Refined structure of villin 14T and a detailed comparison with other actin‐severing domains

Villin 14T is the amino terminal actin monomer binding domain from the actin‐severing and bundling protein villin. Its structure has been determined in solution using heteronuclear multidimensional nuclear magnetic resonance (NMR) spectroscopy (Markus MA, Nakayama T, Matsudaira P, Wagner G. 1994. Solution structure of villin 14T, a domain conserved among actin‐severing proteins. Protein Science 3:70‐81). An additional nuclear Overhauser effect (NOE) spectroscopy data set, acquired using improved gradient techniques, and further detailed analysis of existing data sets, produced an additional 601 NOE restraints for structure calculations. The overall fold does not change significantly with the additional NOE restraints but the definition of the structure is improved, as judged by smaller deviations among an ensemble of calculated structures that adequately satisfy the NMR restraints. Some of the side chains, especially those in the hydrophobic core of the domain, are much more defined. This improvement in the detail of the solution structure of villin 14T makes it interesting to compare the structure with the crystal structure of gelsolin segment 1, which shares 58% sequence identity with villin 14T, in an effort to gain insight into villin 14T's weaker affinity for actin monomers. Villin 14T has smaller side chains at several positions that make hydrophobic contacts with actin in the context of gelsolin segment 1. The structure is also compared with the structure of the related actin‐severing domain, severin domain 2.

[1]  M. Way,et al.  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. , 1988, Journal of molecular biology.

[2]  Kurt Wüthrich,et al.  Stereospecific assignment of the methyl 1H NMR lines of valine and leucine in polypeptides by nonrandom 13C labelling , 1989 .

[3]  P. Janmey,et al.  Identification of critical functional and regulatory domains in gelsolin , 1989, The Journal of cell biology.

[4]  H. Mannherz,et al.  Structure of gelsolin segment 1-actin complex and the mechanism of filament severing , 1993, Nature.

[5]  P. Matsudaira,et al.  Villin sequence and peptide map identify six homologous domains. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[6]  Localization of the calcium-sensitive actin monomer binding site in gelsolin to segment 4 and identification of calcium binding sites. , 1995, Biochemistry.

[7]  L. Kay,et al.  Overcoming the overlap problem in the assignment of 1H NMR spectra of larger proteins by use of three-dimensional heteronuclear 1H-15N Hartmann-Hahn-multiple quantum coherence and nuclear Overhauser-multiple quantum coherence spectroscopy: application to interleukin 1 beta. , 1989, Biochemistry.

[8]  Sensitivity improvement in 2D and 3D HCCH spectroscopy using heteronuclear cross-polarization , 1993 .

[9]  J. Bryan,et al.  Gelsolin has three actin-binding sites , 1988, The Journal of cell biology.

[10]  T F Havel,et al.  The solution structure of eglin c based on measurements of many NOEs and coupling constants and its comparison with X‐ray structures , 1992, Protein science : a publication of the Protein Society.

[11]  W. Kabsch,et al.  Dictionary of protein secondary structure: Pattern recognition of hydrogen‐bonded and geometrical features , 1983, Biopolymers.

[12]  J. Richardson,et al.  Amino acid preferences for specific locations at the ends of alpha helices. , 1988, Science.

[13]  G. Marius Clore,et al.  Stereospecific assignment of β-methylene protons in larger proteins using 3D15N-separated Hartmann-Hahn and13C-separated rotating frame Overhauser spectroscopy , 1991, Journal of biomolecular NMR.

[14]  Ad Bax,et al.  Quantitative J correlation: a new approach for measuring homonuclear three-bond J(HNH.alpha.) coupling constants in 15N-enriched proteins , 1993 .

[15]  T. Holak,et al.  Structure of severin domain 2 in solution. , 1995, Journal of molecular biology.

[16]  D. DeRosier,et al.  Determination of the alpha-actinin-binding site on actin filaments by cryoelectron microscopy and image analysis , 1994, The Journal of cell biology.

[17]  K. Wüthrich,et al.  Stereospecific nuclear magnetic resonance assignments of the methyl groups of valine and leucine in the DNA-binding domain of the 434 repressor by biosynthetically directed fractional 13C labeling. , 1989, Biochemistry.

[18]  M. Way,et al.  Molecular model of an actin filament capped by a severing protein. , 1995, Journal of structural biology.

