ATP with ADP affects the dynamic and conformational properties of actin monomer. Biochemistry. 38, 12885-12892. 131 George, S.P., Wang, Y., Mathew, S., Srinivasan, K., and Khurana, S. (2007). Dimerization and actin-bundling properties of villin and its role in the assembly of epithelial cell brush borders. J Biol Chem. 282, 26528-26541. Gershman, L.C., Selden, L.A., and Estes, J.E. (1986). High affinity binding of divalent cation to actin monomer is much stronger than previously reported. Biochem Biophys Res Commun. 135, 607-614. Gieni, R.S., and Hendzel, M.J. (2009). Actin dynamics and functions in the interphase nucleus: moving toward an understanding of nuclear polymeric actin. Biochem Cell Biol. 87, 283-306. Gloss, A., Rivero, F., Khaire, N., Muller, R., Loomis, W.F., Schleicher, M., and Noegel, A.A. (2003). Villidin, a novel WD-repeat and villin-related protein from Dictyostelium, is associated with membranes and the cytoskeleton. Mol Biol Cell. 14, 2716-2727. Gonsior, S.M., Platz, S., Buchmeier, S., Scheer, U., Jockusch, B.M., and Hinssen, H. (1999). Conformational difference between nuclear and cytoplasmic actin as detected by a monoclonal antibody. J Cell Sci. 112 ( Pt 6), 797-809. Gourlay, C.W., and Ayscough, K.R. (2005). The actin cytoskeleton: a key regulator of apoptosis and ageing? Nat Rev Mol Cell Biol. 6, 583-589. Gremm, D., and Wegner, A. (2000). Gelsolin as a calcium-regulated actin filament-capping protein. Eur J Biochem. 267, 4339-4345. Guettler, S., Vartiainen, M.K., Miralles, F., Larijani, B., and Treisman, R. (2008). RPEL motifs link the serum response factor cofactor MAL but not myocardin to Rho signaling via actin binding. Mol Cell Biol. 28, 732-742. Gunning, P., O'Neill, G., and Hardeman, E. (2008). Tropomyosin-based regulation of the actin cytoskeleton in time and space. Physiol Rev. 88, 1-35. Hasegawa, H., Abbott, S., Han, B.X., Qi, Y., and Wang, F. (2007). Analyzing somatosensory axon projections with the sensory neuron-specific Advillin gene. J Neurosci. 27, 1440414414. Hellweg, T., Hinssen, H., and Eimer, W. (1993). The Ca(2+)-induced conformational change of gelsolin is located in the carboxyl-terminal half of the molecule. Biophys J. 65, 799-805. Herz, R., Weber, A., and Reiss, I. (1969). The role of magnesium in the relaxation of myofibrils. Biochemistry. 8, 2266-2271. Hesterberg, L.K., and Weber, K. (1983). Ligand-induced conformational changes in villin, a calcium-controlled actin-modulating protein. J Biol Chem. 258, 359-364. Hesterberg, L.K., and Weber, K. (1986). Isolation of a domain of villin retaining calciumdependent interaction with G-actin, but devoid of F-actin fragmenting activity. Eur J Biochem. 154, 135-140. Holmes, K.C., Popp, D., Gebhard, W., and Kabsch, W. (1990). Atomic model of the actin filament. Nature. 347, 44-49. 132 Irobi, E., Burtnick, L.D., Urosev, D., Narayan, K., and Robinson, R.C. (2003). From the first to the second domain of gelsolin: a common path on the surface of actin? FEBS Lett. 552, 8690. Isaacson, R.L., Weeds, A.G., and Fersht, A.R. (1999). Equilibria and kinetics of folding of gelsolin domain and mutants involved in familial amyloidosis-Finnish type. Proc Natl Acad Sci U S A. 96, 11247-11252. Janmey, P.A., Chaponnier, C., Lind, S.E., Zaner, K.S., Stossel, T.P., and Yin, H.L. (1985). Interactions of gelsolin and gelsolin-actin complexes with actin. Effects of calcium on actin nucleation, filament severing, and end blocking. Biochemistry. 24, 3714-3723. Janmey, P.A., Iida, K., Yin, H.L., and Stossel, T.P. (1987). Polyphosphoinositide micelles and polyphosphoinositide-containing vesicles dissociate endogenous gelsolin-actin complexes and promote actin assembly from the fast-growing end of actin filaments blocked by gelsolin. J Biol Chem. 262, 12228-12236. Janmey, P.A., Lamb, J., Allen, P.G., and Matsudaira, P.T. (1992). Phosphoinositide-binding peptides derived from the sequences of gelsolin and villin. J Biol Chem. 267, 11818-11823. Janmey, P.A., and Matsudaira, P.T. (1988). Functional comparison of villin and gelsolin. Effects of Ca2+, KCl, and polyphosphoinositides. J Biol Chem. 263, 16738-16743. Janmey, P.A., and Stossel, T.P. (1987). Modulation phosphatidylinositol 4,5-bisphosphate. Nature. 