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The platelet integrin αIIbβ3 is widely accepted as a structural and a functional model of the broad integrin protein family. The four calcium-binding sites in the αIIb subunit contribute to biogenesis and stability of the protein. Mansour et al. (J Thromb Haemost 9:192-200, 2011) showed that the natural Asn2Asp mutation causing Glanzmann thrombasthenia, prevented surface expression of αIIbβ3, whereas the artificial Asn2Gln mutation only decreased its level. Molecular dynamics simulations and EDTA chelation assay were used here to explore the mechanism of these structural deformations. We show a considerable expansion of the calcium-binding site 3 in Asn2Asp mutation, whereas the Asn2Gln toggles between normal and expanded conformations. The αIIbβ3 surface expression level correlates to the relative spending time in the expanded conformation. By a comparison to other calcium-binding sites of αIIb and of other α integrins we show that the size of a calcium-binding loop is conserved. EDTA chelation assay shows a sensitivity to calcium removal, which correlates with the reduction in αIIbβ3 surface expression and with the calcium binding site expansion, thus verifying the simulation data. Here we indicate that Asn2 mutation affects a calcium-binding site 3 of αIIb, which structural deformation is proposed to deprive calcium binding and interfere with an integrin intracellular trafficking and its surface expression.

The calcium- and integrin-binding protein 1 (CIB1) is a ubiquitous Ca(2+)-binding protein and a specific binding partner for the platelet integrin αIIb cytoplasmic domain, which confers the key role of CIB1 in hemostasis. CIB1 is also known to be involved in apoptosis, embryogenesis, and the DNA damage response. In this study, the solution structures of both Ca(2+)-CIB1 and Mg(2+)-CIB1 were determined using solution-state NMR spectroscopy. The methyl groups of Ile, Leu, and Val were selectively protonated to compensate for the loss of protons due to deuteration. The solution structure of Ca(2+)-CIB1 possesses smaller opened EF-hands in its C-domain compared with available crystal structures. Ca(2+)-CIB1 and Mg(2+)-CIB1 have similar structures, but the N-lobe of Mg(2+)-CIB1 is slightly more opened than that of Ca(2+)-CIB1. Additional NMR experiments, such as chemical shift perturbation and methyl group solvent accessibility as measured by a nitroxide surface probe, were carried out to further characterize the structures of Ca(2+)-CIB1 and Mg(2+)-CIB1 as well as their interactions with the integrin αIIb cytoplasmic domain. NMR measurements of backbone amide proton slow motion (microsecond to millisecond) dynamics confirmed that the C-terminal helix of Ca(2+)-CIB1 is displaced upon αIIb binding. The EF-hand III of both Ca(2+)-CIB1 and Mg(2+)-CIB1 was identified to be directly involved in the interaction of CIB1 with αIIb. Together, these data illustrate that CIB1 behaves quite differently from related EF-hand regulatory calcium-binding proteins, such as calmodulin or neuronal calcium sensor proteins.

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).

Calcium-binding domains in the alpha-subunit of integrins contain a central loop structure. To examine the importance of the loop structure, a series of alphaIIb mutants containing changes to the calcium-liganding amino acids have been constructed. Significantly, none of the mutant alphaIIbbeta3 complexes was detected on the surface of transfected cells, but mutant pro-alphaIIb was detected in cell lysates in complex with beta3. To study the importance of the regions flanking the second calcium-binding domain for ligand-binding and ligand-binding specificity, three alphaIIb/alpha5 chimaeras containing alpha5 sequences flanking or flanking and including the second calcium-binding domain were constructed. The chimaera containing both alpha5-flanking regions was not expressed on the cell surface, but FR1 and FR2, substituting either the first or second flanking region, were expressed. FR1beta3-transfected cells lost the ability to adhere to fibrinogen and to support aggregation and had minimal fibrinogen-binding ability. The heterodimer complex was less stable than the wild-type. FR2beta3-transfected cells adhered to fibrinogen and bound soluble fibrinogen with higher affinity when compared with wild-type. In addition, the heterodimer complex was more stable than wild-type. These results indicate that the conformation of the second calcium-binding domain is critical for maturation of the alphaIIbbeta3 complex and expression on the cell surface and that the surrounding sequences are critical for alphaIIbbeta3 function.

The recently published crystal structure of the external domains of alphaVbeta3 confirms the prediction that the aminoterminal portion of alphaV, which shares 40% homology with alphaIIb, folds into a beta-propeller structure and that the 4 calcium-binding domains are positioned on the bottom of the propeller. To gain insight into the role of the calcium-binding domains in alphaIIb biogenesis, we characterized mutations in the second and third calcium-binding domains of alphaIIb in 2 patients with Glanzmann thrombasthenia. One patient inherited a Val298Phe mutation in the second domain, and the other patient inherited an Ile374Thr mutation in the third domain. Mammalian cell expression studies were performed with normal and mutant alphaIIb and beta3 cDNA constructs. By flow cytometry, expression of alphaIIb Val298Phe/beta3 in transfected cells was 28% of control, and expression of alphaIIbIle374Thr/beta3 was 11% of control. Pulse-chase analyses showed that both mutant pro-alphaIIb subunits are retained in the endoplasmic reticulum and degraded. Mutagenesis studies of the Val298 and Ile374 residues showed that these highly conserved, branch-chained hydrophobic residues are essential at these positions and that biogenesis and expression of alphaIIbbeta3 is dramatically affected by structural variations in these regions of the calcium-binding domains. Energy calculations derived from a new model of the alphaIIb beta-propeller indicate that these mutations interfere with calcium binding. These data suggest that the alphaIIb calcium-binding domains play a key structural role in the beta-propeller, and that the structural integrity of the calcium-binding domains is critical for integrin biogenesis.