The Interaction between Calcium- and Integrin-binding Protein 1 and the αIIb Integrin Cytoplasmic Domain Involves a Novel C-terminal Displacement Mechanism

Abstract

Calcium-and integrin-binding protein 1 (CIB1) regulates platelet aggregation in hemostasis through a specific interaction with the ␣IIb cytoplasmic domain of platelet integrin ␣IIb␤ 3 . In this work we report the structural characteristics of CIB1 in solution and the mechanistic details of its interaction with a synthetic peptide derived from the ␣IIb cytoplasmic domain. NMR spectroscopy experiments using perdeuterated CIB1 together with heteronuclear nuclear Overhauser effect experiments have revealed a well folded ␣-helical structure for both the ligand-free and ␣IIb-bound forms of the protein. Residual dipolar coupling experiments have shown that the N and C domains of CIB1 are positioned side by side, and chemical shift perturbation mapping has identified the ␣IIb-binding site as a hydrophobic channel spanning the entire C domain and part of the N domain. Data obtained with a truncated version of CIB1 suggest that the extreme C-terminal end of the protein weakly interacts with this channel in the absence of a biological target, but it is displaced by the ␣IIb cytoplasmic domain, suggesting a novel mechanism to increase binding specificity.The platelet-specific heterodimeric transmembrane integrin receptor ␣IIb␤ 3 plays a central role in hemostasis and thrombosis (1). At the site of vascular injury, platelet agonists such as thrombin trigger "inside-out" signaling events that activate ␣IIb␤ 3 , resulting in ligand binding by the integrin extracellular domains, integrin cross-linking, and ultimately, platelet aggregation. Ligand occupancy also generates "outside-in" signals that lead to granular secretion of ADP, cytoskeletal reorganization, and platelet spreading (2). The small EF-hand calciumbinding protein CIB1, 4 (calcium-and integrin-binding protein 1, also known as CIB, calmyrin, KIP) binds specifically to the ␣IIb cytoplasmic domain (3), and the interaction has been implicated in both inside-out and outside-in signaling events (4 -6). The binding of CIB1 to synthetic ␣IIb peptides can occur in vitro with a dissociation constant in the high nanomolar range (7,8); however, the mechanistic details of the interaction are not well understood. Because inappropriate platelet activation is a major contributor to cardiovascular disease (9), understanding the interaction between CIB1 and ␣IIb could be an important step toward the development of novel anti-platelet therapeutics.CIB1 shares significant sequence homology with calcineurin B (CnB), calcineurin homologous protein-1, and the neuronal calcium sensor (NCS) family of EF-hand proteins. Like these proteins, CIB1 is myristoylated on its N-terminal glycine residue and is membrane-associated in vivo (10, 11). However, myristoylation is not required for ␣IIb binding (7,8,10). Recent x-ray crystal structures (Protein Data Bank codes 1XO5 and 1Y1A) have shown that like its homologs, calcium-bound CIB1 (Ca 2ϩ -CIB1) folds into Nand C-terminal globular domains, each composed of two EF-hand motifs, with extended N-and C-terminal regions (12, 13). Ca 2ϩ is bound to th...

Structural Analysis of a Calmodulin Variant from Rice

“…Chemical shift differences between the holo and apo forms of full-length OsCaM61 as well as between the full-length and truncated forms of holo-and apo-OsCaM61 were analyzed using Equation 2 (32). Chemical shifts obtained for deuterated samples through transverse relaxation optimized spectroscopy-based experiments were corrected for isotope shifts before further analysis.…”

“…Papers were washed with 3 ϫ 500 ml of 0.5% (v/v) H 3 PO 4 (5 min each) and once with 500 ml of acetone (5 min) and left to dry. Subsequently, 32 P was quantified by Cerenkov counting (41). Calmodulin kinase II (CaMKII) was also purified from chicken gizzards (42,43).…”

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

“…These two crystal structures have a similar arrangement of the secondary structural elements, but large differences exist in their relative orientations of the N-and C-domains as well as the secondary structure of their N-and C-terminal extensions. Our initial residual dipolar coupling (RDC) 3 NMR study (12) suggested that the monomeric crystal structure of CIB1 (1XO5) more closely resembles the overall conformation of the protein in solution, except for its N-terminal extension.…”

“…In addition, CIB1 contains a myristoylated N-terminal extension in vivo (16), and a short C-terminal extension that was postulated to be involved in a displacement mechanism to increase its target-binding specificity (12). So far, two x-ray crystal structures of Ca 2ϩ -CIB1 have been deposited in the Protein Data Bank, in which 1XO5 (15) is a monomer and 1Y1A (14) is a head-to-tail homodimer.…”

“…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).…”