Dataset Information

Opening and closing of the periplasmic gate in lactose permease.

ABSTRACT: X-ray crystal structures of lactose permease (LacY) reveal pseudosymmetrically arranged N- and C-terminal six-transmembrane helix bundles surrounding a deep internal cavity open on the cytoplasmic side and completely closed on the periplasmic side. The residues essential for sugar recognition and H(+) translocation are located at the apex of the cavity and are inaccessible from the outside. On the periplasmic side, helices I/II and VII from the N- and C- six helix bundles, respectively, participate in sealing the cavity from the outside. Three paired double-Cys mutants-Ile-40 --> Cys/Asn-245 --> Cys, Thr-45 --> Cys/Asn-245 --> Cys, and Ile-32 --> Cys/Asn-245 --> Cys-located in the interface between helices I/II and VII on the periplasmic side of LacY were constructed. After cross-linking with homobifunctional reagents less than approximately 15 A in length, all three mutants lose the ability to catalyze lactose transport. Strikingly, however, full or partial activity is observed when cross-linking is mediated by flexible reagents greater than approximately 15 A in length. The results provide direct support for the argument that transport via LacY involves opening and closing of a large periplasmic cavity.

SUBMITTER: Zhou Y

PROVIDER: S-EPMC2268789 | biostudies-literature | 2008 Mar

REPOSITORIES: biostudies-literature

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Opening and closing of the periplasmic gate in lactose permease.

Zhou Yonggang Y Guan Lan L Freites J Alfredo JA Kaback H Ronald HR

Proceedings of the National Academy of Sciences of the United States of America 20080304 10

X-ray crystal structures of lactose permease (LacY) reveal pseudosymmetrically arranged N- and C-terminal six-transmembrane helix bundles surrounding a deep internal cavity open on the cytoplasmic side and completely closed on the periplasmic side. The residues essential for sugar recognition and H(+) translocation are located at the apex of the cavity and are inaccessible from the outside. On the periplasmic side, helices I/II and VII from the N- and C- six helix bundles, respectively, particip ...[more]

PMID: 18319336

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Project description:Based on the crystal structure of lactose permease (LacY) open to the cytoplasm, a hybrid molecular simulation approach with self-guided Langevin dynamics is used to describe conformational changes that lead to a periplasmic-open state. This hybrid approach consists of implicit (IM) and explicit (EX) membrane simulations and requires self-guided Langevin dynamics to enhance protein motions during the IM simulations. The pore radius of the lumen increases by 3.5 Å on the periplasmic side and decreases by 2.5 Å on the cytoplasmic side (relative to the crystal structure), suggesting a lumen that is fully open to the periplasm to allow for extracellular sugar transport and closed to the cytoplasm. Based on our simulations, the mechanism that triggers this conformational change to the periplasmic-open state is the protonation of Glu269 and binding of the disaccharide. Then, helix packing is destabilized by breaking of several side chains involved in hydrogen bonding (Asn245, Ser41, Glu374, Lys42, and Gln242). For the periplasmic-open conformations obtained from our simulations, helix-helix distances agree well with experimental measurements using double electron-electron resonance, fluorescence resonance energy transfer, and varying sized cross-linkers. The periplasmic-open conformations are also in compliance with various substrate accessibility/reactivity measurements that indicate an opening of the protein lumen on the periplasmic side on sugar binding. The comparison with these measurements suggests a possible incomplete closure of the cytoplasmic half in our simulations. However, the closure is sufficient to prevent the disaccharide from transporting to the cytoplasm, which is in accordance with the well-established alternating access model. Ser53, Gln60, and Phe354 are determined to be important in sugar transport during the periplasmic-open stage of the sugar transport cycle and the sugar is found to undergo an orientational change in order to escape the protein lumen.

Dataset Information

Opening and closing of the periplasmic gate in lactose permease.

Publications

Opening and closing of the periplasmic gate in lactose permease.

Similar Datasets

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