Helix Straightening as an Activation Mechanism in the Gelsolin Superfamily of Actin Regulatory Proteins

Villin and gelsolin consist of six homologous domains of the gelsolin/cofilin fold (V1–V6 and G1–G6, respectively). Villin differs from gelsolin in possessing at its C terminus an unrelated seventh domain, the villin headpiece. Here, we present the crystal structure of villin domain V6 in an environment in which intact villin would be inactive, in the absence of bound Ca2+ or phosphorylation. The structure of V6 more closely resembles that of the activated form of G6, which contains one bound Ca2+, rather than that of the calcium ion-free form of G6 within intact inactive gelsolin. Strikingly apparent is that the long helix in V6 is straight, as found in the activated form of G6, as opposed to the kinked version in inactive gelsolin. Molecular dynamics calculations suggest that the preferable conformation for this helix in the isolated G6 domain is also straight in the absence of Ca2+ and other gelsolin domains. However, the G6 helix bends in intact calcium ion-free gelsolin to allow interaction with G2 and G4. We suggest that a similar situation exists in villin. Within the intact protein, a bent V6 helix, when triggered by Ca2+, straightens and helps push apart adjacent domains to expose actin-binding sites within the protein. The sixth domain in this superfamily of proteins serves as a keystone that locks together a compact ensemble of domains in an inactive state. Perturbing the keystone initiates reorganization of the structure to reveal previously buried actin-binding sites. Villin and gelsolin consist of six homologous domains of the gelsolin/cofilin fold (V1–V6 and G1–G6, respectively). Villin differs from gelsolin in possessing at its C terminus an unrelated seventh domain, the villin headpiece. Here, we present the crystal structure of villin domain V6 in an environment in which intact villin would be inactive, in the absence of bound Ca2+ or phosphorylation. The structure of V6 more closely resembles that of the activated form of G6, which contains one bound Ca2+, rather than that of the calcium ion-free form of G6 within intact inactive gelsolin. Strikingly apparent is that the long helix in V6 is straight, as found in the activated form of G6, as opposed to the kinked version in inactive gelsolin. Molecular dynamics calculations suggest that the preferable conformation for this helix in the isolated G6 domain is also straight in the absence of Ca2+ and other gelsolin domains. However, the G6 helix bends in intact calcium ion-free gelsolin to allow interaction with G2 and G4. We suggest that a similar situation exists in villin. Within the intact protein, a bent V6 helix, when triggered by Ca2+, straightens and helps push apart adjacent domains to expose actin-binding sites within the protein. The sixth domain in this superfamily of proteins serves as a keystone that locks together a compact ensemble of domains in an inactive state. Perturbing the keystone initiates reorganization of the structure to reveal previously buried actin-binding sites. Actin is crucial to such processes as cell movement, cell division, and apoptosis, which are regulated by numerous actin-binding proteins, including gelsolin, Arp2/3, and profilin (for review, see Ref. 1dos Remedios C.G. Chhabra D. Kekic M. Dedova I.V. Tsubakihara M. Berry D.A. Nosworthy N.J. Physiol. Rev. 2003; 83: 433-473Crossref PubMed Scopus (764) Google Scholar). Gelsolin, the most potent actin filament-severing protein known, can bind to, sever, cap, and nucleate actin filaments in a calcium-, pH-, ATP-, and phospholipid-dependent manner (for review, see Ref. 2McGough A.M. Staiger C.J. Min J.K. Simonetti K.D. FEBS Lett. 2003; 552: 75-81Crossref PubMed Scopus (157) Google Scholar). Villin, found in microvilli of absorptive epithelium, is a second member of the gelsolin family of actin-binding proteins. In addition to standard gelsolin-type activities, villin is able to bundle actin filaments and is subject to regulation by tyrosine phosphorylation as well as by Ca2+ and phosphatidylinositol 4,5-bisphosphate (for review, see Ref. 3Khurana S. George S.P. FEBS Lett. 2008; 582: 2128-2139Crossref PubMed Scopus (118) Google Scholar). Many comparisons have been made between gelsolin and villin. The two share 50% amino acid sequence identity and show similar proteolytic cleavage patterns (4Janmey P.A. Matsudaira P.T. J. Biol. Chem. 1988; 263: 16738-16743Abstract Full Text PDF PubMed Google Scholar). Both contain six similarly folded domains, but villin possesses a seventh domain at its C terminus, the headpiece (HP) 2The abbreviations used are: HPheadpieceG1–G6gelsolin domains 1–6V1–V6villin domains 1–6. domain, which folds into a compact structure that introduces a second F-actin-binding site into the protein. Recent studies indicate that villin uses the HP F-actin-binding sites to achieve bundling (5George S.P. Wang Y. Mathew S. Srinivasan K. Khurana S. J. Biol. Chem. 2007; 282: 26528-26541Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). In an environment devoid of free Ca2+, gelsolin and villin assume inactive conformations. After binding Ca2+, both undergo conformational rearrangements that expose their binding sites for F-actin. In villin, this includes revealing the HP actin-binding site through a “hinge mechanism” (6Hesterberg L.K. Weber K. J. Biol. Chem. 1983; 258: 365-369Abstract Full Text PDF PubMed Google Scholar). headpiece gelsolin domains 1–6 villin domains 1–6. Biochemical and structural studies have revealed eight Ca2+-binding sites of two types in gelsolin (for review, see Ref. 7Burtnick L.D. Urosev D. Irobi E. Narayan K. Robinson R.C. EMBO J. 2004; 23: 2713-2722Crossref PubMed Scopus (97) Google Scholar). Each of the six domains contains a complete and evolutionarily conserved site, termed type 2, whereas G1 and G4 provide partial Ca2+ coordination at interfaces with actin through sites termed type 1. Sequential mutagenesis of these sites in villin has identified six functional Ca2+-binding sites (8Kumar N. Tomar A. Parrill A.L. Khurana S. J. Biol. Chem. 2004; 279: 45036-45046Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar): two major sites, one each of type 1 and type 2, in V1, plus four type 2 sites in V2–V6. The type 1 site in V1 regulates F-actin-capping and F-actin-severing activities, whereas the lower affinity type 2 site in V1 only affects severing (9Northrop J. Weber A. Mooseker M.S. Franzini-Armstrong C. Bishop M.F. Dubyak G.R. Tucker M. Walsh T.P. J. Biol. Chem. 1986; 261: 9274-9281Abstract Full Text PDF PubMed Google Scholar). The other four sites are involved in stabilizing villin conformation, but they do not directly influence actin-severing activity. NMR studies of a fragment of villin that consists of V6 and the HP domain have implicated V6 residues Asn647, Asp648, and Glu670 in binding Ca2+ (10Smirnov S.L. Isern N.G. Jiang Z.G. Hoyt D.W. McKnight C.J. Biochemistry. 2007; 46: 7488-7496Crossref PubMed Scopus (12) Google Scholar). These experiments also revealed the first 80 residues of V6 to undergo significant conformational change as a result of Ca2+ binding. Nanomolar to micromolar concentrations of free Ca2+ govern the actin-binding activities of gelsolin. In contrast, micromolar and millimolar concentrations of calcium ions are required for villin to exhibit capping and severing, respectively. However, after tyrosine phosphorylation, villin can sever actin filaments even at nanomolar Ca2+ concentrations (11Kumar N. Khurana S. J. Biol. Chem. 2004; 279: 24915-24918Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Furthermore, although the actin-severing ability of the N-terminal half of villin is calcium-dependent, that by the N-terminal half of gelsolin is not. In contrast, the binding of G-actin of the C-terminal half of both villin and gelsolin requires Ca2+. Creation of hybrid proteins demonstrated that the domains of villin and gelsolin are not interchangeable (12Finidori J. Friederich E. Kwiatkowski D.J. Louvard D. J. Cell Biol. 1992; 116: 1145-1155Crossref PubMed Scopus (36) Google Scholar). Abundant x-ray crystallographic structural information exists for gelsolin, including the calcium ion-free (Ca2+-free), inactive structure of the intact protein (13Burtnick L.D. Koepf E.K. Grimes J. Jones E.Y. Stuart D.I. McLaughlin P.J. Robinson R.C. Cell. 1997; 90: 661-670Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar), the activated N- and C-terminal halves, each in a bimolecular complex with actin (7Burtnick L.D. Urosev D. Irobi E. Narayan K. Robinson R.C. EMBO J. 2004; 23: 2713-2722Crossref PubMed Scopus (97) Google Scholar, 14Choe H. Burtnick L.D. Mejillano M. Yin H.L. Robinson R.C. Choe S. J. Mol. Biol. 2002; 324: 691-702Crossref PubMed Scopus (115) Google