Title
Direct Visualization of Interaction between Calmodulin and Connexin45
Author
Juan Zou, Mani Salarian, Yanyi Chen, You Zhuo, Nicole E Brown, John R Hepler, Jenny Yang
Year
2017
Journal
Biochemical Journal
Abstract
Calmodulin (CaM) is an intracellular Ca2+ transducer involved in numerous activities in a broad Ca2+signaling network. Previous studies have suggested that the Ca2+/CaM complex may participate in gap junction regulation via interaction with putative CaM-binding motifs in connexins, however, evidence of direct interactions between CaM and connexins has remained elusive to date due to challenges related to the study of membrane proteins. Here we report the first direct interaction of CaM with Cx45 of g-family in living cells under physiological conditions by monitoring bioluminescence resonance energy transfer (BRET). The interaction between CaM and Cx45 in cells is strongly dependent on intracellular Ca2+ concentration and can be blocked by the CaM inhibitor, N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W7). We further reveal a CaM binding site at the cytosolic loop (residues 164-186) of Cx45 using a peptide model. The strong binding ( K d ~5 nM) observed between CaM and Cx45 peptide, monitored by fluorescence-labeled CaM, is found to be Ca2+- dependent. Furthermore, high resolution NMR spectroscopy reveals that CaM and Cx45 peptide binding leads to global chemical shift changes of 15N labeled CaM but does not alter the size of the structure. Observations involving both N- and C-domains of CaM to interact with the Cx45 peptide differs from the embraced interaction with Cx50 from another connexin family. Such interaction further increases Ca2+sensitivity of CaM especially at the N-terminal domain. Results of this study suggest that both helicity and the interaction mode of the cytosolic loop are likely to contribute to CaM's modulation of connexins.
Instrument
J-810
Keywords
Circular dichroism, Secondary structure, Chemical stability, Ligand binding, Biochemistry