325, 362-364. of gelsolin function by Jia, S., Omelchenko, M., Garland, D., Vasiliou, V., Kanungo, J., Spencer, M., Wolf, Y., Koonin, E., and Piatigorsky, J. (2007). Duplicated gelsolin family genes in zebrafish: a novel scinderin-like gene (scinla) encodes the major corneal crystallin. FASEB J. 21, 3318-3328. Jiang, P., Enomoto, A., and Takahashi, M. (2009). Cell biology of the movement of breast cancer cells: intracellular signalling and the actin cytoskeleton. Cancer Lett. 284, 122-130. Jones, L.J., Carballido-Lopez, R., and Errington, J. (2001). Control of cell shape in bacteria: helical, actin-like filaments in Bacillus subtilis. Cell. 104, 913-922. Kabsch, W., Mannherz, H.G., Suck, D., Pai, E.F., and Holmes, K.C. (1990). Atomic structure of the actin:DNase I complex. Nature. 347, 37-44. Kasai, M., Asakura, S., and Oosawa, F. (1962). The G-F equilibrium in actin solutions under various conditions. Biochim Biophys Acta. 57, 13-21. Kawamoto, S., Suzuki, T., Aki, T., Katsutani, T., Tsuboi, S., Shigeta, S., and Ono, K. (2002). Der f 16: a novel gelsolin-related molecule identified as an allergen from the house dust mite, Dermatophagoides farinae. FEBS Lett. 516, 234-238. Kazmirski, S.L., Isaacson, R.L., An, C., Buckle, A., Johnson, C.M., Daggett, V., and Fersht, A.R. (2002). Loss of a metal-binding site in gelsolin leads to familial amyloidosis-Finnish type. Nat Struct Biol. 9, 112-116. Keller, J.W., Haigis, K.M., Franklin, J.L., Whitehead, R.H., Jacks, T., and Coffey, R.J. (2007). Oncogenic K-RAS subverts the antiapoptotic role of N-RAS and alters modulation of the N-RAS:gelsolin complex. Oncogene. 26, 3051-3059. 133 Khaitlina, S., Walloscheck, M., and Hinssen, H. (2004). Calcium-Induced Conformational Changes in the C-Terminal Half of Gelsolin Stabilize Its Interaction with the Actin Monomer. Biochemistry. 43, 12838-12845. Khatau, S.B., Hale, C.M., Stewart-Hutchinson, P.J., Patel, M.S., Stewart, C.L., Searson, P.C., Hodzic, D., and Wirtz, D. (2009). A perinuclear actin cap regulates nuclear shape. Proc Natl Acad Sci U S A. 106, 19017-19022. Khurana, S., and George, S.P. (2008). Regulation of cell structure and function by actinbinding proteins: villin's perspective. FEBS Lett. 582, 2128-2139. Kinosian, H.J., Newman, J., Lincoln, B., Selden, L.A., Gershman, L.C., and Estes, J.E. (1998). Ca2+ regulation of gelsolin activity: binding and severing of F-actin. Biophys J. 75, 3101-3109. Kinosian, H.J., Selden, L.A., Estes, J.E., and Gershman, L.C. (1993). Nucleotide binding to actin. Cation dependence of nucleotide dissociation and exchange rates. J Biol Chem. 268, 8683-8691. Kinosian, H.J., Selden, L.A., Estes, J.E., and Gershman, L.C. (1996). Kinetics of gelsolin interaction with phalloidin-stabilized F-actin. Rate constants for binding and severing. Biochemistry. 35, 16550-16556. Kiselar, J.G., Janmey, P.A., Almo, S.C., and Chance, M.R. (2003a). Structural Analysis of Gelsolin Using Synchrotron Protein Footprinting. Molecular and Cellular Proteomics. 2, 1120–1132. Kiselar, J.G., Janmey, P.A., Almo, S.C., and Chance, M.R. (2003b). Visualizing the Ca2+dependent activation of gelsolin by using synchrotron footprinting. Proceedings of the National Academy of Sciences. 100, 3942-3947. Kitazawa, T., Shuman, H., and Somlyo, A.P. (1982). Calcium and magnesium binding to thin and thick filaments in skinned muscle fibres: electron probe analysis. J Muscle Res Cell Motil. 