[19]  D. Kwiatkowski,et al.  In vivo analysis of functional domains from villin and gelsolin , 1992, The Journal of cell biology.

[20]  P. Janmey,et al.  Isolation and properties of two actin-binding domains in gelsolin. , 1985, The Journal of biological chemistry.

[21]  P. Matsudaira,et al.  The secrets of severing? , 1993, Current Biology.

[22]  A. Noegel,et al.  The 100 kDa F‐actin capping protein of Dictyostelium amoebae is a villin prototype (‘protovillin’) , 1993, FEBS letters.

[23]  L. Kay,et al.  Comparison of different modes of two-dimensional reverse-correlation NMR for the study of proteins , 1990 .

[24]  P. Janmey,et al.  Pieces in the actin-severing protein puzzle , 1988, Cell.

[25]  A. Bax,et al.  An alternative 3D NMR technique for correlating backbone 15N with side chain Hβ resonances in larger proteins , 1991 .

[26]  P. Janmey,et al.  Identification of a polyphosphoinositide-modulated domain in gelsolin which binds to the sides of actin filaments , 1988, The Journal of cell biology.

[27]  J. Thornton,et al.  Conformational analysis of protein structures derived from NMR data , 1993, Proteins.

[28]  K. Sharp,et al.  Protein folding and association: Insights from the interfacial and thermodynamic properties of hydrocarbons , 1991, Proteins.

[29]  S. Grzesiek,et al.  Improved 3D triple-resonance NMR techniques applied to a 31 kDa protein , 1992 .

[30]  W. Kabsch,et al.  Atomic model of the actin filament , 1990, Nature.

[31]  Timothy F. Havel.  An evaluation of computational strategies for use in the determination of protein structure from distance constraints obtained by nuclear magnetic resonance. , 1991, Progress in biophysics and molecular biology.

[32]  Robert Powers,et al.  Three-dimensional triple-resonance NMR of 13C/15N-enriched proteins using constant-time evolution , 1991 .

[33]  P. Janmey,et al.  Functional comparison of villin and gelsolin. Effects of Ca2+, KCl, and polyphosphoinositides. , 1988, The Journal of biological chemistry.

[34]  K. Wüthrich,et al.  Support of1H NMR assignments in proteins by biosynthetically directed fractional13C-labeling , 1992 .

[35]  Richard R. Ernst,et al.  Multiple quantum filters for elucidating NMR coupling networks , 1982 .

[36]  J. Vandekerckhove,et al.  Sequence of human villin: a large duplicated domain homologous with other actin-severing proteins and a unique small carboxy-terminal domain related to villin specificity , 1988, The Journal of cell biology.

[37]  M Ikura,et al.  An efficient 3D NMR technique for correlating the proton and15N backbone amide resonances with the α-carbon of the preceding residue in uniformly15N/13C enriched proteins , 1991, Journal of biomolecular NMR.

[38]  M. Way,et al.  Evidence for functional homology in the F-actin binding domains of gelsolin and alpha-actinin: implications for the requirements of severing and capping , 1992, The Journal of cell biology.

[39]  Gerhard Wagner,et al.  Solution structure of villin 14T, a domain conserved among actin‐severing proteins , 1994, Protein science : a publication of the Protein Society.

[40]  P. Kraulis.  A program to produce both detailed and schematic plots of protein structures , 1991 .

[41]  K. Ogura,et al.  Tertiary Structure of Destrin and Structural Similarity between Two Actin-Regulating Protein Families , 1996, Cell.

[42]  M. Way,et al.  Two of the three actin‐binding domains of gelsolin bind to the same subdomain of actin Implications for capping and severing mechanisms , 1991, FEBS letters.

[43]  Gerhard Wagner,et al.  1H, 15N, 13C and 13CO resonance assignments and secondary structure of villin 14T, a domain conserved among actin-severing proteins , 1994, Journal of biomolecular NMR.

[44]  L. Kay,et al.  A Gradient-Enhanced HCCH-TOCSY Experiment for Recording Side-Chain 1H and 13C Correlations in H2O Samples of Proteins , 1993 .

[45]  Richard R. Ernst,et al.  Coherence transfer by isotropic mixing: Application to proton correlation spectroscopy , 1983 .

[46]  Ad Bax,et al.  Three-dimensional heteronuclear NMR of nitrogen-15 labeled proteins , 1989 .