title: 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 C2221, with a = 75.7 A, b = 80.7 A, and c = 42.1 A. Data were collected from a single crystal grown in 15% glycerol, which served 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 reflections in the range 8 to 1.65 A [1 > 2sigma(I)]. The refined model includes 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) simulations focused primarily on the dynamics of the ligands coordinating the Ca2+ ions in the CD and EF sites. MD simulations were performed on both the fully 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 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.
authors.affiliation: Gustaf H. Carlson School of Chemistry, Clark University, Worcester, Massachusetts 01610, USA.
authors.forename: R C
authors.initials: RC
authors.lastname: Richardson
authors.forename: N M
authors.initials: NM
authors.lastname: King
authors.forename: D J
authors.initials: DJ
authors.lastname: Harrington
authors.forename: H
authors.initials: H
authors.lastname: Sun
authors.forename: W E
authors.initials: WE
authors.lastname: Royer
authors.forename: D J
authors.initials: DJ
authors.lastname: Nelson
mesh_terms: D000818:Animals; D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D018360:Crystallography, X-Ray; D005399:Fishes; D006860:Hydrogen Bonding; D008958:Models, Molecular; D010320:Parvalbumins / Q000737:chemistry*; D011487:Protein Conformation; D020033:Protein Isoforms / Q000737:chemistry; D012997:Solvents; D013696:Temperature
chemical_list: D002135:Calcium-Binding Proteins; D010320:Parvalbumins; D020033:Protein Isoforms; D012997:Solvents