3, 437-454. Klaavuniemi, T., Yamashiro, S., and Ono, S. (2008). Caenorhabditis elegans gelsolin-like protein is a novel actin filament-severing protein with four gelsolin-like repeats. J Biol Chem. 283, 26071-26080. Kolappan, S., Gooch, J.T., Weeds, A.G., and McLaughlin, P.J. (2003). Gelsolin domains 4-6 in active, actin-free conformation identifies sites of regulatory calcium ions. J Mol Biol. 329, 85-92. Kopecki, Z., Arkell, R., Powell, B.C., and Cowin, A.J. (2009). Flightless I regulates hemidesmosome formation and integrin-mediated cellular adhesion and migration during wound repair. J Invest Dermatol. 129, 2031-2045. Kothakota, S., Azuma, T., Reinhard, C., Klippel, A., Tang, J., Chu, K., McGarry, T.J., Kirschner, M.W., Koths, K., Kwiatkowski, D.J., et al. (1997). Caspase-3-generated fragment of gelsolin: effector of morphological change in apoptosis. Science. 278, 294-298. Kouyama, T., Mihashi, K. (1981). Fluorimetry study of N-(1-pyrenyl)iodoacetamide-labelled F-actin. Local structural change of actin protomer both on polymerization and on binding of heavy meromyosin. European Journal of Biochemistry. 114, 33-38 134 Krissinel, E., and Henrick, K. (2004), Acta Cryst. D. 60, 2256-2268. Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions. Kudryashov, D.S., Sawaya, M.R., Adisetiyo, H., Norcross, T., Hegyi, G., Reisler, E., and Yeates, T.O. (2005). The crystal structure of a cross-linked actin dimer suggests a detailed molecular interface in F-actin. Proc Natl Acad Sci U S A. 102, 13105-13110. Kumar, N., and Khurana, S. (2004). Identification of a functional switch for actin severing by cytoskeletal proteins. J Biol Chem. 279, 24915-24918. Kumar, N., Tomar, A., Parrill, A.L., and Khurana, S. (2004). Functional dissection and molecular characterization of calcium-sensitive actin-capping and actin-depolymerizing sites in villin. J Biol Chem. 279, 45036-45046. Kurth, M.C., Wang, L.L., Dingus, J., and Bryan, J. (1983). Purification and characterization of a gelsolin-actin complex from human platelets. Evidence for Ca2+-insensitive functions. J Biol Chem. 258, 10895-10903. Kwiatkowski, D.J. (1999). Functions of gelsolin: Motility, signaling, apoptosis, cancer. current Opinion in Cell Biology. 11, 103-108. Kwiatkowski, D.J., Janmey, P.A., Mole, J.E., and Yin, H.L. (1985). Isolation and properties of two actin-binding domains in gelsolin. J Biol Chem. 260, 15232-15238. Kwiatkowski, D.J., Janmey, P.A., and Yin, H.L. (1989). Identification of critical functional and regulatory domains in gelsolin. J Cell Biol. 108, 1717-1726. Kwiatkowski, D.J., Stossel, T.P., Orkin, S.H., Mole, J.E., Colten, H.R., and Yin, H.L. (1986). Plasma and cytoplasmic gelsolins are encoded by a single gene and contain a duplicated actin-binding domain. Nature. 323, 455-458. Kwiatkowski, D.J., Westbrook, C.A., Bruns, G.A., and Morton, C.C. (1988). Localization of gelsolin proximal to ABL on chromosome 9. Am J Hum Genet. 42, 565-572. Kwiatkowski, D.J., and Yin, H.L. (1987). Molecular biology of gelsolin, a calcium-regulated actin filament severing protein. Biorheology. 24, 643-647. Lagarrigue, E., Maciver, S.K., Fattoum, A., Benyamin, Y., and Roustan, C. (2003a). Cooperation of domain-binding and calcium-binding sites in the activation of gelsolin. European Journal of Biochemistry. 270, 2236-2243. Lagarrigue, E., Ternent, D., Maciver, S.K., Fattoum, A., Benyamin, Y., and Roustan, C. (2003b). The activation of gelsolin by low pH: the calcium latch is sensitive to calcium but not pH. Eur J Biochem. 270, 4105-4112. Lamb, J.A., Allen, P.G., Tuan, B.Y., and Janmey, P.A. (1993). Modulation of gelsolin function. Activation at low pH overrides Ca2+ requirement. J Biol Chem. 268, 8999-9004. Langer, G.G., Cohen, S.X., Perrakis, A. & Lamzin, V.S. (2008). Automated macromolecular model building for X-ray crystallography using ARP/wARP version 7. Nature Protocols 3, 1171-1179. Laskowski, R. A., MacArthur, M. W., Moss, D. S. and Thornton, J. M. (1993) PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26, 283–291 135 Lejen, T., Pene, T.D., Rose, S.D., and Trifaro, J.M. (2002). The role of different Scinderin domains in the control of F-actin cytoskeleton during exocytosis. Ann N Y Acad Sci. 971, 248-250. Liepina, I., Czaplewski, C., Janmey, P., and Liwo, A. (2003). Molecular dynamics study of a gelsolin-derived peptide binding to a lipid bilayer containing phosphatidylinositol 4,5bisphosphate. Biopolymers. 