title: Auxiliary Ca2+ binding sites can influence the structure of CIB1.
abstract: Recent X-ray crystal structures and solution NMR spectroscopy data for calcium- and integrin-binding protein 1 (CIB1) have all revealed a common EF-hand domain structure for the protein. However, the orientation of the two protein domains, the oligomerization state, and the conformations of the N- and C-terminal extensions differ among the structures. In this study, we examine whether the binding of glutathione or auxiliary Ca(2+) ions as observed in the crystal structures, occur in solution, and whether these interactions can influence the structure or dimerization of CIB1. In addition, we test the potential phosphatase activity of CIB1, which was hypothesized based on the glutathione binding site geometry observed in one of the crystal structures of the protein. Biophysical and biochemical experiments failed to detect glutathione binding, protein dimerization, or phosphatase activity for CIB1 under several solution conditions. However, our data identify low affinity (K(d), 10(-2)M) Ca(2+) binding events that influence the structures of the N- and C-terminal extensions of CIB1 under high (300 mM) Ca(2+) crystallization conditions. In addition to providing a rationale for differences amongst the various solution and crystal structures of CIB1, our results show that the impact of low affinity Ca(2+) binding events should be considered when analyzing and interpreting protein crystallographic structures determined in the presence of very high Ca(2+) concentrations.
authors.affiliation: Structural Biology Research Group, Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada.
authors.forename: Aaron P
authors.initials: AP
authors.lastname: Yamniuk
authors.forename: Kathryn L
authors.initials: KL
authors.lastname: Anderson
authors.forename: Marie E
authors.initials: ME
authors.lastname: Fraser
authors.forename: Hans J
authors.initials: HJ
authors.lastname: Vogel
mesh_terms: D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray; D005978:Glutathione / Q000378:metabolism; D008027:Light; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D055503:Protein Multimerization; D012542:Scattering, Radiation
chemical_list: C109198:CIB1 protein, human; D002135:Calcium-Binding Proteins; D010744:Phosphoric Monoester Hydrolases; D005978:Glutathione; D002118:Calcium