X‐ray crystal structure and molecular dynamics simulations of silver hake parvalbumin (Isoform B)

Abstract

Parvalbumins constitute a class of calcium‐binding proteins characterized by the presence of several helix‐loop‐helix (EF‐hand) motifs. In a previous study (Revett SP, King G, Shabanowitz J, Hunt DF, Hartman KL, Laue TM, Nelson DJ, 1997, Protein Sci 7 :2397–2408), we presented the sequence of the major parvalbumin isoform from the silver hake ( Merluccius bilinearis ) and presented spectroscopic and structural information on the excised “EF‐hand” portion of the protein. In this study, the X‐ray crystal structure of the silver hake major parvalbumin has been determined to high resolution, in the frozen state, using the molecular replacement method with the carp parvalbumin structure as a starting model. The crystals are orthorhombic, space group C222 1 , with a = 75.7 Å, b = 80.7 Å, and c = 42.1 Å. Data were collected from a single crystal grown in 15% glycerol, which served as a cryoprotectant for flash freezing at ‐188 °C. The structure refined to a conventional R ‐value of 21% (free R 25%) for observed reflections in the range 8 to 1.65 Å [ I > 2σ( I )]. The refined model includes an acetylated amino terminus, 108 residues (characteristic of a β parvalbumin lineage), 2 calcium ions, and 114 water molecules per protein molecule. The resulting structure was used in molecular dynamics (MD) simulations 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 fully Ca 2+ loaded protein and on a Ca 2+ deficient variant, with Ca 2+ only in the CD site. There was substantial agreement between the MD and X‐ray results in addressing the issue of mobility of key residues in the calcium‐binding sites, especially with regard to the side chain of Ser55 in the CD site and Asp92 in the EF site.