Abstract
Calcium- and Integrin-Binding protein 2 (CIB2) is a small and ubiquitously expressed protein with largely unknown biological function but ascertained role in hearing physiology and disease. Recent studies found that CIB2 binds Ca2+ with moderate affinity and dimerizes under conditions mimicking the physiological ones. Here we provided new lines of evidence on CIB2 oligomeric state and the mechanism of interaction with the α7B integrin target. Based on a combination of native mass spectrometry, chemical cross-linking/mass spectrometry, analytical gel filtration, dynamic light scattering and molecular dynamics simulations we conclude that CIB2 is monomeric under all tested conditions and presents uncommon hydrodynamic properties, most likely due to the high content of hydrophobic solvent accessible surface. Surface plasmon resonance shows that the interaction with α7B occurs with relatively low affinity and is limited to the cytosolic region proximal to the membrane, being kinetically favored in the presence of physiological Mg2+ and in the absence of Ca2+. Although CIB2 binds to an α7B peptide in a 1:1 stoichiometry, the formation of the complex might induce binding of another CIB2 molecule.
Highlights
Calcium- and Integrin-Binding protein 2 (CIB2) is a small (21.7 kDa) Ca2+ and Mg2+-binding protein initially discovered as a DNA-dependent protein kinase interacting protein[1]
CIB2 knockout mice showed abolished mechanoelectrical transduction in auditory cells leading to profound hearing loss[9], missense mutations in the gene encoding for CIB2 have been found to be associated with hereditary non-syndromic deafness (DFNB48) and possibly Usher Syndrome type 1J, a genetic disorder characterized by hearing loss and progressive blindness[10,11]
Native electrospray ionization (ESI)-mass spectrometry (MS) served to investigate the oligomeric state of CIB2
Summary
Calcium- and Integrin-Binding protein 2 (CIB2) is a small (21.7 kDa) Ca2+ and Mg2+-binding protein initially discovered as a DNA-dependent protein kinase interacting protein[1]. The apparently conservative E64D mutation abolishes the conformational switch and significantly lowers the affinity for both cations[8], indicating that even a small difference such as the presence or absence of a methylene bridge between acidic amino acids can be critical to whether an essential interaction can take place or not Both wild type (WT) and E64D-CIB2 were found to recognize and bind a peptide from the known target integrin α7B3,7 with similar affinity in the micromolar range, and based on analytical size exclusion chromatography (SEC), dynamic light scattering (DLS) and non-denaturing electrophoresis assays we concluded that CIB2 is dimeric, in line with independent previous observations[11,12]. The MS-based techniques as well as surface plasmon resonance (SPR) analysis exclude dimerization of CIB2 over the broad range of conditions tested in this study, and highlight a specific interaction only with the membrane proximal segment of the cytoplasmic domain of α7B integrin, in line with previous results by us[8] and others[7], revealing no significant interaction with the C-terminus proximal region
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