Solution Structures of Ca2+-CIB1 and Mg2+-CIB1 and Their Interactions with the Platelet Integrin αIIb Cytoplasmic Domain

Abstract

The calcium-and integrin-binding protein 1 (CIB1) is a ubiquitous Ca 2؉ -binding protein and a specific binding partner for the platelet integrin ␣IIb 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 ␣IIb 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 ␣IIb 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 ␣IIb. 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.The calcium-and integrin-binding protein 1 (CIB1) is a member of the regulatory Ca 2ϩ -binding helix-loop-helix or EFhand protein family (1). CIB1 is a ubiquitous 191-residue (22 kDa) protein with several functions in cell signaling (2, 3). CIB1 has been reported to interact with a number of protein targets, including the platelet integrin ␣IIb subunit (4), sphingosine kinase 1 (5), p21-activated kinase (6), apoptosis signal-regulating kinase (7), polo-like protein kinases (8), and a recently reported new target in the regulation of cardiac hypertrophy, calcineurin B (9). The interaction of CIB1 with the integrin ␣IIb subunit has been studied extensively (10 -13). The integrin ␣IIb subunit usually associates with the integrin ␤3 subunit, and the heterodimeric ␣IIb␤3 protein is involved in both so-called "inside-out" and "outside-in" signaling pathways (3). CIB1 is believed to be capable of specifically binding to the ␣IIb cytoplasmic domain and dissociating the ␣IIb␤3 dimer, which in turn triggers integrin activation (11,12). The interactions between Ca 2ϩ -CIB1 and a fragment of the integrin ␣IIb subunit (residues 983-1008, hereafter referred to as the ␣IIb peptide) have been suggested to mainly involve a hydrophobic pocket in the C-domain of Ca 2ϩ -CIB1 with a dissociation constant in the submicromolar range (10).The calcium-bound CIB1 (Ca 2ϩ -CIB1) structure consists of four helix-loop-helix (EF-hand) Ca 2ϩ -binding m...

Novel and recurrent CIB2 variants, associated with nonsyndromic deafness, do not affect calcium buffering and localization in hair cells

“…Therefore, an amino acid change at p.Arg66 would likely destabilize the interaction with integrin and thus attenuate integrin activation (Figures 4a and b). 22,34 In consistence with our calcium-imaging data, Arg66 is located away from the Ca 2+ binding pockets, 20,21 and thus the substitution of tryptophan for arginine at position 66 is predicted not to alter calcium-buffering abilities of CIB2. Similarly, the substitution of serine for phenylalanine at position 91 is also predicted to weaken the interaction of CIB2 with αIIβ integrin.…”

“…Molecular modeling suggests impaired interaction of CIB2 with effector molecules due to the c.196C4T and c.272T4C variants When modeled using the known crystal structure of CIB1 as backbone 4,21,34 in CIB2, the p.Arg66 maps to the N-terminal domain of CIB2 within a region that has been implicated in the interaction of CIB protein with the carboxy terminal, unstructured, negatively charged tail of αIIβ integrin. CIB1 is involved in the activation of integrin αIIβ3.…”

Structural Analysis of a Calmodulin Variant from Rice

“…NMR residual dipolar couplings take advantage of the restriction on the molecular tumbling introduced by anisotropic media to address the relative orientation of secondary structural elements inside proteins (45). Hence, RDCs were measured for the apo and holo forms of the protein in the presence of phage pf1 (45,46). Also, model structures of the N-lobe and C-lobe of OsCaM61 were created from available mCaM structures (apo N-lobe from Protein Data Bank code 1CFD, apo C-lobe from Protein Data Bank code 1CFD, holo N-lobe from Protein Data Bank code 1J7O, and holo C-lobe from Protein Data Bank code 1J7P) using the SWISS-MODEL server (Fig.…”

The Emerging Roles of CIB1 in Cancer

“…As we known, structure determines function. To get insight into the function of CIB1, the structure of CIB1 has been explored extensively by sequence analysis, nuclear magnetic resonance spectroscopy (NMR), circular dichroism or X-ray crystallography [6-8, 10, 80]. Thus, we believed that along with the further analysis of CIB1 structure, more and more CIB1 partners and the precise mechanisms for multiple function of CIB1 will be identified in the future.…”

“…Yamniuk et al identified that low affinity Ca 2+ binding events that influence the structures of the N- and C-terminal extensions of CIB1 under high Ca 2+ crystallization conditions [10]. Studies suggested that Mg 2+ -CIB1 and Ca 2+ -CIB1 exhibit a more ordered 4-EF-hand structure with smaller hydrodynamic radius than a highly flexible molten globule-like CIB1 (not bound to any metals)[6, 8]. A consensus sequence 5’-MGXXXS/T-3’ at the CIB1 N terminus appears to be crucial for its myristoylation modification and plasma membrane localization [4, 7, 13, 14].…”