Ion and inhibitor binding of the double-ring ion selectivity filter of the mitochondrial calcium uniporter
Ion and inhibitor binding of the double-ring ion selectivity filter of the mitochondrial calcium uniporter
复制标题
线粒体钙单向转运蛋白双环离子选择性过滤器的离子和抑制剂结合。
DOI：
10.1073/pnas.1620316114
复制
发表时间：
2017-04-04
影响因子：
11.1
通讯作者：
Chou, James J.
中科院分区：
文献类型：
--
作者：
Cao, Chan;Wang, Shuqing;Chou, James J.
线粒体的钙（Ca 2+）单向转运体是由称为线粒体钙单向转运体（MCU）的Ca 2+传导通道和若干辅助和调节组分组成的全复合物。先前的电生理学研究发现，单向转运体具有高的Ca 2+选择性和电导，这主要取决于MCU的保守氨基酸序列基序DXXE（Asp-X-X-Glu）。最近的NMR结构的MCU通道从秀丽隐杆线虫显示，DXXE形成两个平行的羧酸环的通道入口处，似乎作为离子选择性过滤器，虽然直接离子相互作用的结构基序还没有得到解决。在这里，我们使用顺磁探针，锰（Mn 2+），调查离子和抑制剂结合这个假定的选择性过滤器。我们的顺磁NMR数据显示，具有单个羧酸环的突变体NXXE（Asn-X-X-Glu）和DXXQ（Asp-X-X-Gln）各自可以特异性结合Mn 2+，而在WT中，两个环协同结合Mn 2+，导致类似于1，000倍的表观亲和力。Ca ~（2+）可以特异性地置换通道中DXXE位点上结合的Mn ~（2+）。此外，用已知的通道抑制剂钌360（Ru 360）滴定样品可以从溶剂可接近的Asp位点而不是内部Glu位点置换Mn 2+结合。的NMR滴定数据，连同DXXE基序和分子动力学模拟的结构分析，表明在MCU孔的顶点的双羧酸环构成的离子选择性过滤器和Ru 360直接阻止离子进入过滤器通过绑定到外部羧酸环。
The calcium (Ca2+) uniporter of mitochondria is a holocomplex consisting of the Ca2+-conducting channel, known as mitochondrial calcium uniporter (MCU), and several accessory and regulatory components. A previous electrophysiology study found that the uniporter has high Ca2+ selectivity and conductance and this depends critically on the conserved amino acid sequence motif, DXXE (Asp-X-X-Glu) of MCU. A recent NMR structure of the MCU channel from Caenorhabditis elegans revealed that the DXXE forms two parallel carboxylate rings at the channel entrance that seem to serve as the ion selectivity filter, although direct ion interaction of this structural motif has not been addressed. Here, we use a paramagnetic probe, manganese (Mn2+), to investigate ion and inhibitor binding of this putative selectivity filter. Our paramagnetic NMR data show that mutants with a single carboxylate ring, NXXE (Asn-X-X-Glu) and DXXQ (Asp-X-X-Gln), each can bind Mn2+ specifically, whereas in the WT the two rings bind Mn2+ cooperatively, resulting in similar to 1,000-fold higher apparent affinity. Ca2+ can specifically displace the bound Mn2+ at the DXXE site in the channel. Furthermore, titrating the sample with the known channel inhibitor ruthenium 360 (Ru360) can displace Mn2+ binding from the solvent-accessible Asp site but not the inner Glu site. The NMR titration data, together with structural analysis of the DXXE motif and molecular dynamics simulation, indicate that the double carboxylate rings at the apex of the MCU pore constitute the ion selectivity filter and that Ru360 directly blocks ion entry into the filter by binding to the outer carboxylate ring.