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

Autor(en): Finkenwirth, Friedrich
Sippach, Michael
Landmesser, Heidi
Kirsch, Franziska
Ogienko, Anastasia
Grunzel, Miriam
Kiesler, Cornelia
Steinhoff, Heinz-Juergen 
Schneider, Erwin 
Eitinger, Thomas
Stichwörter: BINDING CASSETTE TRANSPORTER; Biochemistry & Molecular Biology; CATALYTIC CYCLE; COUPLING FACTOR TRANSPORTER; CRYSTAL-STRUCTURE; ESCHERICHIA-COLI; MALK DIMER; MALTOSE ABC TRANSPORTER; MEMBRANE-PROTEINS; P-GLYCOPROTEIN; S COMPONENTS
Erscheinungsdatum: 2015
Herausgeber: AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
Journal: JOURNAL OF BIOLOGICAL CHEMISTRY
Volumen: 290
Ausgabe: 27
Startseite: 16929
Seitenende: 16942
Zusammenfassung: 
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 (BioM(2)) 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 [H-3] 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.
DOI: 10.1074/jbc.M115.654343

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