71, 49-70. Lin, K.M., Mejillano, M., and Yin, H.L. (2000). Ca2+ regulation of gelsolin by its C-terminal tail. J Biol Chem. 275, 27746-27752. Lindberg, U., Schutt, C.E., Goldman, R.D., Nyakern-Meazza, M., Hillberg, L., Rathje, L.S., and Grenklo, S. (2008). Tropomyosins regulate the impact of actin binding proteins on actin filaments. Adv Exp Med Biol. 644, 223-231. Lorenz, K., Grashoff, C., Torka, R., Sakai, T., Langbein, L., Bloch, W., Aumailley, M., and Fassler, R. (2007). Integrin-linked kinase is required for epidermal and hair follicle morphogenesis. J Cell Biol. 177, 501-513. Lueck, A., Yin, H.L., Kwiatkowski, D.J., and Allen, P.G. (2000). Calcium regulation of gelsolin and adseverin: a natural test of the helix latch hypothesis. Biochemistry. 39, 52745279. Maekawa, S., and Sakai, H. (1990). Inhibition of actin regulatory activity of the 74-kDa protein from bovine adrenal medulla (adseverin) by some phospholipids. J Biol Chem. 265, 10940-10942. Marcu, M.G., Zhang, L., Elzagallaai, A., and Trifaro, J.M. (1998). Localization by segmental deletion analysis and functional characterization of a third actin-binding site in domain of scinderin. J Biol Chem. 273, 3661-3668. Marks, P.W., Arai, M., Bandura, J.L., and Kwiatkowski, D.J. (1998). Advillin (p92): a new member of the gelsolin/villin family of actin regulatory proteins. J Cell Sci. 111 ( Pt 15), 2129-2136. Markus, M.A., Matsudaira, P., and Wagner, G. (1997). Refined structure of villin 14T and a detailed comparison with other actin-severing domains. Protein Sci. 6, 1197-1209. Matsudaira, P., Jakes, R., and Walker, J.E. (1985). A gelsolin-like Ca2+-dependent actinbinding domain in villin. Nature. 315, 248-250. Maury, C.P. (1991). Gelsolin-related amyloidosis. Identification of the amyloid protein in Finnish hereditary amyloidosis as a fragment of variant gelsolin. Journal of Clinical Investigation. 87, 1195-1199. McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Storoni L.C., and Read. R.J. (2007). PHASER crystallographic software. J. Appl. Cryst. 40, 658-674. McDonald, D., Carrero, G., Andrin, C., de Vries, G., and Hendzel, M.J. (2006). Nucleoplasmic beta-actin exists in a dynamic equilibrium between low-mobility polymeric species and rapidly diffusing populations. J Cell Biol. 172, 541-552. McGough, A., Pope, B., Chiu, W., and Weeds, A. (1997). Cofilin changes the twist of Factin: implications for actin filament dynamics and cellular function. J Cell Biol. 138, 771781. 136 McGough, A., Staiger, C.J., Mina, J.-K., and Simonetti, K.D. (2003). The gelsolin family of actin regulatory proteins: Modular structures, versatile function. FEBS Letters. 552, 75-81. McLaughlin, P.J., Gooch, J.T., Mannherz, H.G., and Weeds, A.G. (1993). Structure of gelsolin segment 1-actin complex and the mechanism of filament severing. Nature. 364, 685692. Mishra, V.S., Henske, E.P., Kwiatkowski, D.J., and Southwick, F.S. (1994). The human actin-regulatory protein cap G: gene structure and chromosome location. Genomics. 23, 560565. Moller-Jensen, J., Jensen, R.B., Lowe, J., and Gerdes, K. (2002). Prokaryotic DNA segregation by an actin-like filament. EMBO J. 21, 3119-3127. Murshudov, G.N., Vagin, A.A., and Dodson, E.J. (1997) Refinement of Macromolecular Structures by the Maximum-Likelihood method. Acta Cryst. D 53, 240-255. Nag, S., Ma, Q., Wang, H., Chumnarnsilpa, S., Lee, W.L., Larsson, M., Kannan, B., Hernandez-Valladares, M., Burtnick, L.D., and Robinson, R.C. (2009). Ca2+ binding by domain plays a critical role in the activation and stabilization of gelsolin. Proc Natl Acad Sci U S A. 106, 