Dataset Information

Sugar binding and protein conformational changes in lactose permease.

ABSTRACT: Lactose permease is an integral membrane protein that uses the cell membrane's proton gradient for import of lactose. Based on extensive biochemical data and a substrate-bound crystal structure, intermediates involved in lactose/H(+) co-transport have been suggested. Yet, the transport mechanism, especially the coupling of protonation states of essential residues and protein conformational changes involved in the transport, is not understood. Here we report molecular-dynamics simulations of membrane-embedded lactose permease in different protonation states, both in the presence and in the absence of lactose. The results analyzed in terms of pore diameter, salt-bridge formation, and substrate motion, strongly implicate Glu(269) as one of the main proton translocation sites, whose protonation state controls several key steps of the transport process. A critical ion pair (Glu(269) and Arg(144)) was found to keep the cytoplasmic entrance open, but via a different mechanism than the currently accepted model. After protonation of Glu(269), the salt bridge between Glu(269) and Arg(144) was found to break, and Arg(144) to move away from Glu(269), establishing a new salt bridge with Glu(126); furthermore, neutralization of Glu(269) and the displacement of Arg(144) and consequently of water molecules from the interdomain region was seen to initiate the closing of the cytoplasmic half channel (2.6-4.0 A reduction in diameter in the cytoplasmic constriction region in 10 ns) by allowing hydrophobic surfaces of the N- and C-domains to fuse. Charged Glu(269) was found to strongly bind the lactose permeant, indicating that proton transfer from water or another residue to Glu(269) is a prerequisite for unbinding of lactose from the binding pocket.

SUBMITTER: Yin Y

PROVIDER: S-EPMC1635680 | biostudies-literature | 2006 Dec

REPOSITORIES: biostudies-literature

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Sugar binding and protein conformational changes in lactose permease.

Yin Ying Y Jensen Morten Ø MØ Tajkhorshid Emad E Schulten Klaus K

Biophysical journal 20060908 11

Lactose permease is an integral membrane protein that uses the cell membrane's proton gradient for import of lactose. Based on extensive biochemical data and a substrate-bound crystal structure, intermediates involved in lactose/H(+) co-transport have been suggested. Yet, the transport mechanism, especially the coupling of protonation states of essential residues and protein conformational changes involved in the transport, is not understood. Here we report molecular-dynamics simulations of memb ...[more]

PMID: 16963502

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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

Sugar binding and protein conformational changes in lactose permease.

Publications

Sugar binding and protein conformational changes in lactose permease.

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