Calcium- and magnesium-dependent interactions between calcium- and integrin-binding protein and the integrin alphaIIb cytoplasmic domain.
Calcium- and integrin-binding protein (CIB) is a small EF-hand calcium-binding protein that is involved in hemostasis through its interaction with the alphaIIb cytoplasmic domain of integrinalphaIIbbeta(3). We have previously demonstrated that CIB lacks structural stability in the absence of divalent metal ions but that it acquires a well-folded conformation upon addition of Ca(2+) or Mg(2+). Here, we have used fluorescence spectroscopy, NMR spectroscopy, and isothermal titration calorimetry to demonstrate that both Ca(2+)-bound CIB (Ca(2+)-CIB) and the Mg(2+)-bound protein (Mg(2+)-CIB) bind with high affinity and through a similar mechanism to alphaIIb cytoplasmic domain peptides, but that metal-free CIB (apo-CIB) binds in a different manner. The interactions are thermodynamically distinct for Ca(2+)-CIB and Mg(2+)-CIB, but involve hydrophobic interactions in each case. Since the Mg(2+) concentration inside the cell is sufficient to saturate CIB at all times, our results imply that CIB would be capable of binding to the alphaIIb cytoplasmic domain independent of an intracellular Ca(2+) stimulus in vivo. This raises the question of whether CIB can act as a Ca(2+) sensor in alphaIIbbeta(3) signaling or if other regulatory mechanisms such as fibrinogen-induced conformational changes in alphaIIbbeta(3), post-translational modifications, or the binding of other accessory proteins mediate the interactions between CIB and alphaIIbbeta(3). Differences in NMR spectra do suggest, however, that Ca(2+)-binding to the Mg(2+)- CIB-alphaIIb complex induces subtle structural changes that could further modulate the activity of alphaIIbbeta(3).
Yamniuk
Aaron P
AP
Structural Biology Research Group, Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, AB, Canada T2N 1N4.
Vogel
Hans J
HJ
D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D040201:Platelet Membrane Glycoprotein IIb / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary
C109198:CIB1 protein, human; D002135:Calcium-Binding Proteins; D040201:Platelet Membrane Glycoprotein IIb; D002118:Calcium