X-ray crystal structure and molecular dynamics simulations of silver hake parvalbumin (isoform B)

Parvalbumins constitute a class of calcium-binding proteins characterized b y the presence of several helix-loop-helix (EF-hand) motifs. In a previous study (Revett SP, King G, Shabanowitz J, Hunt DE Hartman KL, Laue TM, Nelso n DJ, 1997, Protein Sci 7:2397-2408), we presented the sequence of the majo r parvalbumin isoform from the silver hake (Merluccius bilinearis) and pres ented spectroscopic and structural information on the excised "EF-hand" por tion of the protein. In this study, the X-ray crystal structure of the silv er hake major parvalbumin has been determined to high resolution, in the fr ozen state, using the molecular replacement method with the carp parvalbumi n structure as a starting model. The crystals are orthorhombic, space group C222(1), with a = 75.7 Angstrom, b = 80.7 Angstrom, and c = 42.1 Angstrom. Data were collected from a single crystal grown in 15% glycerol, which ser ved as a cryoprotectant for flash freezing at -188 degrees C. The structure refined to a conventional R-value of 21% (free R 25%) for observed reflect ions in the range 8 to 1.65 Angstrom [I > 2 sigma(I)]. The refined model in cludes an acetylated amino terminus, 108 residues (characteristic of a beta parvalbumin lineage), 2 calcium ions, and 114 water molecules per protein molecule. The resulting structure was used in molecular dynamics (MD) simul ations focused primarily on the dynamics of the Ligands coordinating the Ca 2+ ions in the CD and EF sites. MD simulations were performed on both the f ully Ca2+ loaded protein and on a Ca2+ deficient variant, with Ca2+ only in the CD site. There was substantial agreement between the MD and X-ray resu lts in addressing the issue of mobility of key residues in the calcium-bind ing sites, especially with regard to the side chain of Ser55 in the CD site and Asp92 in the EF site.