Department of Chemistry, Brooklyn College of the City University of New York, Brooklyn, NY 11210, United States.
Arch Biochem Biophys. 2012 Oct 1;526(1):1-8. doi: 10.1016/j.abb.2012.05.027. Epub 2012 Jun 14.
The amino terminus of gap junction proteins, connexins, plays a fundamental role in voltage gating and ion permeation. We have previously shown with (1)H NMR that the structure of the N-terminus of functional connexin molecules contains a flexible turn around G12 (Arch. Biochem. Biophys.490:9,2009) allowing the N-terminus to form a portion of the channel pore near the cytoplasmic entrance. The mutants of nonfunctional connexin molecules G12S and G12Y were found to prevent this turn. Previous functional studies of loci at which Cx32 mutations cause a peripheral neuropathy, Charcot-Marie-Tooth disease, have shown that G12S is not plasma membrane inserted. Presently, we solve the structure of nonfunctional Connexin 32 mutants W3D and Y7D which do not appear to be membrane inserted. Using 2D (1)H NMR, we report that similar to G12S and G12Y, alterations in hydrophobic sidechain interactions disrupt (Y7D) or constrain (W3D) the flexible turn around G12. The alteration in the open turn around residue 12, observed in all nonfunctional mutants G12S, G12Y, W3D and Y7D correlates with loss of function. We propose that loss of the open turn causes the N-terminus to extend out of the channel pore and this misfolding may target mutants for destruction in the endoplasmic reticulum.
间隙连接蛋白的氨基末端在电压门控和离子渗透中起着至关重要的作用。我们之前已经通过（1）H NMR 表明，功能性连接蛋白分子的 N 末端结构包含一个围绕 G12 的灵活转弯（Arch Biochem Biophys 490：9,2009），允许 N 末端形成靠近细胞质入口的通道孔的一部分。已经发现非功能性连接蛋白分子 G12S 和 G12Y 的突变体阻止了这种转弯。先前对 Cx32 突变导致周围神经病（Charcot-Marie-Tooth 病）的基因座的功能研究表明，G12S 未插入质膜。目前，我们解决了非功能性Connexin 32 突变体 W3D 和 Y7D 的结构问题，这些突变体似乎没有插入膜内。使用 2D（1）H NMR，我们报告说，类似于 G12S 和 G12Y，改变疏水性侧链相互作用会破坏（Y7D）或限制（W3D）围绕 G12 的灵活转弯。在所有非功能性突变体 G12S、G12Y、W3D 和 Y7D 中观察到的开放转弯周围残基 12 的改变与功能丧失相关。我们提出，开放转弯的丧失导致 N 末端从通道孔中伸出，这种错误折叠可能会导致突变体在内质网中被破坏。
