Structure of LacY with an α-substituted galactoside: Connecting the binding site to the protonation site

Abstract

The X-ray crystal structure of a conformationally constrained mutant of the Escherichia coli lactose permease (the LacY double-Trp mutant Gly-46→Trp/Gly-262→Trp) with bound p-nitrophenyl-α-D-galactopyranoside (α-NPG), a high-affinity lactose analog, is described. With the exception of Glu-126 (helix IV), side chains Trp-151 (helix V), Glu-269 (helix VIII), Arg-144 (helix V), His-322 (helix X), and Asn-272 (helix VIII) interact directly with the galactopyranosyl ring of α-NPG to provide specificity, as indicated by biochemical studies and shown directly by X-ray crystallography. In contrast, are within van der Waals distance of the benzyl moiety of the analog and thereby increase binding affinity nonspecifically. Thus, the specificity of LacY for sugar is determined solely by side-chain interactions with the galactopyranosyl ring, whereas affinity is increased by nonspecific hydrophobic interactions with the anomeric substituent.X-ray structure | membrane protein | transport | major facilitator superfamily | conformational change T he lactose permease of Escherichia coli (LacY) binds and catalyzes the coupled stoichiometric transport of D-galactose or β-D-galactopyranosides and H + (galactoside/H + symport) but does not interact with glucopyranosides. Biochemical studies (1-7) indicate that affinity and specificity are distinct properties determined by different interactions with LacY. Specificity is determined entirely by interactions with the galactopyranosyl ring, whereas affinity is better with α-than β-galactopyranosides (anomeric at C1) and can be increased dramatically by hydrophobic anomeric substituents with no effect on specificity.By using the free energy released from the energetically downhill movement of H + in response to the electrochemical H + gradient (ΔμH+), LacY catalyzes uphill (active) transport of galactosides against a concentration gradient. Because coupling between sugar and H + translocation is obligatory, in the absence of ΔμH+, LacY can also transduce the free energy released from the downhill transport of sugar to drive uphill H + transport with the generation of ΔμH+, the polarity of which depends upon the direction of the sugar gradient (reviewed in refs. 8-10).Rates of equilibrium exchange and counterflow (exchange of one substrate molecule for another labeled molecule from the other side of the membrane) are unaffected by imposition of Δμ̃H+. Therefore, it is apparent that alternating accessibility of sugar-and H + -binding sites to either side of the membrane is the result of galactoside binding and dissociation and not ΔμH+ (reviewed in refs. 8-10). Moreover, downhill lactose/H + symport from a high to a low lactose concentration in the absence of ΔμH+ exhibits a primary deuterium isotope effect that is not observed for ΔμH+-driven lactose/H + symport, equilibrium exchange, or counterflow (11, 12). Thus, it is likely that the rate-limiting step for downhill symport is deprotonation (13,14), whereas in the presence of ΔμH+, opening of a cavity on the other side of the membrane ...

Crystal structure of a LacY–nanobody complex in a periplasmic-open conformation

“…Data collection and refinement statistics are summarized in Table S1. Crystals of the apo LacY ww -Nb complex are in space group P4 1 The structure is in an outward (periplasmic)-facing conformation with a tightly sealed cytoplasmic side that is very similar to two previous LacY ww structures with bound substrates (18,19). Thus, the apo LacY ww -Nb structure can be superimposed on the LacY ww -TDG structure (PDB ID code 4OAA) with an rmsd of 0.6 Å and on the LacY ww -NPG structure (PDB ID code 4ZYR) with an rmsd of 0.5 Å.…”

“…The densities for the side chains forming the sugar-binding site are well-defined. Thus, close inspection of the residues directly involved in binding the sugar indicates that they are positioned almost identically in the apo LacY ww -Nb structure presented here and the structures of LacY ww with bound substrate (PDB ID codes 4OAA and 4ZYR) 18,19). The side chains involved in binding either TDG (Fig.…”

“…2A). Changes in the current structure relative to that of LacY ww with bound sugar (18,19) involve the area of a kink in transmembrane helix 1 (TM1) around Phe27. In contrast, organization of the central cavity, which contains the substrate-binding site, is virtually identical in the apo LacY ww -Nb complex and LacY ww -TDG structures ( Fig.…”

“…More recently, a stable outward-open, nontransporting mutant was engineered with Trp replacements for two periplasmic Gly residues in the N-and C-terminal halves of LacY (17). X-ray structures of the doubleTrp mutant G46W/G262W (LacY ww ) with either of two bound lactose homologs, 4-nitrophenyl-α-D-galactopyranoside (NPG) or β-D-galactopyranosyl-1-thio-β-D-galactopyranoside (TDG), display a conformation with a narrowly open periplasmic pathway and a tightly sealed cytoplasmic side (18,19). These structures confirm the location of the sugar-binding site in the middle of the molecule, define the side chains that determine specificity for galactopyranosides, and indicate that an occluded conformation(s) is an intermediate in the transport cycle.…”

Understanding phospholipid function: Why are there so many lipids?

“…Ron eventually established that an electrochemical proton gradient was the driving force for lactose accumulation, which was the culmination of what Ron termed the "Chemiosmotic Wars." Ron of course went on to establish the detailed molecular structure and mechanism of LacY (14,15). It was during these Gordon Conferences debates that I got to know Ron (Fig.…”