Crystal Structure of a ligand-bound LacY–Nanobody Complex
| Autor: | Irina Smirnova, H. Ronald Kaback, Xiaoxu Jiang, Els Pardon, Robert M. Stroud, Jan Steyaert, Vladimir N. Kasho, Janet Finer-Moore, Hemant Kumar |
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| Přispěvatelé: | Department of Bio-engineering Sciences, Structural Biology Brussels |
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Lactose permease Single-Domain Antibodies/chemistry Conformational change Monosaccharide Transport Proteins Escherichia coli/chemistry Substrate analog Crystallography X-Ray 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Protein structure Symporters/chemistry Escherichia coli Major facilitator superfamily Protein Structure Quaternary Multidisciplinary Symporters Chemistry Escherichia coli Proteins Monosaccharide Transport Proteins/chemistry Periplasmic space Biological Sciences Single-Domain Antibodies Ligand (biochemistry) carbohydrates (lipids) Transmembrane domain Crystallography 030104 developmental biology general Membrane protein Escherichia coli Proteins/chemistry 030220 oncology & carcinogenesis transport bacteria X-ray structure mutation |
| Zdroj: | Proceedings of the National Academy of Sciences. 115:8769-8774 |
| ISSN: | 1091-6490 0027-8424 |
| DOI: | 10.1073/pnas.1801774115 |
| Popis: | 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 (LacY(WW)) 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 LacY(WW), 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 LacY(WW), which implicates dynamic flexibility of the Nb–LacY(WW) complex. Nb9047-binding neither changes the overall structure of LacY(WW) 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-LacY(WW)/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. |
| Databáze: | OpenAIRE |
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