Pinpoint Dual Chemical Cross-Linking Explores the Structural Dynamics of the Ubiquinone Reaction Site in Mitochondrial Complex I
Pinpoint Dual Chemical Cross-Linking Explores the Structural Dynamics of the Ubiquinone Reaction Site in Mitochondrial Complex I
复制标题
精确双化学交联探索线粒体复合物 I 中泛醌反应位点的结构动力学
DOI：
10.1021/acs.biochem.0c00991
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发表时间：
2021
期刊：
影响因子：
2.9
通讯作者：
Miyoshi Hideto
中科院分区：
文献类型：
--
作者：
Masuya Takahiro;Uno Shinpei;Murai Masatoshi;Miyoshi Hideto
NADH-泛醌氧化还原酶（复合物I）中的泛醌还原步骤是触发其膜部分质子转运的关键。虽然存在一个长而窄的醌通道已被确定，它仍然是有争议的通道模型是否可以解释结合的各种配体（泛醌和抑制剂）的酶。我们以前提出，基质侧的界面区域的49 kDa，ND 1，PSST，和39 kDa的亚基，这是由一个环连接跨膜螺旋（TMH）1和2的ND 3，可能是一些庞大的配体进入醌反应腔的面积。然而，这个命题缺乏直接的证据表明，空腔是从假定的基质侧区域，这使得配体通过。为了解决这个问题，我们研究了ND 3的Cys 39和49 kDa的Asp 160是否可以通过双功能交联剂（四嗪-马来酰亚胺杂合物，称为TMBC）特异性交联。基于复合物I的结构模型，这种双重交联是出乎意料的，因为ND 3 Cys 39和49 kDa Asp 160分别位于TMH 1 -2环上和通道内部深处，因此，它们被形成通道壁的肽链物理分离。然而，具有不同间隔区长度的三个TMBC确实交联了这两个残基，导致形成新的交联的ND 3/49 kDa亚基。ND 3 Cys 39或49 kDa Asp 160的化学修饰阻断了双交联，确保了交联的特异性。总而言之，这项研究提供了直接的证据，醌反应腔确实是从拟议的基质侧区域覆盖的ND 3 TMH 1 -2环访问。
The ubiquinone reduction step in NADH-ubiquinone oxidoreductase (complex I) is the key to triggering proton translocation in its membrane part. Although the existence of a long and narrow quinone-access channel has been identified, it remains debatable whether the channel model can account for binding of various ligands (ubiquinones and inhibitors) to the enzyme. We previously proposed that the matrix-side interfacial region of the 49 kDa, ND1, PSST, and 39 kDa subunits, which is covered by a loop connecting transmembrane helices (TMHs) 1 and 2 of ND3, may be the area for entry of some bulky ligands into the quinone reaction cavity. However, this proposition lacks direct evidence that the cavity is accessible from the putative matrix-side region, which allows ligands to pass. To address this, we examined whether Cys39of ND3 and Asp160of 49 kDa can be specifically cross-linked by bifunctional cross-linkers (tetrazine-maleimide hybrid, named TMBC). On the basis of the structural models of complex I, such dual cross-linking is unexpected because ND3 Cys39and 49 kDa Asp160are located on the TMH1–2 loop and deep inside the channel, respectively, and hence, they are physically separated by peptide chains forming the channel wall. However, three TMBCs with different spacer lengths did cross-link the two residues, resulting in the formation of new cross-linked ND3/49 kDa subunits. Chemical modification of either ND3 Cys39or 49 kDa Asp160blocked the dual cross-linking, ensuring the specificity of the cross-linking. Altogether, this study provides direct evidence that the quinone reaction cavity is indeed accessible from the proposed matrix-side region covered by the ND3 TMH1–2 loop.