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：16
页码：E1571-E1580
DOI：10.1073/pnas.1404446111
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：The lactose permease (LacY) of Escherichia coli, a paradigm for the major facilitator superfamily, catalyzes the coupled stoichiometric translocation of a galactopyranoside and an H+ across the cytoplasmic membrane. To catalyze transport, LacY undergoes large conformational changes that allow alternating access of sugar- and H+-binding sites to either side of the membrane. Despite strong evidence for an alternating access mechanism, it remains unclear how H+- and sugar-binding trigger the cascade of interactions leading to alternating conformational states. Here we used dynamic single-molecule force spectroscopy to investigate how substrate binding induces this phenomenon. Galactoside binding strongly modifies kinetic, energetic, and mechanical properties of the N-terminal 6-helix bundle of LacY, whereas the C-terminal 6-helix bundle remains largely unaffected. Within the N-terminal 6-helix bundle, the properties of helix V, which contains residues critical for sugar binding, change most radically. Particularly, secondary structures forming the N-terminal domain exhibit mechanically brittle properties in the unbound state, but highly flexible conformations in the substrate-bound state with significantly increased lifetimes and energetic stability. Thus, sugar binding tunes the properties of the N-terminal domain to initiate galactoside/H+ symport. In contrast to wild-type LacY, the properties of the conformationally restricted mutant Cys154Gly do not change upon sugar binding. It is also observed that the single mutation of Cys154Gly alters intramolecular interactions so that individual transmembrane helices manifest different properties. The results support a working model of LacY in which substrate binding induces alternating conformational states and provides insight into their specific kinetic, energetic, and mechanical properties.
关键词：atomic force microscopy ; membrane ; transport protein ; membrane protein structure ; membrane protein folding ; membrane transport
