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The extracellular gate shapes the energy profile of an ABC exporter


Hutter, Cedric A J; Timachi, M Hadi; Hürlimann, Lea M; Zimmermann, Iwan; Egloff, Pascal; Göddeke, Hendrik; Kucher, Svetlana; Štefanić, Saša; Karttunen, Mikko; Schäfer, Lars V; Bordignon, Enrica; Seeger, Markus A (2019). The extracellular gate shapes the energy profile of an ABC exporter. Nature Communications, 10(1):2260.

Abstract

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.

Abstract

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.

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Item Type:Journal Article, refereed, original work
Communities & Collections:05 Vetsuisse Faculty > Institute of Parasitology
04 Faculty of Medicine > Institute of Parasitology

04 Faculty of Medicine > Institute of Medical Microbiology
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
600 Technology
Language:English
Date:May 2019
Deposited On:13 Feb 2020 15:41
Last Modified:16 Feb 2020 19:55
Publisher:Nature Publishing Group
ISSN:2041-1723
OA Status:Gold
Free access at:Publisher DOI. An embargo period may apply.
Publisher DOI:https://doi.org/10.1038/s41467-019-09892-6

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