Protein Science, Vol 3, Issue 1 70-81, Copyright © 1994 by Cold Spring Harbor Laboratory Press

ARTICLE

Solution structure of villin 14T, a domain conserved among actin-severing proteins

M. A. MARKUS, T. NAKAYAMA, P. MATSUDAIRA and G. WAGNER
Committee on Higher Degrees in Biophysics, Harvard University, Cambridge, Massachusetts 02138, and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115

The solution structure of the N-terminal domain of the actin-severing protein villin has been determined by multidimensional heteronuclear resonance spectroscopy. Villin is a member of a family of actin-severing proteins that regulate the organization of actin in the eukaryotic cytoskeleton. Members of this family are built from 3 or 6 homologous repeats of a structural domain of approximately 130 amino acids that is unrelated to any previously known structure. The N-terminal domain of villin (14T) contains a central {beta}-sheet with 4 antiparallel strands and a fifth parallel strand at one edge. This sheet is sandwiched between 2 helices on one side and a 2-stranded parallel {beta}-sheet with another helix on the other side. The strongly conserved sequence characteristic of the protein family corresponds to internal hydrophobic residues. Calcium titration experiments suggest that there are 2 binding sites for Ca(2+), a stronger site near the N-terminal end of the longest helix, with a K(d) of 1.8 +/- 0.4 mM, and a weaker site near the C-terminal end of the same helix, with a K(d) of 11 +/- 2 mM. Mutational and biochemical studies of this domain in several members of the family suggest that the actin monomer binding site is near the parallel strand at the edge of the central {beta}-sheet.
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