Preferential Binding of Mg2+ Over Ca2+ to CIB2 Triggers an Allosteric Switch Impaired in Usher Syndrome Type 1J

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.

Usher Syndrome: Genetics and Molecular Links of Hearing Loss and Directions for Therapy

“…In contrast to previous studies, Vallone et al (2018) showed that CIB2 is unable to function as a calcium sensor under physiological conditions due to a low affinity for Ca 2+ ions. However, they demonstrated that CIB2 does have a high affinity for Mg 2+ under the same conditions, and is therefore much more likely to function as a magnesium sensor (Vallone et al, 2018).…”

“…A small number of mutations in CIB2 have been associated with USH1J, including a conservative point mutation, c.192G>C (p.E64D; Riazuddin et al, 2012). The E64 residue is in the N-terminal domain and is thought to communicate with the EF3 cation binding site in the C-terminal domain (Vallone et al, 2018). Therefore, mutations at this site prohibit this inter-domain communication, impairing the cation responsiveness of CIB2.…”

“…Therefore, mutations at this site prohibit this inter-domain communication, impairing the cation responsiveness of CIB2. In the presence of physiological Mg 2+ , the p.E64D mutation causes CIB2 to adopt a less stable partially unfolded conformation that is more prone to aggregation (Vallone et al, 2018). Alternative splicing gives rise to four CIB2 isoforms, and disease-causing mutations in exon 4-6 affect all isoforms (Riazuddin et al, 2012;Michel et al, 2017).…”

Atypical and ultra-rare Usher syndrome: a review

“…In 2012, Riazuddin et al reported a homozygous pathogenic variant of CIB2 in USH1 patients (50) which affected the function of CIB2 as a calcium sensor. Vallone et al later demonstrated that, in physiological conditions, CIB2 can function as a magnesium sensor and the variant described by Riazuddin et al affects this capacity (88). Studies show that knockdown of Cib2 in mice eliminates mechanotransduction in cochlear hair cells (89,90).…”

Constitutive Activation of Guanylate Cyclase by the G86R GCAP1 Variant Is Due to “Locking” Cation-π Interactions that Impair the Activator-to-Inhibitor Structural Transition

“…The molecular weight and Stokes radius of all GCAP1 variants (20 µL injection volume at a concentration of 50 µg/µL) was estimated in the presence of 2 mM Ca 2+ , 3.5 mM Mg 2+ or 2 mM EGTA, according to refs. [51,52].…”