Scholar), and the activated C-terminal half on its own (15Kolappan S. Gooch J.T. Weeds A.G. McLaughlin P.J. J. Mol. Biol. 2003; 329: 85-92Crossref PubMed Scopus (21) Google Scholar, 16Narayan K. Chumnarnsilpa S. Choe H. Irobi E. Urosev D. Lindberg U. Schutt C.E. Burtnick L.D. Robinson R.C. FEBS Lett. 2003; 552: 82-85Crossref PubMed Scopus (26) Google Scholar). Structural data for intact villin are unavailable and are limited to fragment V1 (17Markus M.A. Matsudaira P. Wagner G. Protein Sci. 1997; 6: 1197-1209Crossref PubMed Scopus (29) Google Scholar), solved using NMR methods, and the HP domain, solved by NMR and x-ray crystallography (18Vardar D. Buckley D.A. Frank B.S. McKnight C.J. J. Mol. Biol. 1999; 294: 1299-1310Crossref PubMed Scopus (63) Google Scholar, 19Meng J. Vardar D. Wang Y. Guo H.C. Head J.F. McKnight C.J. Biochemistry. 2005; 44: 11963-11973Crossref PubMed Scopus (47) Google Scholar). NMR experiments also indicate that HP is connected to V6 by a 40-residue disordered linker. As a result, HP has been proposed to bind actin independently of the remainder of the protein (10Smirnov S.L. Isern N.G. Jiang Z.G. Hoyt D.W. McKnight C.J. Biochemistry. 2007; 46: 7488-7496Crossref PubMed Scopus (12) Google Scholar). In this report, we present the structure of Ca2+-free, isolated villin V6, which exhibits a typical gelsolin domain fold. The long helix in V6 in this structure is straight, unlike the corresponding helix in G6 of intact Ca2+-free gelsolin, which is bent, and only straightens on calcium activation of the intact protein. Hence, V6 appears to be in an active conformation in the absence of Ca2+. Molecular dynamics simulations indicate that the preferred state of the long helix is also straight for isolated G6 in the absence of Ca2+. Furthermore, they suggest a bistable mechanism of helix conformational change regulated by the presence of the remaining domains, by calcium ions, and by other interactants. We therefore propose a mechanism for the gelsolin family proteins whereby Ca2+ triggers the straightening of the domain 6 helix in the native conformation of the inactive proteins to propagate more widespread conformational changes. Villin domains V4–V6 with an N-terminal 8-histidine tag was expressed in Escherichia coli from the expression vector pSY5 and purified through sequential Ni2+ affinity and gel filtration chromatographies (see supplemental “Experimental Methods”). Actin was purified from rabbit skeletal muscle by a modified method of Spudich and Watt (20Spudich J.A. Watt S. J. Biol. Chem. 1971; 246: 4866-4871Abstract Full Text PDF PubMed Google Scholar). Villin V4–V6 in 10 mm Tris-HCl, 150 mm NaCl, and 5 mm CaCl2, pH 7.5, was added to actin in 2 mm Tris-HCl, 0.2 mm CaCl2, 0.2 mm ATP, 1 mm dithiothreitol, pH 7.6–7.8 (buffer A), to a molar ratio of 1:1. The resulting solution was held at 4 °C overnight prior to gel filtration (Bio-Rad Sephacryl S300, 90 × 2.5 cm) with elution by buffer A. The fractions containing the protein complex were pooled and concentrated to 10 mg/ml, as determined by UV absorbance at 280 nm (PerkinElmer Life Sciences Lamda 4B) using a calculated absorption coefficient of 1.4 ml mg−1 cm−1. Crystals were grown at 4 °C using the hanging-drop vapor diffusion method. The protein solution (10 mg/ml) was mixed in a 1:1 ratio (v/v) with a reservoir solution containing 15% (w/v) polyethylene glycol 8000, 100 mm sodium acetate buffer, pH 5.0. Crystals required 3–4 weeks of incubation to grow to a suitable quality for diffraction analysis. Prior to x-ray data collection, the crystals were transferred into a cryoprotectant solution, 25% (v/v) glycerol, 15% (w/v) polyethylene glycol 8000, 100 mm sodium acetate buffer, pH 5.0, and flash-frozen in liquid nitrogen. Diffraction data initially were collected to a resolution of 3.0 Å using a Rigaku MM007HF rotating copper anode source with OSMIC VariMaxHR mirrors and a MAR345 image plate detector at the University of British Columbia Centre for Blood Research (Vancouver, Canada). Data were indexed, integrated, and scaled using HKL2000 software (21Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38572) Google Scholar). Subsequently, data were collected to a resolution of 2.0 Å using an Area Detector Systems Corp. Quantum-315 charge-coupled device detector on beamline BL31B1 at the National Synchrotron Radiation Research Center (Taiwan, China; supplemental Table 1). Structural analysis was initiated by molecular replacement using single domains G4–G6 (from Protein Data Bank code 1p8x) as a model. An unambiguous solution showed that the diffraction originated solely from V6, with the V4 and V5 domains and actin not evident. The crystallographic asymmetric unit contains two V6 domains, which permits calculation of a solvent content of 55%. Refinement was carried out using the CCP4 suite of crystallographic programs (22Collaborative Computational Project 4 Acta Crystallogr. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19769) Google Scholar). To monitor the relaxation of G6 isolated from the context of intact Ca2+-free gelsolin, five simulations were initiated from residues 640–731 (G6) excised from the inactive structure of gelsolin (Protein Data Bank code 1d0n) (13Burtnick L.D. Koepf E.K. Grimes J. Jones E.Y. Stuart D.I. McLaughlin P.J. Robinson R.C. Cell. 1997; 90: 661-670Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). Two control runs, meant to assure that the long helix does not bend spontaneously in the isolated domain, were initiated from residues 640–742 (G6) excised from the activated form of the C-terminal half of gelsolin (Protein Data Bank code 1h1v) (14Choe H. Burtnick L.D. Mejillano M. Yin H.L. Robinson R.C. Choe S. J. Mol. Biol. 2002; 324: 691-702Crossref PubMed Scopus (115) Google Scholar). All simulations were performed using GROMACS version 3.3.3. The force field used was OPLS-AA, with time steps of 2 fs, at a temperature of 300 K, with Berendsen pressure coupling and a pressure of 1 bar. Water was simulated explicitly using the simple point charge model, and Na+ cations were added as counterions, as needed to render the system electrostatically neutral. The water box size was set to 0.9 nm from the protein surface. Energy minimization lasted 6 ps, and frames were sampled every 100 ps. the structure of V6 determined at gelsolin G6, V6 consists of a at its between a of a long helix that and a helix that to the is calcium bound to V6 in this the amino acid sequence of V6 is with that of G6, the and of these two domains the six domains of gelsolin, the structure of V6 most closely resembles that of G6 in its calcium form of for residues The long helix in V6 and the form of G6 is in an in to the kinked state in G6 in the context of its intact Ca2+-free protein of the of the residues in binding Ca2+ in G6 Asn647, and with that of the homologous residues from V6 Asp648, and the structural to contrast, in Ca2+-free G6, the and of these residues are These comparisons suggest that the present structure of V6 isolated from the of the villin domains is that of an activated state the absence of bound Ca2+. G6 is the domain in inactive Ca2+-free gelsolin, with other domains (13Burtnick L.D. Koepf E.K. Grimes J. Jones E.Y. Stuart D.I. McLaughlin P.J. Robinson R.C. Cell. 1997; 90: 661-670Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). G6 most with G2 and G4 is a between G4 and G6 that the G4–G6 the actin-binding site on G4. binding Ca2+ triggers of this only at the G6 with G4 2, and is on the of V6 a similar to that on Ca2+-free G6, as in to suggest that interaction with the of the of V4 as between G6 and G4. However, for the that the domain 4 indicate with both G6 and Ca2+-free V6 and supplemental that binding of domain 6 to domain 4 would be in these two conformations. residues at this including residues involved in the show and 1). these data suggest that the manner in which V6 with V4 in villin is with that in which G6 with G4 in gelsolin, of Ca2+-free V6 in an activated In inactive gelsolin, between a on G6, which includes with and and the the F-actin-binding site of G2 (14Choe H. Burtnick L.D. Mejillano M. Yin H.L. Robinson R.C. Choe S. J. Mol. Biol. 2002; 324: 691-702Crossref PubMed Scopus (115) Google Scholar, R.C. Mejillano M. Burtnick L.D. Yin H.L. Choe S. 1999; PubMed Scopus Google Scholar). of a Ca2+ by G6 requires of the of to the of these D and These residues are conserved in villin and Furthermore, residues at the with the N-terminal half and that V6 form similar but with with between G6 and of the structure of G6 and the Ca2+-free form of V6 the structure of Ca2+-free G6 bound to G2 excised from Protein Data Bank code 1d0n) the of conformational in domain 6 in gelsolin family to be with to interaction with domain The within domain 6 in the presence