Expression of human plasma gelsolin in Escherichia coli and dissection of actin binding sites by segmental deletion mutagenesis

doi:10.1083/jcb.109.2.593

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Expression of human plasma gelsolin in Escherichia coli and dissection of actin binding sites by segmental deletion mutagenesis

M Way, J Gooch, B Pope and AG Weeds
Medical Research Council Laboratory of Molecular Biology, Cambridge, England.

Human plasma gelsolin has been expressed in high yield and soluble form in Escherichia coli. The protein has nucleating and severing activities identical to those of plasma gelsolin and is fully calcium sensitive in its interactions with monomeric actin. A number of deletion mutants have been expressed to explore the function of the three actin binding sites. Their design is based on the sixfold segmental repeat in the protein sequence. (These sites are located in segment 1, segments 2-3, and segments 4-6). Two mutants, S1-3 and S4-6, are equivalent to the NH2- and COOH-terminal halves of the molecule obtained by limited proteolysis. S1-3 binds two actin monomers in the presence or absence of calcium, it severs and caps filaments but does not nucleate polymerization. S4-6 binds a single actin monomer but only in calcium. These observations confirm and extend current knowledge on the properties of the two halves of gelsolin. Two novel constructs have also been studied that provide a different pairwise juxtaposition of the three sites. S2-6, which lacks the high affinity site of segment 1 (equivalent to the 14,000-Mr proteolytic fragment) and S1,4-6, which lacks segments 2-3 (the actin filament binding domain previously identified using the 28,000-Mr proteolytic fragment). S2-6 binds two actin monomers in calcium and nucleates polymerization; it associates laterally with filaments in the presence or absence of calcium and has a weak calcium-dependent fragmenting activity. S1,4-6 also binds two actin monomers in calcium and one in EGTA, has weak severing activity but does not nucleate polymerization. A model is presented for the involvement of the three binding sites in the various activities of gelsolin.
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Chen, Y., Takizawa, N., Crowley, J. L., Oh, S. W., Gatto, C. L., Kambara, T., Sato, O., Li, X.-d., Ikebe, M., Luna, E. J. (2003). F-actin and Myosin II Binding Domains in Supervillin. J. Biol. Chem. 278: 46094-46106 [Abstract] [Full Text]
Kiselar, J. G., Janmey, P. A., Almo, S. C., Chance, M. R. (2003). Visualizing the Ca2+-dependent activation of gelsolin by using synchrotron footprinting. Proc. Natl. Acad. Sci. USA 100: 3942-3947 [Abstract] [Full Text]
Dawson, J. F., Sablin, E. P., Spudich, J. A., Fletterick, R. J. (2003). Structure of an F-actin Trimer Disrupted by Gelsolin and Implications for the Mechanism of Severing. J. Biol. Chem. 278: 1229-1238 [Abstract] [Full Text]
Moolman-Smook, J., Flashman, E., de Lange, W., Li, Z., Corfield, V., Redwood, C., Watkins, H. (2002). Identification of Novel Interactions Between Domains of Myosin Binding Protein-C That Are Modulated by Hypertrophic Cardiomyopathy Missense Mutations. Circ. Res. 91: 704-711 [Abstract] [Full Text]
Nyakern-Meazza, M., Narayan, K., Schutt, C. E., Lindberg, U. (2002). Tropomyosin and Gelsolin Cooperate in Controlling the Microfilament System. J. Biol. Chem. 277: 28774-28779 [Abstract] [Full Text]
Pyle, W. G., Hart, M. C., Cooper, J. A., Sumandea, M. P., de Tombe, P. P., Solaro, R. J. (2002). Actin Capping Protein: An Essential Element in Protein Kinase Signaling to the Myofilaments. Circ. Res. 90: 1299-1306 [Abstract] [Full Text]
Jahraus, A., Egeberg, M., Hinner, B., Habermann, A., Sackman, E., Pralle, A., Faulstich, H., Rybin, V., Defacque, H., Griffiths, G. (2001). ATP-dependent Membrane Assembly of F-Actin Facilitates Membrane Fusion. Mol. Biol. Cell 12: 155-170 [Abstract] [Full Text]
Redwood, C., Lohmann, K., Bing, W., Esposito, G. M., Elliott, K., Abdulrazzak, H., Knott, A., Purcell, I., Marston, S., Watkins, H. (2000). Investigation of a Truncated Cardiac Troponin T That Causes Familial Hypertrophic Cardiomyopathy : Ca2+ Regulatory Properties of Reconstituted Thin Filaments Depend on the Ratio of Mutant to Wild-Type Protein. Circ. Res. 86: 1146-1152 [Abstract] [Full Text]
MOUNZER, K. C., MONCURE, M., SMITH, Y. R., DINUBILE, M. J. (1999). Relationship of Admission Plasma Gelsolin Levels to Clinical Outcomes in Patients after Major Trauma. Am. J. Respir. Crit. Care Med. 160: 1673-1681 [Abstract] [Full Text]
