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 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.
Highlights
Research. □S The on-line version of this article contains supplemental Figs. 1–3 and additional references. 1 Recipient of an Alberta Heritage Foundation for Medical Research Studentship award. 2 Recipient of a scientist award from the Alberta Heritage Foundation for 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]
Even though the amide NH-residual dipolar coupling (RDC) analysis results suggested that the monomer crystal structure of calcium- and integrin-binding protein 1 (CIB1) (Protein Data Bank 1XO5) more closely resembles the conformation of the protein in solution [12], the secondary structure of 1XO5 differs from the solution state conformation in the N- and C-terminal extensions (Fig. 1)
In the solution structure of Ca2ϩ-CIB1, these methyl groups are normally buried in the hydrophobic pocket, and the accessibility to solvent would be shielded by the C-terminal extension of the protein, which folds back over this region in the crystal structure
Summary
Research. □S The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1–3 and additional references. 1 Recipient of an Alberta Heritage Foundation for Medical Research Studentship award. 2 Recipient of a scientist award from the Alberta Heritage Foundation for 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 solution structures of Ca2ϩ-CIB1 and Mg2ϩ-CIB1 NMR were subsequently determined by using chemical shift information, two sets of backbone RDCs (1DCЈN and 1DNH) in combination with the NOEs from backbone amide protons (NH) as well as selectively labeled methyl groups (Ile/Leu/Val) in an otherwise perdeuterated protein sample.
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