title: Outward-facing conformers of LacY stabilized by nanobodies.
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).
authors.affiliation: Department of Physiology.
authors.forename: Irina
authors.initials: I
authors.lastname: Smirnova
authors.affiliation: Department of Physiology.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Kasho
authors.affiliation: Department of Physiology.
authors.forename: Xiaoxu
authors.initials: X
authors.lastname: Jiang
authors.affiliation: Structural Biology Research Center, VIB, Pleinlaan 2, 1050 Brussels, Belgium; and Structural Biology Brussels, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussel, Belgium.
authors.forename: Els
authors.initials: E
authors.lastname: Pardon
authors.affiliation: Structural Biology Research Center, VIB, Pleinlaan 2, 1050 Brussels, Belgium; and Structural Biology Brussels, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussel, Belgium jan.steyaert@vub.ac.be rkaback@mednet.ucla.edu.
authors.forename: Jan
authors.initials: J
authors.lastname: Steyaert
authors.affiliation: Department of Physiology, Department of Microbiology, Immunology, and Molecular Genetics, and Molecular Biology Institute, University of California, Los Angeles, CA 90095-7327; jan.steyaert@vub.ac.be rkaback@mednet.ucla.edu.
authors.forename: H Ronald
authors.initials: HR
authors.lastname: Kaback
mesh_terms: D001693:Biological Transport, Active / Q000502:physiology*; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007785:Lactose / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009004:Monosaccharide Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D046912:Multiprotein Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D019897:Periplasm / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D055550:Protein Stability; D027981:Symporters / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D009004:Monosaccharide Transport Proteins; D046912:Multiprotein Complexes; D027981:Symporters; D007785:Lactose
keywords: fluorescence; major facilitator superfamily; membrane transport proteins