Structural Basis for the Activation of Platelet Integrin αIIbβ3 by Calcium- and Integrin-Binding Protein 1

Abstract

Calcium and integrin binding protein 1 (CIB1) is a specific binding partner for the cytoplasmic domain of the αIIb subunit of the highly abundant platelet integrin αIIbβ3. This protein has been suggested to be involved in the regulation of the activation of αIIbβ3, a process leading to platelet aggregation and blood coagulation. In this work, the solution structure of the deuterated Ca2+-CIB1 protein complexed with an αIIb peptide was first determined through modern RDC-based NMR methods. Next, we generated a complex structure for CIB1 and the αIIb domain (Ca2+-CIB1/αIIb) using the program Haddock, which is based on experimental restraints obtained for the protein–peptide interface from cross-saturation NMR experiments. In this data-driven complex structure, the N-terminal α-helix of the cytoplasmic domain of αIIb is buried in the hydrophobic pocket of the C-lobe of Ca2+-CIB1. The C-terminal acidic tail of αIIb remains unstructured and likely interacts with several positively charged residues in the N-lobe of Ca2+-CIB1. A potential molecular mechanism for the CIB1-mediated activation of the platelet integrin could be proposed on the basis of the model structure of this protein complex. Another feature of this work is that, in the NMR cross-saturation experiments, we applied the selective radio frequency irradiation to the smaller binding partner (the αIIb peptide), and successfully detected the binding interface on the larger binding partner Ca2+-CIB1 through its selectively protonated methyl groups. This ‘reverse’ methodology has a broad potential to be employed to many other complexes where synthetic peptides and a suitably isotope-labeled medium- to large-sized protein are used to study protein–protein interactions.

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.…”

“…20,21 Upon Ca 2+ binding, conformational changes occur that create a hydrophobic pocket that mediates the interaction with the C-terminal tail of integrin. 22,23 CIB2 has a broad expression pattern that includes the inner ear where it is immunohistochemically detected in supporting cells and in stereocilia and cuticular plate of hair cells in the cochlea and the vestibular system. 4 Based on the functional impairment of hair cells in Cib2 zebrafish morphants, it has been suggested that Cib2 is important for Ca 2+ homeostasis in sensory hair cells, and that loss of Cib2 function might affect processes like mechanotransduction, adaptation, electromotility and synaptic transmission.…”

Structural Insights into Calmodulin-regulated L-selectin Ectodomain Shedding

“…The interaction of CaM with a sequence predicted to extend by ϳ6 amino acids into L-selectin's putative membrane-spanning region is not a unique occurrence. Ca 2ϩ -and integrin-binding protein 1 binds the cytoplasmic tail of the ␣ IIb subunit of the platelet integrin ␣ IIb ␤ 3 through a sequence both juxtamembrane to and extending by six amino acids into the predicted membrane-spanning region (53)(54)(55)(56)(57)(58). Furthermore, bioinformatic analysis presented here indicates that several other transmembrane receptors downregulated through Ca 2ϩ /CaM-dependent ectodomain shedding have CaM-binding motifs that extend into the transmembrane domain (Fig.…”

The Solution Structure, Binding Properties, and Dynamics of the Bacterial Siderophore-binding Protein FepB

“…A mixing time of 180 ms was used in the three-dimensional 15 N-edited NOESY-TROSY (44) and 13 C-edited NOESY-HMQC experiments. Additionally, a constant time, 13 C-edited NOESY-HSQC experiment with a 250-ms mixing time was measured by using a non-uniform sampling procedure (30%) and processed using MDDGUI (46,47) with the irradiation radiofrequency field set at 5.5 ppm (4.0 -7.0 ppm). The reference experiment was performed identically except with the irradiation set at Ϫ15 ppm, where there were no proton signals.…”