An Asymmetric Conformational Change in LacY

Abstract

Galactoside/H+ symport by the lactose permease of Escherichia coli (LacY) involves reciprocal opening and closing of periplasmic and cytoplasmic cavities so that sugar- and H+-binding sites become alternatively accessible to either side of the membrane. After reconstitution into proteoliposomes, LacY with the periplasmic cavity sealed by cross-linking paired-Cys residues does not bind sugar from the periplasmic side. However, reduction of the S–S bond restores opening of the periplasmic cavity and galactoside binding. Furthermore, nanobodies that stabilize the double-Cys mutant in a periplasmic-open conformation and allow free access of galactoside to the binding site do so only after reduction of the S–S bond. In contrast, when cross-linked LacY is solubilized in detergent, galactoside binding is observed, indicating that the cytoplasmic cavity is patent. Sugar binding from the cytoplasmic side exhibits nonlinear stopped-flow kinetics, and analysis reveals a two-step process in which a conformational change precedes binding. Because the cytoplasmic cavity is spontaneously closing and opening in the symporter with a sealed periplasmic cavity, it is apparent that an asymmetrical conformational transition controls access of sugar to the binding site.

Engineered occluded apo-intermediate of LacY

“…2, trace 4, black lines). Notably, with WT LacY, the effect of Nb9065 is not altered by Cu 2+ (21). Thus, after oxidation of either paired-Cys mutant, the periplasmic cavity on the opposite face of LacY is not open, the epitope for Nb9065 on the periplasmic surface is not exposed, and the sugar-binding site in the middle of LacY is inaccessible.…”

“…Therefore, both possibilities were tested by either (i) using a conformation-sensitive Nb or (ii) cross-linking the double-Cys mutant containing bound sugar, and measuring the dissociation rate constant (k off ) in displacement experiments. Conformational mobility of the double-Cys mutants was examined with Nb9065, which recognizes the binding epitope in LacY with an open periplasmic cavity and stabilizes WT LacY in an outward-open conformation (18,19,21). Stopped-flow rates of NPG binding, measured with mutants 134C/343C and 135C/ 338C in the presence of TCEP are greatly increased by preincubation of mutants with Nb9065 (Fig.…”

“…LacY with a locked periplasmic cavity (by cross-linking I32C-N245C or H35C-I245C Cys-pairs) binds galactosides exclusively via the cytoplasmic opening, and presteady-state kinetic analysis reveals a two-step process in which opening of the cytoplasmic cavity occurs first followed by galactoside binding due to a conformational selection mechanism (21). The results indicate clearly that the cytoplasmic cavity opens and closes spontaneously in protein with a sealed periplasmic side, and the rate of opening is slower than the rate of closing.…”

“…Kinetic parameters for galactoside binding have been determined with lactose analogs p-nitrophenyl-α-D-galactopyranoside (NPG), an acceptor for FRET from Trp151 (helix V) in the sugar-binding site, and β-D-galactopyranosyl-1-thio-β-D-galactopyranoside (TDG), which does not participate in FRET (22). The data show clearly that the rate of cavity opening limits sugar-binding rates on either side of WT LacY, and that sugar has free access to the binding site when the outward-or inward-facing cavity is open (15,21). Both outward-and inward-open conformations are characterized by a linear concentration dependence of galactoside-binding rates with high association rate constants (k on = 5 to 10 μM −1 s −1 ).…”

“…The turnover number of the overall LacY transport cycle is relatively low (∼20 s −1 ) (20), and a limiting step corresponds to the rate of opening of the periplasmic cavity (15,16), but some conformational transitions are fast (200-300 s −1 ), as measured by fluorescence quenching (16,21). Kinetic parameters for galactoside binding have been determined with lactose analogs p-nitrophenyl-α-D-galactopyranoside (NPG), an acceptor for FRET from Trp151 (helix V) in the sugar-binding site, and β-D-galactopyranosyl-1-thio-β-D-galactopyranoside (TDG), which does not participate in FRET (22).…”