Chemical shift assignments of the connexin45 carboxyl terminal domain: monomer and dimer conformations

Abstract

Connexin45 (Cx45) is a gap junction protein involved in cell-to-cell communication in the heart and other tissues. Here we report the 1H, 15N, and 13C resonance assignments for the monomer and dimer conformations of the Cx45 carboxyl terminal (Cx45CT) domain and provide evidence of dimerization using Diffusion Ordered Spectroscopy. The predicted secondary structure of the Cx45CT domain based on the chemical shifts identified one region of α-helical structure, which corresponds to the residues that broadened beyond detection in the dimer confirmation. Previous biophysical studies from our laboratory characterizing the CT domain from the other major cardiac connexins, Cx40 and Cx43, suggest that the amount of α-helical content may translate into the ability of a protein to dimerize. Even though the CT domain is thought to be the main regulatory domain of most connexins, the physiological role of CT dimerization is currently unknown. Therefore, these assignments will be useful for determining the intermolecular interactions that mediate Cx45CT dimerization, information that will be used to characterize dimerization in functional channels, as well as characterizing the binding sites for molecular partners involved in Cx45 regulation.

Chemical shift assignments of the connexin37 carboxyl terminal domain

“…Consistent with this observation, the online program CISPEPpred (sunflower.kuicr.kyoto-u.ac.jp/~sjn/cispep) predicted this Cx37CT region can undergo cis-trans proline isomerization. This observation is similar, both in the location on the CT and the number of prolines involved, to what was observed for the Cx45CT domain (Kopanic and Sorgen, 2013). A potential novel role for cis-trans proline isomerization in the regulation of gap junction intercellular communication may be to modulate protein-protein interactions (69).…”

“…Data collection of the Cx37CT domain was performed in 1× PBS (pH 5.8), as previously collected for the Cx43, Cx40, and Cx45CT domains (Bouvier et al, 2007; Kopanic and Sorgen, 2013; Sorgen et al, 2004a). Assignments were made for all of the 1 H, 15 N, and 13 C backbone resonances and 92% of the side chain resonances (BioMag ResBank database, accession number 26921).…”

Structural order in Pannexin 1 cytoplasmic domains

“…Meanwhile, there is a correlation between the centrally located α-helices of the mPanx1, Cx40, Cx43, and Cx45 CT domains. [38][39][40] Secondary structural predictions for the cytosolic domains of mPanx2 and mPanx3 were also calculated employing the algorithms used for mPanx1 (Fig. 8).…”

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).…”