Department of Anatomy and Cell Biology, University of Western Ontario, London, ON N6A 5C1, Canada.
Mol Biol Cell. 2012 Sep;23(17):3312-21. doi: 10.1091/mbc.E12-02-0128. Epub 2012 Jul 18.
Mutations in the gene encoding connexin-43 (Cx43) cause the human development disorder known as oculodentodigital dysplasia (ODDD). In this study, ODDD-linked Cx43 N-terminal mutants formed nonfunctional gap junction-like plaques and exhibited dominant-negative effects on the coupling conductance of coexpressed endogenous Cx43 in reference cell models. Nuclear magnetic resonance (NMR) protein structure determination of an N-terminal 23-amino acid polypeptide of wild-type Cx43 revealed that it folded in to a kinked α-helical structure. This finding predicted that W4 might be critically important in intramolecular and intermolecular interactions. Thus we engineered and characterized a W4A mutant and found that this mutant formed a regular, nonkinked α-helix but did not form functional gap junctions. Furthermore, a G2V variant peptide of Cx43 showed a kinked helix that now included V2 interactions with W4, resulting in the G2V mutant forming nonfunctional gap junctions. Also predicted from the NMR structures, a G2S mutant was found to relieve these interactions and allowed the protein to form functional gap junctions. Collectively, these studies suggest that the nature of the mutation conveys loss of Cx43 function by distinctly different mechanisms that are rooted in the structure of the N-terminal region.
编码连接蛋白 43（Cx43）的基因突变会导致人类发育障碍，即眼-牙-指（趾）发育不良（ODDD）。在这项研究中，与 ODDD 相关的 Cx43 N 端突变体形成了无功能的缝隙连接样斑块，并在参照细胞模型中对共表达的内源性 Cx43 的偶联电导表现出显性负效应。野生型 Cx43 N 端 23 个氨基酸多肽的核磁共振（NMR）蛋白结构测定表明，它折叠成一个扭曲的α-螺旋结构。这一发现预测 W4 可能在分子内和分子间相互作用中至关重要。因此，我们设计并表征了 W4A 突变体，发现该突变体形成了规则的、非扭曲的α-螺旋，但不能形成功能性缝隙连接。此外，Cx43 的 G2V 变体肽显示出一个扭曲的螺旋，现在包括 V2 与 W4 的相互作用，导致 G2V 突变体不能形成功能性缝隙连接。同样根据 NMR 结构预测，G2S 突变体被发现可以缓解这些相互作用，使蛋白能够形成功能性缝隙连接。总的来说，这些研究表明，突变的性质通过明显不同的机制导致 Cx43 功能丧失，这些机制根植于 N 端区域的结构。
