In vitro reconstitution of dynamically interacting integral membrane subunits of energy-coupling factor transporters

DOI: 10.7554/eLife.64389

Authors: Inda Setyawati (University of Groningen; Bogor Agricultural Universit) , Weronika K Stanek (University of Groningen) , Maria Majsnerowska (University of Groningen) , Lotteke J. Y. M. Swier (University of Groningen) , Els Pardon (Vrije Universiteit Brussel; VIB-VUB Center for Structural Biology, VIB) , Jan Steyaert (Vrije Universiteit Brussel, VUB) , Albert Guskov (University of Groningen; Moscow Institute of Physics and Technology) , Dirk J. Slotboom (University of Groningen)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Elife , VOL 9

State: Published (Approved)
Published: December 2020
Diamond Proposal Number(s): 19800

Abstract: Energy-coupling factor (ECF) transporters mediate import of micronutrients in prokaryotes. They consist of an integral membrane S-component (that binds substrate) and ECF module (that powers transport by ATP hydrolysis). It has been proposed that different S-components compete for docking onto the same ECF module, but a minimal liposome-reconstituted system, required to substantiate this idea, is lacking. Here, we co-reconstituted ECF transporters for folate (ECF-FolT2) and pantothenate (ECF-PanT) into proteoliposomes, and assayed for crosstalk during active transport. The kinetics of transport showed that exchange of S-components is part of the transport mechanism. Competition experiments suggest much slower substrate association with FolT2 than with PanT. Comparison of a crystal structure of ECF-PanT with previously determined structures of ECF-FolT2 revealed larger conformational changes upon binding of folate than pantothenate, which could explain the kinetic differences. Our work shows that a minimal in vitro system with two reconstituted transporters recapitulates intricate kinetics behaviour observed in vivo.

Subject Areas: Biology and Bio-materials, Chemistry


Instruments: I24-Microfocus Macromolecular Crystallography

Documents:
elife-64389-v1.pdf