Sequence-specific resonance assignment of the carboxyl terminal domain of Connexin43.

Abstract

The gap junction family of integral membrane proteins enables the direct cytoplasmic exchange of ions and small molecules (<1 kDa), including second messengers. Gap junctions are involved in a diverse array of cellular processes including cellular differentiation and development, metabolic homeostasis, and in excitable tissue, electrical coupling. They are formed by the apposition of connexons from adjacent cells, where each connexon is formed of six connexin proteins. Connexins are four transmembrane domain proteins with intracellular Nand C-terminal regions. More than twenty different mammalian connexins exist with the major divergence occurring in the cytoplasmic loop (CL) and carboxyl terminal (CT) domains. The subject of this paper, Cx43, is the most widely expressed gap junction protein and is essential for normal cardiac development and function. Recently, the idea of gap junctions being formed solely of the connexin proteins has been replaced by the concept that connexons may be centerpieces of a macromolecular complex or ‘Nexus’ (Spray et al., 1999). Integral tight junction, tight junctionassociated, cytoskeletal, adhedrens junctional complex and tyrosine kinase proteins bind to and/or modify the CT of Cx43, consistent with a more active role for gap junctions in cellular functions (Duffy et al., 2002a). A 7.5 A resolution structure of a recombinant Cx43 cardiac gap junction channel (Unger et al., 1999) has been solved; however, Cx43 was truncated to remove most of the CT. Such truncated constructs ∗To whom correspondence should be addressed. E-mail: girvin@aecom.yu.edu form functional channels, but pH sensitivity and interactions with the ‘Nexus’ proteins are altered. Separate co-expression of the CT domain partially restores pH sensitivity (Morley et al., 1996), and recombinant CT domain binds to the Nexus proteins (Duffy et al., 2002b). The hypothesis that acidification-induced uncoupling results from the intramolecular interaction between the CT domain and a separate region of the protein affiliated with the pore (Morley et al., 1996), was supported by our studies demonstrating pH dependent binding between the CT domain and a region of CL (L2) (Duffy et al., 2002c). Binding of L2 induced no significant chemical shift changes in 15N labeled CT, suggesting that the CT structure necessary for recognizing and binding CL is pre-formed. To understand the structural bases of connexin regulation, we are studying the Cx43CT in the two protonation states for which binding (pH 5.8) or no binding (pH 7.3) to L2 loop are observed. Here we report the sequence-specific assignments of the loop-binding Cx43CT conformation at pH 5.8. These assignments will be generally useful for mapping the binding sites for all of the Nexus proteins.