Alternating Access Mechanism in the Lactose Permease Derived from its Internal Symmetry

Abstract

The lactose permease (LacY) is the prototype of the major facilitator superfamily (MFS) of transporters, the largest evolutionarily-related collection of secondary transporters. The substrate transport mechanism of MFS proteins has long been postulated to involve alternating accessibility of the substrate binding site from either surface of the membrane. Existing structural data for LacY reveal a state in which the substrate is exposed to the cytoplasm, but occluded from the periplasm. To elucidate the conformational changes that open the permeation pathway, we examined the internal symmetry of the LacY structure. Our analysis revealed two sets of topologically-inverted repeats. Following ideas from our earlier studies on LeuT and GltPh, a model of the outward-facing conformation of LacY was built by exchanging the conformations of the repeats. The resulting model exhibits all required properties of an outward-facing conformation, namely closure of the binding site residues from the cytoplasm and exposure to the periplasm. Furthermore, the model agrees with a large amount of experimental data. Analysis of differences between the two states suggests a role for conserved sequence motifs in aiding the occlusion of the central pathway. In addition, interactions between pore-lining helices may be conducted to the full conformational change by movements of peripheral helices. Finally, predicted re-pairing of critical salt-bridging residues in the binding sites agree with previous proposals. Thus, we have generated a reliable atomistic model of the outward-facing state of LacY, using only its internal symmetry, and providing a model for the conformational change of all MFS transporters.