title: Transient conformers of LacY are trapped by nanobodies.
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.
authors.affiliation: Department of Physiology, University of California, Los Angeles, CA 90095-7327;
authors.forename: Irina
authors.initials: I
authors.lastname: Smirnova
authors.affiliation: Department of Physiology, University of California, Los Angeles, CA 90095-7327;
authors.forename: Vladimir
authors.initials: V
authors.lastname: Kasho
authors.affiliation: Department of Physiology, University of California, Los Angeles, CA 90095-7327;
authors.forename: Xiaoxu
authors.initials: X
authors.lastname: Jiang
authors.affiliation: Structural Biology Research Center; VIB, 1050 Brussel, Belgium, Vrije Universiteit Brussel, 1050 Brussel, Belgium;
authors.forename: Els
authors.initials: E
authors.lastname: Pardon
authors.affiliation: Structural Biology Research Center, VIB, 1050 Brussel, Belgium,  Belgium; Structural Biology Brussels, Vrije Universiteit Brussel, 1050 Brussel, Belgium; rkaback@mednet.ucla.edu jan.steyaert@vub.ac.be.
authors.forename: Jan
authors.initials: J
authors.lastname: Steyaert
authors.affiliation: Department of Physiology, University of California, Los Angeles, CA 90095-7327; Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA 90095-7327; Molecular Biology Institute, University of California, Los Angeles, CA 90095-7327 rkaback@mednet.ucla.edu jan.steyaert@vub.ac.be.
authors.forename: H Ronald
authors.initials: HR
authors.lastname: Kaback
mesh_terms: D029968:Escherichia coli Proteins / Q000737:chemistry*; D009004:Monosaccharide Transport Proteins / Q000737:chemistry*; D011487:Protein Conformation; D061905:Single-Domain Antibodies / Q000737:chemistry*; D027981:Symporters / Q000737:chemistry*
chemical_list: D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D009004:Monosaccharide Transport Proteins; D061905:Single-Domain Antibodies; D027981:Symporters
keywords: alternating access; conformers; lactose permease; membrane transport proteins; nanobodies