13713-13718. Narayan, K., Chumnarnsilpa, S., Choe, H., Irobi, E., Urosev, D., Lindberg, U., Schutt, C.E., Burtnick, L.D., and Robinson, R.C. (2003). Activation in isolation: exposure of the actinbinding site in the C-terminal half of gelsolin does not require actin. FEBS Lett. 552, 82-85. Northrop, J., Weber, A., Mooseker, M.S., Franzini-Armstrong, C., Bishop, M.F., Dubyak, G.R., Tucker, M., and Walsh, T.P. (1986). Different calcium dependence of the capping and cutting activities of villin. J Biol Chem. 261, 9274-9281. Oda, T., Iwasa, M., Aihara, T., Maeda, Y., and Narita, A. (2009). The nature of the globularto fibrous-actin transition. Nature. 457, 441-445. Olave, I.A., Reck-Peterson, S.L., and Crabtree, G.R. (2002). Nuclear actin and actin-related proteins in chromatin remodeling. Annu Rev Biochem. 71, 755-781. Onoda, K., and Yin, H.L. (1993). gCap39 is phosphorylated. Stimulation by okadaic acid and preferential association with nuclei. J Biol Chem. 268, 4106-4112. Onoda, K., Yu, F.X., and Yin, H.L. (1993). gCap39 is a nuclear and cytoplasmic protein. Cell Motil Cytoskeleton. 26, 227-238. Oosawa, F., Asakura, S., Hotta, K., Imai, N., and Ooi, T. (1959). G-F transformation of actin as a fibrous condensation. Journal of Polymer Science. 37, 323-336. Orlova, A., Chen, X., Rubenstein, P.A., and Egelman, E.H. (1997). Modulation of yeast Factin structure by a mutation in the nucleotide-binding cleft. J Mol Biol. 271, 235-243. Orlova, A., and Egelman, E.H. (1995). Structural dynamics of F-actin: I. Changes in the C terminus. J Mol Biol. 245, 582-597. Osborn, T.M., Dahlgren, C., Hartwig, J.H., and Stossel, T.P. (2007). Modifications of cellular responses to lysophosphatidic acid and platelet-activating factor by plasma gelsolin. Am J Physiol Cell Physiol. 292, C1323-1330. 137 Otwinowski, Z., and Minor, W. (1997). Processing for X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276:4866-4871 Otterbein, L.R., Graceffa, P., and Dominguez, R. (2001). The crystal structure of uncomplexed actin in the ADP state. Science. 293, 708-711. Owen, C., and DeRosier, D. (1993). A 13-A map of the actin-scruin filament from the limulus acrosomal process. J Cell Biol. 123, 337-344. Page, L.J., Suk, J.Y., Huff, M.E., Lim, H.J., Venable, J., Yates, J., Kelly, J.W., and Balch, W.E. (2005). Metalloendoprotease cleavage triggers gelsolin amyloidogenesis. EMBO J. 24, 4124-4132. Papakonstanti, E.A., and Stournaras, C. (2008). Cell responses regulated by early reorganization of actin cytoskeleton. FEBS Lett. 582, 2120-2127. Parikh, S.S., Litherland, S.A., Clare-Salzler, M.J., Li, W., Gulig, P.A., and Southwick, F.S. (2003). CapG(-/-) mice have specific host defense defects that render them more susceptible than CapG(+/+) mice to Listeria monocytogenes infection but ot to Salmonella enterica serovar Typhimurium infection. Infect Immun. 71, 6582-6590. Patkowski, A., Seils, J., Hinssen, H., and Dorfmuller, T. (1990). Size, Shape Parameters, and Ca2+-Induced Conformational Change of the Gelsolin Molecule: A Dynamic Light Scattering Study. Biopolymers. 30, 427-435. Paunio, T., Kangas, H., Heinonen, O., Buc-Caron, M.H., Robert, J.J., Kaasinen, S., Julkunen, I., Mallet, J., and Peltonen, L. (1998). Cells of the neuronal lineage play a major role in the generation of amyloid precursor fragments in gelsolin-related amyloidosis. J Biol Chem. 273, 16319-16324. Pestonjamasp, K.N., Pope, R.K., Wulfkuhle, J.D., and Luna, E.J. (1997). Supervillin (p205): A novel membrane-associated, F-actin-binding protein in the villin/gelsolin superfamily. J Cell Biol. 139, 1255-1269. Piatigorsky, J. (2001). Enigma of the abundant water-soluble cytoplasmic proteins of the cornea: the "refracton" hypothesis. Cornea. 20, 853-858 Pollard, T.D. (1986a). Mechanism of Actin Filament Self-Assembly and Regulation of the Process by Actin-Binding Proteins. Biophys J. 49, 149-151. Pollard, T.D. (1986b). Rate constants for the reactions of ATP- and ADP-actin with the ends of actin filaments. J Cell Biol. 103, 2747-2754. Pollard, T.D., Blanchoin, L., and Mullins, R.D. (2000). Molecular mechanisms controlling actin filament dynamics in nonmuscle cells. Annu Rev Biophys Biomol Struct. 