Outward-facing conformers of LacY stabilized by nanobodies.

TitleOutward-facing conformers of LacY stabilized by nanobodies.
Publication TypeJournal Article
Year of Publication2014
AuthorsSmirnova, I., V. Kasho, X. Jiang, E. Pardon, J. Steyaert, and R. H Kaback
JournalProc Natl Acad Sci U S A
Volume111
Issue52
Pagination18548-53
Date Published2014 Dec 30
ISSN1091-6490
KeywordsBiological Transport, Active, Escherichia coli, Escherichia coli Proteins, Lactose, Monosaccharide Transport Proteins, Multiprotein Complexes, Mutation, Periplasm, Protein Binding, Protein Stability, Symporters
Abstract

The lactose permease of Escherichia coli (LacY), a highly dynamic polytopic membrane protein, catalyzes stoichiometric galactoside/H(+) symport by an alternating access mechanism and exhibits multiple conformations, the distribution of which is altered by sugar binding. We have developed single-domain camelid nanobodies (Nbs) against a LacY mutant in an outward (periplasmic)-open conformation to stabilize this state of the WT protein. Twelve purified Nbs inhibit lactose transport in right-side-out membrane vesicles, indicating that the Nbs recognize epitopes on the periplasmic side of LacY. Stopped-flow kinetics of sugar binding by WT LacY in detergent micelles or reconstituted into proteoliposomes reveals dramatic increases in galactoside-binding rates induced by interaction with the Nbs. Thus, WT LacY in complex with the great majority of the Nbs exhibits varied increases in access of sugar to the binding site with an increase in association rate constants (kon) of up to ∼ 50-fold (reaching 10(7) M(-1) ⋅ s(-1)). In contrast, with the double-Trp mutant, which is already open on the periplasmic side, the Nbs have little effect. The findings are clearly consistent with stabilization of WT conformers with an open periplasmic cavity. Remarkably, some Nbs drastically decrease the rate of dissociation of bound sugar leading to increased affinity (greater than 200-fold for lactose).

DOI10.1073/pnas.1422265112
Alternate JournalProc. Natl. Acad. Sci. U.S.A.
PubMed ID25512549
PubMed Central IDPMC4284591
Grant ListR56 DK051131 / DK / NIDDK NIH HHS / United States
GM073210 / GM / NIGMS NIH HHS / United States
DK069463 / DK / NIDDK NIH HHS / United States
DK51131 / DK / NIDDK NIH HHS / United States
R01 DK051131 / DK / NIDDK NIH HHS / United States
P50 GM073210 / GM / NIGMS NIH HHS / United States
R56 DK069463 / DK / NIDDK NIH HHS / United States
R01 DK069463 / DK / NIDDK NIH HHS / United States
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