Lactose Permease: Mechanism through Structures

Abstract

The lactose permease of Escherichia coli (LacY), a member of the major facilitator superfamily (MFS), catalyzes the symport of a galactopyranoside and an H+ across the membrane by a mechanism in which the sugar-binding site in the middle of the protein becomes alternately accessible to either side of the membrane through multiple conformational changes. However, initial X-ray structures showed LacY in an inward-facing conformation with a tightly sealed periplasmic side (1). Here utilizing a double-Trp mutant (LacYww) (2), we describe an almost occluded, outward-open conformation with bound sugars [β-D-galactopyranosyl-1-thio-β-D-galactopyranoside, TDG (3) or p-nitrophenyl-α-D-galactopyranoside, NPG (4)] and a complex of LacYww with a camelid nanobody (LacYww/Nb9047/NPG) (Unpublished data), which confirms many biochemical and biophysical findings. Structural data from the LacYww mutants clearly show how the side chains Glu269 (helix VIII) primarily, and Glu126 (helix IV), Trp151 (helix V), Arg144 (helix V), His322 (helix X), and Asn272 (helix VIII) provide specificity, while Phe20, Met23, and Phe27 (helix I) increase binding affinity for NPG nonspecifically. Analysis of the three structures clearly suggests that Glu325 is linked to the galactoside-binding site via a hydrogen bond with His322Nδ. The His322NεH is hydrogen donor to 3-OH of the galactopyranoside, because if Glu269 is negatively charged, the 3-OH in turn acts as hydrogen donor to Glu269. If the site returns to the occluded state without bound substrate, it must lose an H+, implying a drop in the effective pKa. It does so hypothetically through interaction of protonated Glu325 with Arg302 and water molecules and loses the H+ to the side that is closing. Although Glu325 and Arg302 are essential for protonation/deprotonation and His322 is essential for binding, all three may be required for the pKa change in Glu325 upon closing to the occluded, substrate-free conformation.