Crystal Structure of a ligand-bound LacY–Nanobody Complex

• Published in: Proceedings of the National Academy of Sciences - PNAS 2018-08, Vol.115 (35), p.8769-8774
• Main Authors: Kumar, Hemant, Finer-Moore, Janet S., Jiang, Xiaoxu, Smirnova, Irina, Kasho, Vladimir, Pardon, Els, Steyaert, Jan, Kaback, H. Ronald, Stroud, Robert M.
• Format: Article
• Language: English
• Subjects: Bacteria; Binding sites; Biological Sciences; Coordination compounds; Crystal structure; Crystallography, X-Ray; E coli; Escherichia coli - chemistry; Escherichia coli - genetics; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - genetics; Helices; Lactose; Lactose permease; Ligands; Membrane proteins; Membranes; Monosaccharide Transport Proteins - chemistry; Monosaccharide Transport Proteins - genetics; Mutation; Nanobodies; Permease; Protein Structure, Quaternary; Protein transport; Proteins; Single-Domain Antibodies - chemistry; Substrates; Sugar; Symport; Symporters - chemistry; Symporters - genetics; Vestibules
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1801774115

The lactose permease of Escherichia coli (LacY), a dynamic polytopic membrane transport 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. Camelid nanobodies were made against a double-mutant Gly46 → Trp/Gly262 → Trp (LacYWW) that produces an outward-open conformation, as opposed to the cytoplasmic open-state crystal structure of WT LacY. Nanobody 9047 (Nb9047) stabilizes WT LacY in a periplasmic-open conformation. Here, we describe the X-ray crystal structure of a complex between LacYWW, the high-affinity substrate analog 4-nitrophenyl-α-D-galactoside (NPG), and Nb9047 at 3-Å resolution. The present crystal structure demonstrates that Nb9047 binds to the periplasmic face of LacY, primarily to the C-terminal six-helical bundle, while a flexible loop of the Nb forms a bridge between the N- and C-terminal halves of LacY across the periplasmic vestibule. The bound Nb partially covers the vestibule, yet does not affect the on-rates or off-rates for the substrate binding to LacYWW, which implicates dynamic flexibility of the Nb–LacYWW complex. Nb9047-binding neither changes the overall structure of LacYWW with bound NPG, nor the positions of side chains comprising the galactoside-binding site. The current NPG-bound structure exhibits a more occluded periplasmic vestibule than seen in a previous structure of a (different Nb) apo-LacYWW/Nb9039 complex that we argue is caused by sugar-binding, with major differences located at the periplasmic ends of transmembrane helices in the N-terminal half of LacY.