29, 545-576. Pollard, T.D., and Cooper, J.A. (2009). Actin, a central player in cell shape and movement. Science. 326, 1208-1212. Pope, B., Maciver, S., and Weeds, A. (1995). Localization of the calcium-sensitive actin monomer binding site in gelsolin to segment and identification of calcium binding sites. Biochemistry. 34, 1583-1588. 138 Pope, B., Way, M., and Weeds, A.G. (1991). Two of the three actin-binding domains of gelsolin bind to the same subdomain of actin. Implications of capping and severing mechanisms. FEBS Lett. 280, 70-74. Pope, B.J., Gooch, J.T., and Weeds, A.G. (1997). Probing the effects of calcium on gelsolin. Biochemistry. 36, 15848-15855. Ramakrishnan, R., Fujimura, Y., Zou, J.P., Liu, F., Lee, L., Rao, V.N., and Reddy, E.S. (2004). Role of protein-protein interactions in the antiapoptotic function of EWS-Fli-1. Oncogene. 23, 7087-7094. Ren, G., Crampton, M.S., and Yap, A.S. (2009). Cortactin: Coordinating adhesion and the actin cytoskeleton at cellular protrusions. Cell Motil Cytoskeleton. 66, 865-873. Renz, M., Betz, B., Niederacher, D., Bender, H.G., and Langowski, J. (2008). Invasive breast cancer cells exhibit increased mobility of the actin-binding protein CapG. Int J Cancer. 122, 1476-1482. Reutzel, R., Yoshioka, C., Govindasamy, L., Yarmola, E.G., Agbandje-McKenna, M., Bubb, M.R., and McKenna, R. (2004). Actin crystal dynamics: structural implications for F-actin nucleation, polymerization, and branching mediated by the anti-parallel dimer. J Struct Biol. 146, 291-301. Robinson, R.C., Mejillano, M., Le, V.P., Burtnick, L.D., Yin, H.L., and Choe, S. (1999). Domain Movement in Gelsolin: A Calcium-Activated Switch. Science. 286, 1939-1942. Rodriguez Del Castillo, A., Lemaire, S., Tchakarov, L., Jeyapragasan, M., Doucet, J.P., Vitale, M.L., and Trifaro, J.M. (1990). Chromaffin cell scinderin, a novel calcium-dependent actin filament-severing protein. EMBO J. 9, 43-52. Roustan, C., Ferjani, I., Maciver, S.K., Fattoum, A., Rebiere, B., and Benyamin, Y. (2007). Calcium-induced conformational changes in the amino-terminal half of gelsolin. FEBS Lett. 581, 681-686. Sambrook, J., and Russel, D.W. (1989). Molecular cloning: a laboratory manual, Volume Sakurai, T., Kurokawa, H., and Nonomura, Y. (1991a). The Ca2+-dependent actin filamentsevering activity of 74-kDa protein (adseverin) resides in its NH2-terminal half. J Biol Chem. 266, 4581-4585. Sakurai, T., Kurokawa, H., and Nonomura, Y. (1991b). Comparison between the gelsolin and adseverin domain structure. J Biol Chem. 266, 15979-15983. Sakurai, T., Ohmi, K., Kurokawa, H., and Nonomura, Y. (1990). Distribution of a gelsolinlike 74,000 mol. wt protein in neural and endocrine tissues. Neuroscience. 38, 743-756. Selden, L.A., Kinosian, H.J., Newman, J., Lincoln, B., Hurwitz, C., Gershman, L.C., and Estes, J.E. (1998). Severing of F-Actin by the Amino-Terminal Half of Gelsolin Suggests Internal Cooperativity in Gelsolin. Biophysical Journal. 75, 3092-3100. Sheterline, P., Clayton, J., and McCammon, J.A. (Editors). (1998). Actin. In: Protein Profile (4th ed.). Oxford, UK: Academic. Shibata, M., Ishii, J., Koizumi, H., Shibata, N., Dohmae, N., Takio, K., Adachi, H., Tsujimoto, M., and Arai, H. (2004). Type F scavenger receptor SREC-I interacts with 139 advillin, a member of the gelsolin/villin family, and induces neurite-like outgrowth. J Biol Chem. 279, 40084-40090. Silacci, P., Mazzolai, L., Gauci, C., Stergiopulos, N., Yin, H.L., and Hayoz, D. (2004). Gelsolin superfamily proteins: Key regulators of cellular functions. Cell and Molecular Life Sciences. 