Abstract
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.
Highlights
Lactose permease (LacY) is the best-studied member of the major facilitator superfamily (MFS) of secondary transporters [1]
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
Summary
Lactose permease (LacY) is the best-studied member of the major facilitator superfamily (MFS) of secondary transporters [1]. The water density for the simulation of cytoplasmic open LacY (Fig. 3d and Fig. S4a and b) indicated that the constriction between the cytoplasm and the central cavity remained partially solvated even after the initial closure of the cytoplasmic gate, indicating that protons could still be expected to permeate and the conformation cannot be considered a functionally occluded state.
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