Flexible Gates Generate Occluded Intermediates in the Transport Cycle of LacY

The major facilitator superfamily (MFS) transporter lactose permease (LacY) alternates between cytoplasmic and periplasmic open conformations to co-transport a sugar molecule together with a proton across the plasma membrane. Indirect experimental evidence suggested the existence of an occluded tran...

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Bibliographic Details
Published in:Journal of molecular biology 2014-02, Vol.426 (3), p.735-751
Main Authors: Stelzl, Lukas S., Fowler, Philip W., Sansom, Mark S.P., Beckstein, Oliver
Format: Article
Language:English
Subjects:
Cell Membrane - metabolism
Crystallography, X-Ray
major facilitator superfamily transporters
membrane permeation
Membrane Transport Proteins - chemistry
Membrane Transport Proteins - metabolism
Models, Molecular
Molecular Dynamics Simulation
molecular dynamics simulations
Protein Conformation
protein conformational change
Protein Transport
protons
Symporters - metabolism
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Summary:The major facilitator superfamily (MFS) transporter lactose permease (LacY) alternates between cytoplasmic and periplasmic open conformations to co-transport a sugar molecule together with a proton across the plasma membrane. Indirect experimental evidence suggested the existence of an occluded transition intermediate of LacY, which would prevent leaking of the proton gradient. As no experimental structure is known, the conformational transition is not fully understood in atomic detail. We simulated transition events from a cytoplasmic open conformation to a periplasmic open conformation with the dynamic importance sampling molecular dynamics method and observed occluded intermediates. Analysis of water permeation pathways and the electrostatic free-energy landscape of a solvated proton indicated that the occluded state contains a solvated central cavity inaccessible from either side of the membrane. We propose a pair of geometric order parameters that capture the state of the pathway through the MFS transporters as shown by a survey of available crystal structures and models. We present a model for the occluded state of apo-LacY, which is similar to the occluded crystal structures of the MFS transporters EmrD, PepTSo, NarU, PiPT and XylE. Our simulations are consistent with experimental double electron spin–spin distance measurements that have been interpreted to show occluded conformations. During the simulations, a salt bridge that has been postulated to be involved in driving the conformational transition formed. Our results argue against a simple rigid-body domain motion as implied by a strict “rocker-switch mechanism” and instead hint at an intricate coupling between two flexible gates. [Display omitted] •The transport mechanism of LacY is hypothesized to involve an intermediate “occluded” state.•Such a state is observed in computer simulations of the conformational transitions.•Simulation data are validated with experimental double electron–electron spin resonance measurements.•The structural gating elements of LacY are identified.•Occluded LacY is similar to known occluded structures of homologous proteins.
ISSN:0022-2836
1089-8638
DOI:10.1016/j.jmb.2013.10.024