61, 2614-2623. Siripala, A.D., and Welch, M.D. (2007a). SnapShot: actin regulators I. Cell. 128, 626. Siripala, A.D., and Welch, M.D. (2007b). SnapShot: actin regulators II. Cell. 128, 1014. Southwick, F.S. (1995). Gain-of-function mutations conferring actin-severing activity to human macrophage cap G. J Biol Chem. 270, 45-48. Southwick, F.S., and DiNubile, M.J. (1986). Rabbit alveolar macrophages contain a Ca2+sensitive, 41,000-dalton protein which reversibly blocks the "barbed" ends of actin filaments but does not sever them. J Biol Chem. 261, 14191-14195. Spinardi, L., and Witke, W. (2007). Gelsolin and diseases. Subcell Biochem. 45, 55-69. Stocker, S., Hiery, M., and Marriott, G. (1999). Phototactic migration of Dictyostelium cells is linked to a new type of gelsolin-related protein. Mol Biol Cell. 10, 161-178. Straub, K.L., Stella, M.C., and Leptin, M. (1996). The gelsolin-related flightless I protein is required for actin distribution during cellularisation in Drosophila. J Cell Sci. 109 ( Pt 1), 263-270. Sun, H.Q., Kwiatkowska, K., Wooten, D.C., and Yin, H.L. (1995). Effects of CapG overexpression on agonist-induced motility and second messenger generation. J Cell Biol. 129, 147-156. Sun, H.Q., Yamamoto, M., Mejillano, M., and Yin, H.L. (1999). Gelsolin, a Multifunctional Actin Regulatory Protein. The journal of biological chemistry. 274, 33179–33182. Takiguchi, K., and Matsumura, F. (2005). Role of the basic C-terminal half of caldesmon in its regulation of F-actin: comparison between caldesmon and calponin. J Biochem. 138, 805813. Tilney, L.G., Hatano, S., Ishikawa, H., and Mooseker, M.S. (1973). The polymerization of actin: its role in the generation of the acrosomal process of certain echinoderm sperm. J Cell Biol. 59, 109-126. Tilney, L.G., and Mooseker, M. (1971). Actin in the brush-border of epithelial cells of the chicken intestine. Proc Natl Acad Sci U S A. 68, 2611-2615. Tonomura, Y., and Yoshimura, J. (1961). Removal of bound nucleotide and calcium of Gactin by treatment with ethylenediamine-tetraacetic acid. J Biochem. 50, 79-80. Trifaro, J.M., Lejen, T., Rose, S.D., Pene, T.D., Barkar, N.D., and Seward, E.P. (2002). Pathways that control cortical F-actin dynamics during secretion. Neurochem Res. 27, 13711385. Tuominen, E.K., Holopainen, J.M., Chen, J., Prestwich, G.D., Bachiller, P.R., Kinnunen, P.K., and Janmey, P.A. (1999). Fluorescent phosphoinositide derivatives reveal specific binding of gelsolin and other actin regulatory proteins to mixed lipid bilayers. Eur J Biochem. 263, 85-92. 140 Urosev, D., Ma, Q., Tan, A.L., Robinson, R.C., and Burtnick, L.D. (2006). The structure of gelsolin bound to ATP. J Mol Biol. 357, 765-772. Vagin, A.A., and Teplyakov, A. (1997). MOLREP: an Automated Program for Molecular Replacement. Journal of Applied Crystallography. 30, 1022-1025. van den Ent, F., and Lowe, J. (2000). Crystal structure of the cell division protein FtsA from Thermotoga maritima. EMBO J. 19, 5300-5307. Van Troys, M., Dewitte, D., Goethals, M., Vandekerckhove, J., and Ampe, C. (1996). Evidence for an actin binding helix in gelsolin segment 2; have homologous sequences in segments and of gelsolin evolved to divergent actin binding functions? FEBS Lett. 397, 191-196. Vandekerckhove, J., and Weber, K. (1978). At least six different actins are expressed in a higher mammal: an analysis based on the amino acid sequence of the amino-terminal tryptic peptide. J Mol Biol. 126, 783-802. Vardar, D., Chishti, A.H., Frank, B.S., Luna, E.J., Noegel, A.A., Oh, S.W., Schleicher, M., and McKnight, C.J. (2002). Villin-type headpiece domains show a wide range of F-actinbinding affinities. Cell Motil Cytoskeleton. 52, 9-21. Vartiainen, M.K., Guettler, S., Larijani, B., and Treisman, R. (2007). Nuclear actin regulates dynamic subcellular localization and activity of the SRF cofactor MAL. Science. 316, 17491752. Vasconcellos, C.A., and Lind, S.E. (1993). Coordinated inhibition of actin-induced platelet aggregation by plasma gelsolin and vitamin D-binding protein. Blood. 