Isaacson, R. L., Weeds, A. G., Fersht, A. R. (1999). Equilibria and kinetics of folding of gelsolin domain 2 and mutants involved in familial amyloidosis-Finnish type. Proc. Natl. Acad. Sci. USA 96: 11247-11252 [Abstract] [Full Text]
Cattelino, A., Albertinazzi, C., Bossi, M., Critchley, D. R., de Curtis, I. (1999). A Cell-free System to Study Regulation of Focal Adhesions and of the Connected Actin Cytoskeleton. Mol. Biol. Cell 10: 373-391 [Abstract] [Full Text]
Wulfkuhle, J., Donina, I., Stark, N., Pope, R., Pestonjamasp, K., Niswonger, M., Luna, E. (1999). Domain analysis of supervillin, an F-actin bundling plasma membrane protein with functional nuclear localization signals. J. Cell Sci. 112: 2125-2136 [Abstract]
Stocker, S., Hiery, M., Marriott, G. (1999). Phototactic Migration of Dictyostelium Cells Is Linked to a New Type of Gelsolin-related Protein. Mol. Biol. Cell 10: 161-178 [Abstract] [Full Text]
Laine, R. O., Phaneuf, K. L., Cunningham, C. C., Kwiatkowski, D., Azuma, T., Southwick, F. S. (1998). Gelsolin, a Protein That Caps the Barbed Ends and Severs Actin Filaments, Enhances the Actin-Based Motility of Listeria monocytogenes in Host Cells. Infect. Immun. 66: 3775-3782 [Abstract] [Full Text]
Robbens, J., Louahed, J., De Pestel, K., Van Colen, I., Ampe, C., Vandekerckhove, J., Renauld, J.-C. (1998). Murine Adseverin (D5), a Novel Member of the Gelsolin Family, and Murine Adseverin Are Induced by Interleukin-9 in T-Helper Lymphocytes. Mol. Cell. Biol. 18: 4589-4596 [Abstract] [Full Text]
Marcu, M. G., Zhang, L., Elzagallaai, A., Trifaro, J.-M. (1998). Localization by Segmental Deletion Analysis and Functional Characterization of a Third Actin-binding Site in Domain 5�of Scinderin. J. Biol. Chem. 273: 3661-3668 [Abstract] [Full Text]
Bottinelli, R., Coviello, D. A., Redwood, C. S., Pellegrino, M. A., Maron, B. J., Spirito, P., Watkins, H., Reggiani, C. (1998). A Mutant Tropomyosin That Causes Hypertrophic Cardiomyopathy Is Expressed In Vivo and Associated With an Increased Calcium Sensitivity. Circ. Res. 82: 106-115 [Abstract] [Full Text]
Pestonjamasp, K. N., Pope, R. K., Wulfkuhle, J. D., Luna, E. J. (1997). Supervillin (p205): A Novel Membrane-associated, F-Actin-binding Protein in the Villin/Gelsolin Superfamily. JCB 139: 1255-1269 [Abstract] [Full Text]
Rauch, F., Polzar, B., Stephan, H., Zanotti, S., Paddenberg, R., Mannherz, H. G. (1997). Androgen Ablation Leads to an Upregulation and Intranuclear Accumulation of Deoxyribonuclease I in Rat Prostate Epithelial Cells Paralleling Their Apoptotic Elimination. JCB 137: 909-923 [Abstract] [Full Text]
Allen, P. G., Laham, L. E., Way, M., Janmey, P. A. (1996). Binding of Phosphate, Aluminum Fluoride, or Beryllium Fluoride to F-actin Inhibits Severing by Gelsolin. J. Biol. Chem. 271: 4665-4670 [Abstract] [Full Text]
Southwick, F. S., Southwick, F. S. (1995). Gain-of-Function Mutations Conferring Actin-severing Activity to Human Macrophage Cap G. J. Biol. Chem. 270: 45-48 [Abstract] [Full Text]
Borovikov, Y., Norman, J., Price, L., Weeds, A, Koffer, A (1995). Secretion from permeabilised mast cells is enhanced by addition of gelsolin: contrasting effects of endogenous gelsolin. J. Cell Sci. 108: 657-666 [Abstract]
Ferguson, C, Lakey, A, Hutchings, A, Butcher, G., Leonard, K., Bullard, B (1994). Cytoskeletal proteins of insect muscle: location of zeelins in Lethocerus flight and leg muscle. J. Cell Sci. 107: 1115-1129 [Abstract]
Watt, F., Kubler, M., Hotchin, N., Nicholson, L., Adams, J. (1993). Regulation of keratinocyte terminal differentiation by integrin-extracellular matrix interactions. J. Cell Sci. 106: 175-182 [Abstract]
Yu, F., Johnston, P., Sudhof, T., Yin, H. (1990). gCap39, a calcium ion- and polyphosphoinositide-regulated actin capping protein. Science 250: 1413-1415 [Abstract]
Volkmann, N., Amann, K. J., Stoilova-McPhie, S., Egile, C., Winter, D. C., Hazelwood, L., Heuser, J. E., Li, R., Pollard, T. D., Hanein, D. (2001). Structure of Arp2/3 Complex in Its Activated State and in Actin Filament Branch Junctions. Science 293: 2456-2459 [Abstract] [Full Text]
Elliott, K., Watkins, H., Redwood, C. S. (2000). Altered Regulatory Properties of Human Cardiac Troponin I Mutants That Cause Hypertrophic Cardiomyopathy. J. Biol. Chem. 275: 22069-22074 [Abstract] [Full Text]
Lin, K.-M., Mejillano, M., Yin, H. L. (2000). Ca2+ Regulation of Gelsolin by Its C-terminal Tail. J. Biol. Chem. 275: 27746-27752 [Abstract] [Full Text]
Defacque, H., Bos, E., Garvalov, B., Barret, C., Roy, C., Mangeat, P., Shin, H.-W., Rybin, V., Griffiths, G. (2002). Phosphoinositides Regulate Membrane-dependent Actin Assembly by Latex Bead Phagosomes. Mol. Biol. Cell 13: 1190-1202 [Abstract] [Full Text]