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Calcium- and magnesium-dependent interactions between calcium- and integrin-binding protein and the integrin IIb cytoplasmic domain

Structural Biology Research Group, Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada T2N 1N4

(RECEIVED December 23, 2004; FINAL REVISION February 22, 2005; ACCEPTED March 14, 2005)

Calcium- and integrin-binding protein (CIB) is a small EF-hand calcium-binding protein that is involved in hemostasis through its interaction with the IIb cytoplasmic domain of integrin IIb₃. 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 Ca²⁺ or Mg²⁺. Here, we have used fluorescence spectroscopy, NMR spectroscopy, and isothermal titration calorimetry to demonstrate that both Ca²⁺-bound CIB (Ca²⁺-CIB) and the Mg²⁺-bound protein (Mg²⁺-CIB) bind with high affinity and through a similar mechanism to IIb cytoplasmic domain peptides, but that metal-free CIB (apo-CIB) binds in a different manner. The interactions are thermodynamically distinct for Ca²⁺-CIB and Mg²⁺-CIB, but involve hydrophobic interactions in each case. Since the Mg²⁺ concentration inside the cell is sufficient to saturate CIB at all times, our results imply that CIB would be capable of binding to the IIb cytoplasmic domain independent of an intracellular Ca²⁺ stimulus in vivo. This raises the question of whether CIB can act as a Ca²⁺ sensor in IIb₃ signaling or if other regulatory mechanisms such as fibrinogen-induced conformational changes in IIb₃, post-translational modifications, or the binding of other accessory proteins mediate the interactions between CIB and IIb₃. Differences in NMR spectra that Ca²⁺-binding to the Mg²⁺-CIB-IIb complex induces subtle structural changes that could further modulate the activity of IIb₃.

Keywords: calcium- and integrin-binding protein; calcium; magnesium; integrin; peptide; isothermal titration calorimetry; nuclear magnetic resonance spectroscopy; fluorescence spectroscopy

Abbreviations: CIB, calcium- and integrin-binding protein • Ca²⁺-CIB, calcium-bound CIB • Mg²⁺-CIB, magnesium-bound CIB • apo-CIB, metal-free CIB • CaM, calmodulin • CnB, calcineurin B • can, calcineurin A • NCS, neuronal calcium sensor • TnC, troponin C • TnI, troponin I

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.041312805.

Reprint requests to: Hans J. Vogel, Structural Biology Research Group, Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, AB, Canada T2N 1N4; e-mail: vogel{at}ucalgary.ca; fax: (403) 289-9311.

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