Structural changes in the carboxyl terminus of the gap junction protein connexin43 indicates signaling between binding domains for c-Src and zonula occludens-1

Paul L. Sorgen, Heather S. Duffy, Prangya Sahoo, Wanda Coombs, Mario Delmar, David C. Spray

Research output: Contribution to journalArticle

140 Citations (Scopus)

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 (∼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 α-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.

Original languageEnglish (US)
Pages (from-to)54695-54701
Number of pages7
JournalJournal of Biological Chemistry
Volume279
Issue number52
DOIs
StatePublished - Dec 24 2004

Fingerprint

Connexin 43
Connexins
Tight Junctions
src Homology Domains
PDZ Domains
Nuclear magnetic resonance
Gap Junctions
Communication
Titration
Cell Communication
Down-Regulation
Experiments
Binding Sites
Cells

ASJC Scopus subject areas

  • Biochemistry

Cite this

Structural changes in the carboxyl terminus of the gap junction protein connexin43 indicates signaling between binding domains for c-Src and zonula occludens-1. / Sorgen, Paul L.; Duffy, Heather S.; Sahoo, Prangya; Coombs, Wanda; Delmar, Mario; Spray, David C.

In: Journal of Biological Chemistry, Vol. 279, No. 52, 24.12.2004, p. 54695-54701.

Research output: Contribution to journalArticle

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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 (∼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 α-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.",
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