Abstract
Regulation of cell-cell communication by the gap junction protein connexin43 can be modulated by a variety of connexin-associating proteins. In particular, c-Src can disrupt the connexin43 (Cx43)-zonula occludens-1 (ZO-1) interaction, leading to down-regulation of gap junction intercellular communication. The binding sites for ZO-1 and c-Src correspond to widely separated Cx43 domains (approximately 100 residues apart); however, little is known about the structural modifications that may allow information to be transferred over this distance. Here, we have characterized the structure of the connexin43 carboxyl-terminal domain (Cx43CT) to assess its ability to interact with domains from ZO-1 and c-Src. NMR data indicate that the Cx43CT exists primarily as an elongated random coil, with two regions of alpha-helical structure. NMR titration experiments determined that the ZO-1 PDZ-2 domain affected the last 19 Cx43CT residues, a region larger than that reported to be required for Cx43CT-ZO-1 binding. The c-Src SH3 domain affected Cx43CT residues Lys-264-Lys-287, Ser-306-Glu-316, His-331-Phe-337, Leu-356-Val-359, and Ala-367-Ser-372. Only region Lys-264-Lys-287 contains the residues previously reported to act as an SH3 binding domain. The specificity of these interactions was verified by peptide competition experiments. Finally, we demonstrated that the SH3 domain could partially displace the Cx43CT-PDZ-2 complex. These studies represent the first structural characterization of a connexin domain when integrated in a multimolecular complex. Furthermore, we demonstrate that the structural characteristics of a disordered Cx43CT are advantageous for signaling between different binding partners that may be important in describing the mechanism of channel closure or internalization in response to pathophysiological stimuli.
Highlights
Regulation of cell-cell communication by the gap junction protein connexin43 can be modulated by a variety of connexin-associating proteins
We have characterized the structure of the connexin43 carboxyl-terminal domain (Cx43CT) to assess its ability to interact with domains from zonula occludens-1 (ZO-1) and c-Src
These studies represent the first structural characterization of a connexin domain when integrated in a multimolecular complex
Summary
Cx43, connexin; Cx43CT, connexin carboxyl terminus; Cx43CL, connexin cytoplasmic loop; CL, cytoplasmic loop; CT, carboxyl terminus; HSQC, heteronuclear single-quantum correlation; NMR, nuclear magnetic resonance; NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy; ZO-1, zonula Occludens-1; SH, Src homology; MAP, mitogen-activated protein; MAPK, MAP kinase. The role of connexin-binding proteins is presumably in trafficking to sites of gap junction formation between cells, stabilization of the junctional plaque, or perhaps signal transduction [33, 34]. Whereas site-directed mutagenesis has been used to infer the sites of protein-protein interactions, no characterization of the structural modifications resulting from these interactions has been reported previously. We show the solution of the secondary structure of Cx43CT and report the changes that occur upon binding to the SH3 domain of c-Src and to the second PDZ domain of ZO-1. The regions of structural reorganization include but span beyond those identified previously by site-directed mutagenesis to be necessary for binding. Our data suggest that the Cx43CT ligand binding platforms may represent elaborate three-dimensional conformations that include amino acids distal in the primary sequence of the protein
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