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

Abstract

The lactose permease of Escherichia coli (LacY), a dynamic polytopic membrane protein, catalyzes galactoside-H + symport and operates by an alternating access mechanism that exhibits multiple conformations, the distribution of which is altered by sugar binding. We have developed single-domain camelid nanobodies (Nbs) against a mutant in an outward (periplasmic)-open conformation to stabilize this state of the protein. Here we describe an X-ray crystal structure of a complex between a double-Trp mutant (Gly46→Trp/Gly262→Trp) and an Nb in which free access to the sugar-binding site from the periplasmic cavity is observed. The structure confirms biochemical data indicating that the Nb binds stoichiometrically with nanomolar affinity to the periplasmic face of LacY primarily to the C-terminal six-helix bundle. The structure is novel because the pathway to the sugar-binding site is constricted and the central cavity containing the galactoside-binding site is empty. Although Phe27 narrows the periplasmic cavity, sugar is freely accessible to the binding site. Remarkably, the side chains directly involved in binding galactosides remain in the same position in the absence or presence of bound sugar.X-ray structure | bioenergetics | membrane transporter | fluorescence | kinetics T he lactose permease of Escherichia coli (LacY) catalyzes the coupled transport of a galactopyranoside and an H + (galactoside-H + symport) across the cytoplasmic membrane (reviewed in refs. 1-3). Therefore, in the presence of an H + electrochemical gradient (ΔμH+; interior negative and/or alkaline), galactopyranosides can be concentrated 50-to 100-fold over the external concentration (4, 5). However, due to the activity of β-galactosidase, no free lactose is found within the cell under physiological conditions. So why does E. coli need an active transport system for lactose? The answer lies in the kinetics, as the major effect of ΔμH+ is to decrease the K m for transport (6, 7). By increasing the kinetic efficiency of LacY, ΔμH+ enables the cell to assimilate lactose effectively even when the environmental concentration is low.Initial X-ray crystal structures of conformationally restricted LacY (8-10), as well as WT LacY (11) + and is the poster child for the major facilitator superfamily, the largest family of membrane transport proteins. A detailed mechanism has been postulated involving alternating access of sugar-and H + -binding sites to either side of the membrane that is driven by sugar binding and dissociation and independent of the H + electrochemical gradient, which acts kinetically. To characterize structural intermediates in the transport cycle, stable conformers are essential, and camelid single-domain nanobodies (Nbs) are particularly useful in this context. Described herein is a structure of a LacY-Nb complex in which access to the sugar-binding site from the periplasmic cavity is diffusion-limited.Author contributions: X.J., I.S., V.K., N.Y., and H.R.K. designed research; X.J., I.S., V.K., J.W., K.H., and M.K. performed research...

pK a of Glu325 in LacY

“…LacY is composed of N-and C-terminal domains, each with six mostly irregular transmembrane helices linked by a relatively long cytoplasmic loop with the N and C termini on the cytoplasmic face of the membrane. Structures of two conformations of LacY have been solved: (i) an inward-open conformer with a large aqueous cavity open to the cytoplasmic side and a tightly sealed periplasmic side (3-6); and (ii) an outward-open, occluded conformer with a tightly sealed cytoplasmic side and a bound lactose homolog (7,8) or a nanobody (9). As extensively documented (10), LacY operates by an alternating access mechanism.…”

An Asymmetric Conformational Change in LacY

“…In addition, a number of camelid Nbs developed against the periplasmic-open mutant G46W/G262W LacY (Figure 1C) bind to the periplasmic aspect of WT LacY and stabilize outward-facing conformations with dramatically increased k on values indicating a highly accessible galactoside-binding site. 10,16,23 …”

Membrane transport proteins in melanosomes: Regulation of ions for pigmentation

“…5GXB) as a template (Fig. 4) [92]. The model contains 12 transmembrane domains (TM) with the conserved structural fold of the major facilitator superfamily (MFS).…”