82, 3648-3657. Vouyiouklis, D.A., and Brophy, P.J. (1997). A novel gelsolin isoform expressed by oligodendrocytes in the central nervous system. J Neurochem. 69, 995-1005. Wang, H., Chumnarnsilpa, S., Loonchanta, A., Li, Q., Kuan, Y.M., Robine, S., Larsson, M., Mihalek, I., Burtnick, L.D., and Robinson, R.C. (2009). Helix straightening as an activation mechanism in the gelsolin superfamily of actin regulatory proteins. J Biol Chem. 284, 2126521269. Wang, L.L., and Bryan, J. (1981). Isolation of calcium-dependent platelet proteins that interact with actin. Cell. 25, 637-649. Way, M., Gooch, J., Pope, B., and Weeds, A.G. (1989). Expression of human plasma gelsolin in Escherichia coli and dissection of actin binding sites by segmental deletion mutagenesis. J Cell Biol. 109, 593-605. Way, M., Pope, B., and Weeds, A.G. (1992a). Are the conserved sequences in segment of gelsolin important for binding actin? J Cell Biol. 116, 1135-1143. Way, M., Pope, B., and Weeds, A.G. (1992b). Evidence for functional homology in the Factin binding domains of gelsolin and alpha-actinin: implications for the requirements of severing and capping. J Cell Biol. 119, 835-842. Way, M., and Weeds, A. (1988). Nucleotide sequence of pig plasma gelsolin. Comparison of protein sequence with human gelsolin and other actin-severing proteins shows strong homologies and evidence for large internal repeats. J Mol Biol. 203, 1127-1133. 141 Weeds, A.G., Gooch, J., McLaughlin, P., Pope, B., Bengtsdotter, M., and Karlsson, R. (1995). Identification of the trapped calcium in the gelsolin segment 1-actin complex: implications for the role of calcium in the control of gelsolin activity. FEBS Lett. 360, 227-230. Wegner, A. (1976). Head to tail polymerization of actin. J Mol Biol. 108, 139-150. Wegner, A., and Engel, J. (1975). Kinetics of the cooperative association of actin to actin filaments. Biophys Chem. 3, 215-225. Weis, K. (1998). Importins and exportins: how to get in and out of the nucleus. Trends Biochem Sci. 23, 185-189. Wen, D., Corina, K., Chow, E.P., Miller, S., Janmey, P.A., and Pepinsky, R.B. (1996). The Plasma and Cytoplasmic Forms of Human Gelsolin Differ in Disulfide Structure. Biochemistry. 35, 9700-9709. West, J.J. (1971). Binding of nucleotide to cation-free G-action. Biochemistry. 10, 35473553. Witke, W., Sharpe, A.H., Hartwig, J.H., Azuma, T., Stossel, T.P., and Kwiatkowski, D.J. (1995). Hemostatic, inflammatory, and fibroblast responses are blunted in mice lacking gelsolin. Cell. 81, 41-51. Wulfkuhle, J.D., Donina, I.E., Stark, N.H., Pope, R.K., Pestonjamasp, K.N., Niswonger, M.L., and Luna, E.J. (1999). Domain analysis of supervillin, an F-actin bundling plasma membrane protein with functional nuclear localization signals. J Cell Sci. 112 ( Pt 13), 21252136. Xu, Y.S., Kantorow, M., Davis, J., and Piatigorsky, J. (2000). Evidence for gelsolin as a corneal crystallin in zebrafish. J Biol Chem. 275, 24645-24652. Yin, H.L. (1989). Calcium and polyphosphoinositide regulation of actin network structure by gelsolin. Adv Exp Med Biol. 255, 315-323. Yin, H.L., Hartwig, J.H., Maruyama, K., and Stossel, T.P. (1981). Ca2+ control of actin filament length. Effects of macrophage gelsolin on actin polymerization. J Biol Chem. 256, 9693-9697. Yin, H.L., and Janmey, P.A. (2003). Phosphoinositide regulation of the actin cytoskeleton. Annu Rev Physiol. 65, 761-789. Yin, H.L., and Stossel, T.P. (1979). Control of cytoplasmic actin gel-sol transformation by gelsolin, a calcium-dependent regulatory protein. Nature. 281, 583-586. Yin, H.L., and Stossel, T.P. (1980). Purification and structural properties of gelsolin, a Ca2+activated regulatory protein of macrophages. J Biol Chem. 255, 9490-9493. Zapun, A., Grammatyka, S., Deral, G., and Vernet, T. (2000). Calcium-dependent conformational stability of modules and of human gelsolin. Biochem J. 350 Pt 3, 873-881. Zeelen, J.P. (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