Transient conformers of LacY are trapped by nanobodies.

TitleTransient conformers of LacY are trapped by nanobodies.
Publication TypeJournal Article
Year of Publication2015
AuthorsSmirnova, I., Kasho V., Jiang X., Pardon E., Steyaert J., and H Kaback R.
JournalProc Natl Acad Sci U S A
Volume112
Issue45
Pagination13839-44
Date Published2015 Nov 10
ISSN1091-6490
KeywordsEscherichia coli Proteins, Monosaccharide Transport Proteins, Protein Conformation, Single-Domain Antibodies, Symporters
Abstract

The lactose permease of Escherichia coli (LacY), a highly dynamic membrane protein, catalyzes symport of a galactopyranoside and an H(+) by using an alternating access mechanism, and the transport cycle involves multiple conformational states. Single-domain camelid nanobodies (Nbs) developed against a LacY mutant immobilized in an outward (periplasmic)-open conformation bind to the flexible WT protein and stabilize the open-outward conformation(s). Here, we use site-directed, distance-dependent Trp quenching/unquenching of fluorescent probes inserted on opposite surfaces of LacY to assess the conformational states of the protein complexed with each of eight unique Nbs that bind exclusively to the periplasmic side and block transport, but increase the accessibility of the sugar-binding site. Nb binding involves conformational selection of LacY molecules with exposed binding epitopes. Each of eight Nbs induces quenching with three pairs of cytoplasmic Trp/fluorophore probes, indicating closing of cytoplasmic cavity. In reciprocal fashion, the same Nbs induce unquenching of fluorescence in three pairs of periplasmic probes due to opening of the periplasmic cavity. Because the extent of fluorescence change with various Nbs differs and the differences correlate with changes in the rate of sugar binding, it is also concluded that the Nbs stabilize several different outward-open conformations of LacY.

DOI10.1073/pnas.1519485112
Alternate JournalProc. Natl. Acad. Sci. U.S.A.
PubMed ID26512108
PubMed Central IDPMC4653160
Grant ListR01 DK051131 / DK / NIDDK NIH HHS / United States
R56 DK051131 / DK / NIDDK NIH HHS / United States
DK51131 / DK / NIDDK NIH HHS / United States
Research group: