ATP-dependent Conformational Changes Trigger Substrate Capture and Release by an ECF-type Biotin Transporter

Friedrich Finkenwirth,Michael Sippach,Heidi Landmesser,Franziska Kirsch,Anastasia Ogienko,Miriam Grunzel,Cornelia Kiesler,Heinz-Jürgen Steinhoff,Erwin Schneider,Thomas Eitinger

doi:10.1074/jbc.m115.654343

Friedrich Finkenwirth, Michael Sippach + Show 8 more

Open Access

https://doi.org/10.1074/jbc.m115.654343

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Abstract

Energy-coupling factor (ECF) transporters for vitamins and metal ions in prokaryotes consist of two ATP-binding cassette-type ATPases, a substrate-specific transmembrane protein (S component) and a transmembrane protein (T component) that physically interacts with the ATPases and the S component. The mechanism of ECF transporters was analyzed upon reconstitution of a bacterial biotin transporter into phospholipid bilayer nanodiscs. ATPase activity was not stimulated by biotin and was only moderately reduced by vanadate. A non-hydrolyzable ATP analog was a competitive inhibitor. As evidenced by cross-linking of monocysteine variants and by site-specific spin labeling of the Q-helix followed by EPR-based interspin distance analyses, closure and reopening of the ATPase dimer (BioM2) was a consequence of ATP binding and hydrolysis, respectively. A previously suggested role of a stretch of small hydrophobic amino acid residues within the first transmembrane segment of the S units for S unit/T unit interactions was structurally and functionally confirmed for the biotin transporter. Cross-linking of this segment in BioY (S) using homobifunctional thiol-reactive reagents to a coupling helix of BioN (T) indicated a reorientation rather than a disruption of the BioY/BioN interface during catalysis. Fluorescence emission of BioY labeled with an environmentally sensitive fluorophore was compatible with an ATP-induced reorientation and consistent with a hypothesized toppling mechanism. As demonstrated by [(3)H]biotin capture assays, ATP binding stimulated substrate capture by the transporter, and subsequent ATP hydrolysis led to substrate release. Our study represents the first experimental insight into the individual steps during the catalytic cycle of an ECF transporter in a lipid environment.

Highlights

Substrate-binding integral membrane proteins of Energy-coupling factor (ECF) transporters are predicted to undergo reversible rotation during the transport cycle
Energy-coupling factor (ECF) transporters for vitamins and metal ions in prokaryotes consist of two ATP-binding cassettetype ATPases, a substrate-specific transmembrane protein (S component) and a transmembrane protein (T component) that physically interacts with the ATPases and the S component
We show that (i) binding of ATP induces a reorientation of the BioM2 ATPase dimer and eventually of the BioY S unit as a requirement for biotin capture, and (ii) subsequent hydrolysis of ATP leads to biotin release

Summary

Background

Substrate-binding integral membrane proteins of ECF transporters are predicted to undergo reversible rotation during the transport cycle. This work demonstrated that the vitamin transport systems consisted of a specific transmembrane binding protein (later called S component) plus an unknown part, the ECF. The latter was required for energization of substrate transport and was shown to be shared by various S components [1]. A model was proposed according to which the S units undergo a rotation by almost 90° within the membrane (see the schematic shown in Fig. 1) in order to allow substrate binding at the outer surface of the membrane This model, is highly speculative because biochemical evidence is lacking, and the crystal structure of a single state cannot provide information on dynamic rearrangements

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