of the straight kinked version of its long helix and C with of the to from the Ca2+-free to the form of G6 is with binding of G6 to G2 and and is a major in the of G6 and G2 activation of gelsolin by Ca2+. the in Ca2+-free isolated V6 in the in a in the activation for gelsolin and villin. of the be in with of the long helix, for V6 to an inactive conformation within villin. In the complex (Protein Data Bank code is interaction between the of G6 and an of in actin and the actin filament-severing and to of that (14Choe H. Burtnick L.D. Mejillano M. Yin H.L. Robinson R.C. Choe S. J. Mol. Biol. 2002; 324: 691-702Crossref PubMed Scopus (115) Google Scholar, R.C. Mejillano M. Burtnick L.D. Yin H.L. Choe S. 1999; PubMed Scopus Google Scholar). of the for Ca2+-free G6 from inactive intact gelsolin for G6 in the activated structure of the complex a of G6 in this complex to a Ca2+-free state would the interaction with However, of the of Ca2+-free V6 for G6 in that between V6 and the actin helix be we that domain 6 from this family of proteins, of that straightening of its long helix, bind actin and severing at calcium by the apparent that Ca2+-free V6 more closely the form of G6 rather than the Ca2+-free we G6 would such a conformation when isolated from the of the gelsolin structure and free of calcium We a set of molecular dynamics simulations using the inactive conformation of G6 as the of five in the conformation with the long helix straight in than 100 and whereas the bent for the 100 In the set of control using the active conformation of G6 as the the helix was to to the bent state and that the conformation with the long helix straight is the preferred state for the isolated in the present structure of V6, in the absence of V4 is for the helix to assume the kinked The data are with the actin and of gelsolin and villin The of binding Ca2+, in the context of the protein, is to of this is the straightening of bent which we in that would actin-binding The in the helix of the Ca2+-free G6 can be in of the context in which was folded within the compact structure of intact Ca2+-free gelsolin (13Burtnick L.D. Koepf E.K. Grimes J. Jones E.Y. Stuart D.I. McLaughlin P.J. Robinson R.C. Cell. 1997; 90: 661-670Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). The from sequence that the of gelsolin and the of villin are As the long helix of V6 within Ca2+-free villin well be the between G4 and G6 and the interaction between G2 and G6 with are The to do be from the binding of calcium ions from the of such as between and G6 (14Choe H. Burtnick L.D. Mejillano M. Yin H.L. Robinson R.C. Choe S. J. Mol. Biol. 2002; 324: 691-702Crossref PubMed Scopus (115) Google Scholar, R.C. Mejillano M. Burtnick L.D. Yin H.L. Choe S. 1999; PubMed Scopus Google Scholar), and from the straightening of the kinked helix of G6 by helix straightening is gelsolin family activation of their to sever and actin the proteins to of their activation of the villin HP domain to bundle actin filaments a from the of the protein (6Hesterberg L.K. Weber K. J. Biol. Chem. 1983; 258: 365-369Abstract Full Text PDF PubMed Google Scholar). this is with other of activation to be calcium such as present in gelsolin to be at severing actin concentrations of free Ca2+ are not gelsolin is able to actin filaments in this In at gelsolin is able to sever actin in the complete absence of free Ca2+. Furthermore, villin has calcium for activation from of gelsolin (8Kumar N. Tomar A. Parrill A.L. Khurana S. J. Biol. Chem. 2004; 279: 45036-45046Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, J. Weber A. Mooseker M.S. Franzini-Armstrong C. Bishop M.F. Dubyak G.R. Tucker M. Walsh T.P. J. Biol. Chem. 1986; 261: 9274-9281Abstract Full Text PDF PubMed Google Scholar). The of Ca2+ in activation of of this protein superfamily appears to with both protein identity and the of in Ca2+-binding The structure of Ca2+-free V6 in this and its to that of G6 suggest an for the of V6 and G6 active in the absence of Ca2+, when isolated from the other domains that their proteins, as a result of a mechanism that their long by Ca2+ or that of domain 6 from the remaining domains allow domain 6 to an active conformation and the protein to with We suggest that the long helix in domain 6 by the one in domain within this superfamily of proteins can be triggered to a of with

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