Structural studies of N-terminal mutants of Connexin 32 using 1H NMR spectroscopy
Structural studies of N-terminal mutants of Connexin 32 using 1H NMR spectroscopy
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DOI：
10.1016/j.abb.2012.05.027
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发表时间：
2012-10-01
影响因子：
3.9
通讯作者：
Dowd, T. L.
中科院分区：
文献类型：
--
作者：
Kalmatsky, B. D.;Batir, Y.;Dowd, T. L.
缝隙连接蛋白的氨基末端连接蛋白在电压门控和离子渗透中起着重要作用。我们以前已经用H-1核磁共振证明，功能性连接蛋白分子的N端结构包含一个柔性的G12(Arch.生物化学。BiPhys.490：9,2009)允许N-末端在细胞质入口附近形成通道孔的一部分。非功能连接蛋白分子G12S和GUE的突变体被发现阻止了这种转变。以前对Cx32突变导致周围神经病变Charcot-Marie-Tooth病的基因座的功能研究表明，G12S不是质膜插入的。目前，我们解决了无功能连接蛋白32突变体W3D和Y7D的结构，这些突变体似乎不是膜插入的。利用2D H-1核磁共振，我们报告了类似于G12S和G12Y，疏水侧链相互作用的改变中断(Y7D)或限制(W3D)G12的柔性转动。在所有非功能突变体G12S、G12Y、W3D和Y7D中观察到的开放转角残基12的变化与功能丧失有关。我们认为，开放转弯的丢失会导致N-末端延伸出通道孔，这种错误折叠可能会以突变体为目标，破坏内质网。(C)2012 Elsevier Inc.保留所有权利。
The amino terminus of gap junction proteins, connexins, plays a fundamental role in voltage gating and ion permeation. We have previously shown with H-1 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 GUY 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 H-1 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. (C) 2012 Elsevier Inc. All rights reserved.