Structural basis for calcium binding by uteroglobins.
Uteroglobins, i.e. proteins with similar three-dimensional structure and ligand binding specificity to uteroglobin from rabbit uterus, have been found in rat, mouse and human lung. We have recently demonstrated the binding of calcium by human uteroglobin, and we have therefore tried to find potential binding sites for metals in the three-dimensional structure of uteroglobin by the use of two different computational procedures. A putative binding site for calcium in uteroglobin was identified by means of a hydrophobic contrast function. The spatial disposition of atoms that could ligand calcium in the putative calcium-binding site appears similar to that of the primary calcium-binding site of secretory phospholipase A2 enzymes, consisting of the carboxyl group of an aspartic acid residue and a loop providing three backbone carbonyl oxygens. From inspection of their primary sequences and three-dimensional structures, it became clear that this putative calcium-binding motif is conserved among uteroglobins from different species. The potential significance of the predicted site was investigated by site-directed point mutagenesis of human uteroglobin in which Asp46 was replaced by Asn or Lys. In both mutants, the ruthenium red and 45Ca2+ binding was significantly reduced. Sodium dodecyl sulphate/polyacrylamide gel electrophoresis under non-reducing conditions indicated that the mutant proteins had the expected molecular masses and that their ability to dimerize was not disturbed by these mutations. Valence calculations also identified the putative calcium-binding site, but only after optimization of its conformation by the use of molecular dynamics with a restrained calcium ion. Our results support the notion that Asp46 of uteroglobins acts as a "cap" residue in a calcium-binding site structurally similar to the primary calcium binding sites of phospholipases A2.
Barnes
H J
HJ
Center for Structural Biochemistry, NOVUM, S-141 57, Huddinge, Sweden.
Nordlund-Möller
L
L
Nord
M
M
Gustafsson
J
J
Lund
J
J
Gillner
M
M
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003198:Computer Simulation; D004247:DNA / Q000235:genetics; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D051381:Rats; D013329:Structure-Activity Relationship; D014598:Uteroglobin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D002135:Calcium-Binding Proteins; D004247:DNA; D014598:Uteroglobin; D002118:Calcium