The extracellular gate shapes the energy profile of an ABC exporter
| Autor: | Pascal Egloff, Markus A. Seeger, Saša Štefanić, Lea M. Hürlimann, Cedric A. J. Hutter, Enrica Bordignon, Hendrik Göddeke, Mikko Karttunen, Lars V. Schäfer, Iwan Zimmermann, M. Hadi Timachi, Svetlana Kucher |
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| Přispěvatelé: | University of Zurich, Seeger, Markus A |
| Jazyk: | angličtina |
| Rok vydání: | 2019 |
| Předmět: |
10078 Institute of Parasitology
0301 basic medicine AAA Domain Dimer General Physics and Astronomy 02 engineering and technology Biochemistry chemistry.chemical_compound 0302 clinical medicine ATP hydrolysis 600 Technology lcsh:Science 0303 health sciences Multidisciplinary biology 10179 Institute of Medical Microbiology 021001 nanoscience & nanotechnology 3100 General Physics and Astronomy Energy profile Membrane Structural biology 0210 nano-technology Science 610 Medicine & health 1600 General Chemistry Molecular Dynamics Simulation Article General Biochemistry Genetics and Molecular Biology 03 medical and health sciences Bacterial Proteins 1300 General Biochemistry Genetics and Molecular Biology Extracellular Atpase activity Thermotoga maritima 030304 developmental biology Electron Spin Resonance Spectroscopy Transporter General Chemistry Single-Domain Antibodies biology.organism_classification Single-domain antibody 030104 developmental biology Membrane protein chemistry Mutation Biophysics 570 Life sciences ATP-Binding Cassette Transporters lcsh:Q Protein Multimerization 030217 neurology & neurosurgery |
| Zdroj: | Nature Communications, Vol 10, Iss 1, Pp 1-13 (2019) Nature Communications |
| ISSN: | 2041-1723 |
| DOI: | 10.1038/s41467-019-09892-6 |
| Popis: | ABC exporters harness the energy of ATP to pump substrates across membranes. Extracellular gate opening and closure are key steps of the transport cycle, but the underlying mechanism is poorly understood. Here, we generated a synthetic single domain antibody (sybody) that recognizes the heterodimeric ABC exporter TM287/288 exclusively in the presence of ATP, which was essential to solve a 3.2 Å crystal structure of the outward-facing transporter. The sybody binds to an extracellular wing and strongly inhibits ATPase activity by shifting the transporter’s conformational equilibrium towards the outward-facing state, as shown by double electron-electron resonance (DEER). Mutations that facilitate extracellular gate opening result in a comparable equilibrium shift and strongly reduce ATPase activity and drug transport. Using the sybody as conformational probe, we demonstrate that efficient extracellular gate closure is required to dissociate the NBD dimer after ATP hydrolysis to reset the transporter back to its inward-facing state. ABC exporters hydrolyze ATP to pump substrates across membranes, but critical steps of the transport mechanism remain poorly understood. Here, the authors solve the crystal structure of outward-facing TM287/288 with the help of a state-specific sybody and gain insights into the role of the extracellular gate. |
| Databáze: | OpenAIRE |
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