Dynamic interactions of CbiN and CbiM trigger activity of a cobalt energy-coupling-factor transporter

Autor(en): Finkenwirth, Friedrich
Sippach, Michael
Pecina, Sinah N.
Gaede, Mario
Ruta, Julia
Ricke, Adrian
Bondarenko, Elena
Klare, Johann P.
Zinke, Maximilian
Lange, Sascha
Lange, Adam
Steinhoff, Heinz-Jurgen 
Eitinger, Thomas
Stichwörter: ATP-binding cassette transporter; Biochemistry & Molecular Biology; Biophysics; Electron paramagnetic resonance (EPR); EPR; MECHANISM; Metal ion-protein interaction; MOTION; NICKEL; PERMEASES; PREDICTION; Protein conformation; Protein-protein interaction; PROTEINS; Solid-state NMR; SPECIFICITY; SPIN; SUBSTRATE CAPTURE
Erscheinungsdatum: 2020
Herausgeber: ELSEVIER
Journal: BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES
Volumen: 1862
Ausgabe: 2
Zusammenfassung: 
Energy-coupling factor (ECF) transporters for uptake of vitamins and transition-metal ions into prokaryotic cells share a common architecture consisting of a substrate-specific integral membrane protein (S), a transmembrane coupling protein (T) and two cytoplasmic ATP-binding-cassette-family ATPases. S components rotate within the membrane to expose their binding pockets alternately to the exterior and the cytoplasm. In contrast to vitamin transporters, metal-specific systems rely on additional proteins with essential but poorly understood functions. CbiN, a membrane protein composed of two transmembrane helices tethered by an extracytoplasmic loop of 37 amino-acid residues represents the auxiliary component that temporarily interacts with the CbiMQO(2) Co2+ transporter. CbiN was previously shown to induce significant Co2+ transport activity in the absence of CbiQO(2) in cells producing the S component CbiM plus CbiN or a Cbi(MN) fusion. Here we analyzed the mode of interaction between the two protein domains. Any deletion in the CbiN loop abolished transport activity. In silico predicted protein-protein contacts between segments of the CbiN loop and loops in CbiM were confirmed by cysteine-scanning mutagenesis and crosslinking. Likewise, an ordered structure of the CbiN loop was observed by electron paramagnetic resonance analysis after site-directed spin labeling. The N-terminal loop of CbiM containing three of four metal ligands was partially immobilized in wild-type Cbi(MN) but completely immobile in inactive variants with CbiN loop deletions. Decreased dynamics of the inactive form was also detected by solid-state nuclear magnetic resonance of isotope-labeled protein in proteoliposomes. In conclusion, CbiM-CbiN loop-loop interactions facilitate metal insertion into the binding pocket.
ISSN: 00052736
DOI: 10.1016/j.bbamem.2019.183114

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