Metal ion binding properties and conformational states of calcium- and integrin-binding protein.
Calcium- and integrin-binding protein (CIB) is a novel member of the helix-loop-helix family of regulatory calcium-binding proteins which likely has a specific function in hemostasis through its interaction with platelet integrin alphaIIbbeta(3). The significant amino acid sequence homology between CIB and other regulatory calcium-binding proteins such as calmodulin, calcineurin B, and recoverin suggests that CIB may undergo a calcium-induced conformational change; however, the mechanism of calcium binding and the details of a structural change have not yet been investigated. Consequently, we have performed a variety of spectroscopic and microcalorimetric studies of CIB to determine its calcium binding characteristics, and the subsequent conformational changes that occur. Furthermore, we provide the first evidence for magnesium binding to CIB and determine the structural consequences of this interaction. Our results indicate that in the absence of any bound metal ions, apo-CIB adopts a folded yet highly flexible molten globule-like structure. Both calcium and magnesium binding induce conformational changes which stabilize both the secondary and tertiary structure of CIB, resulting in considerable increases in the thermal stability of the proteins. CIB was found to bind two Ca(2+) ions in a sequential manner with dissociation constants (K(d)) near 0.54 and 1.9 microM for sites EF-4 and EF-3, respectively. In contrast, CIB bound only one Mg(2+) ion to EF-3 with a K(d) near 120 microM. Together, our results suggest that CIB may exist in multiple structural and metal ion-bound states in vivo which may play a role in its regulation of target proteins such as platelet integrin.
Structural Biology Research Group, Department of Biological Sciences, University of Calgary, 2500 University Drive Northwest, Calgary, Alberta, Canada T2N 1N4.
Aaron P
AP
Yamniuk
Leonard T
LT
Nguyen
Tung T
TT
Hoang
Hans J
HJ
Vogel
D000817:Anilino Naphthalenesulfonates; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D002151:Calorimetry; D002152:Calorimetry, Differential Scanning; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D002413:Cations, Divalent / Q000378:metabolism; D002942:Circular Dichroism; D020832:EF Hand Motifs; D005456:Fluorescent Dyes; D006801:Humans; D016023:Integrins / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism*; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence
D000817:Anilino Naphthalenesulfonates; C109198:CIB1 protein, human; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D002413:Cations, Divalent; D005456:Fluorescent Dyes; D016023:Integrins; C027132:1-anilino-8-naphthalenesulfonate; D008274:Magnesium; D002118:Calcium