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