期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2014
卷号：111
期号：5
页码：1819-1824
DOI：10.1073/pnas.1317890111
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：CLC transporters catalyze transmembrane exchange of chloride for protons. Although a putative pathway for Cl- has been established, the pathway of H+ translocation remains obscure. Through a highly concerted computational and experimental approach, we characterize microscopic details essential to understanding H+-translocation. An extended (0.4 {micro}s) equilibrium molecular dynamics simulation of membrane-embedded, dimeric ClC-ec1, a CLC from Escherichia coli, reveals transient but frequent hydration of the central hydrophobic region by water molecules from the intracellular bulk phase via the interface between the two subunits. We characterize a portal region lined by E202, E203, and A404 as the main gateway for hydration. Supporting this mechanism, site-specific mutagenesis experiments show that ClC-ec1 ion transport rates decrease as the size of the portal residue at position 404 is increased. Beyond the portal, water wires form spontaneously and repeatedly to span the 15-A hydrophobic region between the two known H+ transport sites [E148 (Gluex) and E203 (Gluin)]. Our finding that the formation of these water wires requires the presence of Cl- explains the previously mystifying fact that Cl- occupancy correlates with the ability to transport protons. To further validate the idea that these water wires are central to the H+ transport mechanism, we identified I109 as the residue that exhibits the greatest conformational coupling to water wire formation and experimentally tested the effects of mutating this residue. The results, by providing a detailed microscopic view of the dynamics of water wire formation and confirming the involvement of specific protein residues, offer a mechanism for the coupled transport of H+ and Cl- ions in CLC transporters.
