Abstract
ABC importers are membrane proteins responsible for the transport of nutrients into the cells of prokaryotes. Although the structures of ABC importers vary, all contain four conserved domains: two nucleotide-binding domains (NBDs), which bind and hydrolyze ATP, and two transmembrane domains (TMDs), which help translocate the substrate. ABC importers are also dependent on an additional protein component, a high-affinity substrate-binding protein (SBP) that specifically binds the target ligand for delivery to the appropriate ABC transporter. AbnE is a SBP belonging to the ABC importer for arabino-oligosaccharides in the Gram-positive thermophilic bacterium Geobacillus stearothermophilus. Using isothermal titration calorimetry (ITC), purified AbnE was shown to bind medium-sized arabino-oligosaccharides, in the range of arabino-triose (A3) to arabino-octaose (A8), all with Kd values in the nanomolar range. We describe herein the 3D structure of AbnE in its closed conformation in complex with a wide range of arabino-oligosaccharide substrates (A2-A8). These structures provide the basis for the detailed structural analysis of the AbnE-sugar complexes, and together with complementary quantum chemical calculations, site-specific mutagenesis, and isothermal titration calorimetry (ITC) experiments, provide detailed insights into the AbnE-substrate interactions involved. Small-angle X-ray scattering (SAXS) experiments and normal mode analysis (NMA) are used to study the conformational changes of AbnE, and these data, taken together, suggest clues regarding its binding mode to the full ABC importer.
| Original language | American English |
|---|---|
| Pages (from-to) | 2099-2120 |
| Number of pages | 22 |
| Journal | Journal of Molecular Biology |
| Volume | 432 |
| Issue number | 7 |
| DOIs | |
| State | Published - 27 Mar 2020 |
Bibliographical note
Funding Information:This work was supported by the Israel Science Foundation Grants 500/10 , 152/11 and 1505/15 , the Israeli Ministry of Science and Technology (MOST) Grant 3-12484 , the I-CORE Program of the Planning and Budgeting Committee, the Israeli Ministry of Environmental Protection , the Israeli Ministry of Science , and the Grand Technion Energy Program (GTEP) , which comprises part of The Leona M. and Harry B. Helmsley Charitable Trust reports on Alternative Energy series of the Technion, Israel Institute of Technology , and the Weizmann Institute of Science . The research also received funding from the European Community’s Seventh Framework Programme ( FP7/2007–2013 ) under BioStruct-X (grant agreement N° 283570 ). Y.S. acknowledges partial support by the Russell Berrie Nanotechnology Institute and The Lorry I. Lokey Interdisciplinary Center for Life Science and Engineering, Technion . I.S. gratefully acknowledges partial funding by European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (grant agreement No 678169 "PhotoMutant"). S.L. is grateful to the Azrieli Foundation for the award of an Azrieli Fellowship. We thank the staff of the Technion Center for Structural Biology (TCSB) for their helpful support in the crystallization and X-ray data collection. We also thank the staff at the European Synchrotron Research Facility (ESRF; BM14, BM30A, ID30A-3 beamlines) for their helpful support in the X-ray synchrotron data measurement and analysis. The synchrotron experiments at ESRF were supported also by the ESRF internal funding program. Dr. Hay Dvir is acknowledged for his helpful assistance in the crystal freezing procedures of AbnE-A7. Y.S. holds the Erwin and Rosl Pollak Chair in Biotechnology at the Technion.
Funding Information:
This work was supported by the Israel Science Foundation Grants 500/10, 152/11 and 1505/15, the Israeli Ministry of Science and Technology (MOST) Grant 3-12484, the I-CORE Program of the Planning and Budgeting Committee, the Israeli Ministry of Environmental Protection, the Israeli Ministry of Science, and the Grand Technion Energy Program (GTEP), which comprises part of The Leona M. and Harry B. Helmsley Charitable Trust reports on Alternative Energy series of the Technion, Israel Institute of Technology, and the Weizmann Institute of Science. The research also received funding from the European Community's Seventh Framework Programme (FP7/2007?2013) under BioStruct-X (grant agreement N?283570). Y.S. acknowledges partial support by the Russell Berrie Nanotechnology Institute and The Lorry I. Lokey Interdisciplinary Center for Life Science and Engineering, Technion. I.S. gratefully acknowledges partial funding by European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (grant agreement No 678169 ?PhotoMutant?). S.L. is grateful to the Azrieli Foundation for the award of an Azrieli Fellowship. We thank the staff of the Technion Center for Structural Biology (TCSB) for their helpful support in the crystallization and X-ray data collection. We also thank the staff at the European Synchrotron Research Facility (ESRF; BM14, BM30A, ID30A-3 beamlines) for their helpful support in the X-ray synchrotron data measurement and analysis. The synchrotron experiments at ESRF were supported also by the ESRF internal funding program. Dr. Hay Dvir is acknowledged for his helpful assistance in the crystal freezing procedures of AbnE-A7. Y.S. holds the Erwin and Rosl Pollak Chair in Biotechnology at the Technion.
Publisher Copyright:
© 2020
Keywords
- ABC transporter
- Arabinan utilization system
- Geobacillus stearothermophilus
- arabinose
- substrate-binding protein