Characterization of the Connexin45 Carboxyl-Terminal Domain Structure and Interactions with Molecular Partners

Abstract

Mechanisms underlying the initiation and persistence of lethal cardiac rhythms are of significant clinical and scientific interests. Gap junctions are principally involved in forming the electrical connections between myocytes, and changes in distribution, density, and properties are consistent characteristics in arrhythmic heart disease. Therefore, understanding the structure and function of gap junctions during normal and abnormal impulse propagation are essential in the control of arrhythmias. For example, Cx45 is predominately expressed in the specialized myocytes of the impulse generation and conduction system. In both ventricular and atrial human working myocytes, Cx45 is present in very low quantities. However, a reduction in Cx43 coupled with an increased Cx45 protein levels within the ventricles have been observed after myocardial infarction and end-stage heart failure. Cx45 may influence electrical and/or metabolic coupling as a result of pathophysiological overexpression. Our goal was to identify mechanisms that could cause cellular coupling to be different between the cardiac connexins. Based upon the conserved transmembrane and extracellular loop segments, our focus was on identifying features within the divergent cytoplasmic portions. Here, we biophysically characterize the carboxyl-terminal domain of Cx45 (Cx45CT). Purification revealed the possibility of oligomeric species, which was confirmed by analytical ultracentrifugation experiments. Sedimentation equilibrium and circular dichroism studies of different Cx45CT constructs identified one region of α-helical structure (A333-N361) that mediates CT dimerization through hydrophobic contacts. Interestingly, the binding affinity of Cx45CT dimerization is 1000-fold stronger than Cx43CT dimerization. Cx45CT resonance assignments were also used to identify the binding sites and affinities of molecular partners involved in the Cx45 regulation; although none disrupted dimerization, many of these proteins interacted within one intrinsically disordered region (P278-P285). This domain has similarities with other cardiac connexins, and we propose they constitute a master regulatory domain, which contains overlapping molecular partner binding, cis-trans proline isomerization, and phosphorylation sites.

Intramolecular signaling in a cardiac connexin: Role of cytoplasmic domain dimerization

“…Cx45CT dimerization was initially identified through the use of a recombinantly expressed soluble peptide in vitro [24]. We posited that regulatory and structural features of the isolated CT peptides would be recapitulated in cells.…”

“…Previous work from our laboratory identified high affinity dimerization ( K D ~100 nM) of soluble Cx45CT domains mediated by 6 hydrophobic residues aligned on one face of an α-helical region (A333-N361) [24, 25]. When each hydrophobic residue was replaced by glutamic acid (L335E, L338E, I324E, L349E, I353E, Y356E; construct named Cx45CT-6E; Fig.…”

“…When each hydrophobic residue was replaced by glutamic acid (L335E, L338E, I324E, L349E, I353E, Y356E; construct named Cx45CT-6E; Fig. 1) dimerization was disrupted while the α-helical conformation was retained [24]. Based upon dimerization of cytosolic domains as a key regulatory mechanism of ion channels (e.g., IP3 receptor, [26]) and the role of connexin CT domains as important for proper channel function, mediated in part through protein partner interactions, here we set forth to determine the biological significance of Cx45CT dimerization in the function of Cx45.…”

Degradation of gap junction connexins is regulated by the interaction with Cx43-interacting protein of 75 kDa (CIP75)

“…The UBA domain of CIP75 (M549-S596) [26], Cx32CT (C217-C283) [33], Cx37CT (C233-V333) [34], Cx40CT (S251-V355) [10], Cx43CT (S255-I382) [35], Cx45CT (K265-I396) [13] and the Cx45CT dimerization domain (A333-N361) [12] were expressed and purified as previously described. For the UBA and Cx32CT domains, site-directed mutagenesis was performed to change the linker amino acid Leu3 to Trp in order to more precisely determine the protein concentration; using circular dichroism and NMR, no change in structure was observed in either of these constructs (data not shown).…”

Quarterly intrinsic disorder digest (April–May–June, 2014)

“…They found that multiple regulatory partners bound with an intrinsically disordered region in the C-terminal, and propose that this could be a master regulatory domain. 19 Chandrababu et al used NMR to investigate the folding of the tooth enamel protein amelogenin in the presence of SDS micelles. They confirmed that the complex forms in the N-terminal region and that amelogenin increases in structure upon forming a complex with SDS.…”