Crystal structure of a LacY-nanobody complex in a periplasmic-open conformation. Author Xin Jiang, Irina Smirnova, Vladimir Kasho, Jianping Wu, Kunio Hirata, Meng Ke, Els Pardon, Jan Steyaert, Nieng Yan, H Ronald Kaback Publication Year 2016 Type Journal Article 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. Keywords Binding Sites, Protein Binding, Mutation, Models, Molecular, Crystallography, X-Ray, Protein Conformation, Escherichia coli Proteins, Periplasm, Symporters, Monosaccharide Transport Proteins, Single-Domain Antibodies Journal Proc Natl Acad Sci U S A Volume 113 Issue 44 Pages 12420-12425 Date Published 2016 Nov 01 ISSN Number 1091-6490 DOI 10.1073/pnas.1615414113 Alternate Journal Proc Natl Acad Sci U S A PMCID PMC5098631 PMID 27791182 PubMedPubMed CentralGoogle ScholarBibTeXEndNote X3 XML