The extracellular gate shapes the energy profile of an ABC exporter

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 heterodimeri...

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Veröffentlicht in:Nature communications 2019-05, Vol.10 (1), p.2260-2260, Article 2260
Hauptverfasser: 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.
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Sprache:eng
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Zusammenfassung: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.
ISSN:2041-1723
2041-1723
DOI:10.1038/s41467-019-09892-6