Abstract
Calcium and integrin binding protein 2 (CIB2) shares with the other members of the CIB family the ability to bind Ca2+ and Mg2+ via two functional EF-hand motifs, namely EF3 and EF4. As a cation sensor, CIB2 is able to switch to a conformation likely associated with specific biological functions yet to be clarified. Recent findings demonstrate the involvement of CIB2 in hearing physiology and a single, conservative point mutation (p.E64D) has been related to Usher Syndrome type 1J (USH1J) and non-syndromic hearing loss. We present an exhaustive biochemical and biophysical characterization of human wild type (WT) and E64D CIB2. We found that CIB2 does not possibly work as a calcium sensor under physiological conditions, its affinity for Ca2+ (Kdapp = 0.5 mM) being too low for detecting normal intracellular levels. Instead, CIB2 displays a significantly high affinity for Mg2+ (Kdapp = 290 μM), and it is probably Mg2+ -bound under physiological conditions. At odds with the homologous protein CIB1, CIB2 forms a non-covalent dimer under conditions that mimic the physiological ones, and as such it interacts with its physiological target α7B integrin. NMR spectroscopy revealed a long-range allosteric communication between the residue E64, located at the N-terminal domain, and the metal cation binding site EF3, located at the C-terminal domain. The conservative E64D mutation breaks up such inter-domain communication resulting in the impaired ability of CIB2 to switch to its Mg2+-bound form. The ability to bind the target integrin peptide was substantially conserved for E64D CIB2, thus suggesting that the molecular defect associated with USH1J resides in its inability to sense Mg2+ and adopt the required conformation.
Highlights
Calcium and integrin binding protein 2 (CIB2) is a 21.6 kDa protein sharing with the other members of the CIB family the ability to bind Ca2+ and Mg2+ via two functional EF-hand motifs, namely EF3 and EF4, switching to a specific conformation likely associated with specific biological functions (Huang et al, 2012)
We investigated the oligomeric state of both wild type (WT) and E64D CIB2 by three different approaches, namely PolyAcrylamide Gel Electrophoresis under non-denaturing conditions, analytical size exclusion chromatography (SEC) and Dynamic light scattering (DLS)
Besides being involved in hearing physiology and pathology (Riazuddin et al, 2012; Jan, 2013; Patel et al, 2015; Seco et al, 2016; Wang et al, 2017), CIB2 has been found to play a role in congenital muscular dystrophy type 1A (Häger et al, 2008), in the N-methyl-D-aspartate receptor-mediated Ca2+ signaling in cultured hippocampal neurons (Blazejczyk et al, 2009), in the promotion of HIVviral infection (Godinho-Santos et al, 2016), and very recently it was found to act as a negative regulator of sphingosine kinase 1-mediated oncogenic signaling in ovarian cancer (Zhu et al, 2017)
Summary
Calcium and integrin binding protein 2 (CIB2) is a 21.6 kDa protein sharing with the other members of the CIB family the ability to bind Ca2+ and Mg2+ via two functional EF-hand motifs, namely EF3 and EF4, switching to a specific conformation likely associated with specific biological functions (Huang et al, 2012). R186W (Patel et al, 2015)] have been found to be associated with non-syndromic deafness (DFNB48) while a single, conservative point mutation (p.E64D) (Riazuddin et al, 2012) has been related to Usher Syndrome type 1J (USH1J, OMIM entry: 614869), a genetic disorder characterized by hearing loss and progressive vision loss due to retinitis pigmentosa. These recent findings suggest that CIB2 is an essential component for the normal development of both hair cells and photoreceptor cells. The E64D mutation associated with USH1J, conservative, apparently breaks up such inter-domain communication resulting in the impaired ability of CIB2 to switch to its Mg2+ (and Ca2+)-bound form, suggesting that the molecular defect associated with CIB2 and causing USH1J resides in its inability to sense Mg2+ and adopt the required conformation
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