Journal of Biological Chemistry
Volume 290, Issue 9, 27 February 2015, Pages 5555-5565
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Membrane Biology
In Vitro Reassembly of the Ribose ATP-binding Cassette Transporter Reveals a Distinct Set of Transport Complexes*

https://doi.org/10.1074/jbc.M114.621573Get rights and content
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Bacterial ATP-binding cassette (ABC) importers are primary active transporters that are critical for nutrient uptake. Based on structural and functional studies, ABC importers can be divided into two distinct classes, type I and type II. Type I importers follow a strict alternating access mechanism that is driven by the presence of the substrate. Type II importers accept substrates in a nucleotide-free state, with hydrolysis driving an inward facing conformation. The ribose transporter in Escherichia coli is a tripartite complex consisting of a cytoplasmic ATP-binding cassette protein, RbsA, with fused nucleotide binding domains; a transmembrane domain homodimer, RbsC2; and a periplasmic substrate binding protein, RbsB. To investigate the transport mechanism of the complex RbsABC2, we probed intersubunit interactions by varying the presence of the substrate ribose and the hydrolysis cofactors, ATP/ADP and Mg2+. We were able to purify a full complex, RbsABC2, in the presence of stable, transition state mimics (ATP, Mg2+, and VO4); a RbsAC complex in the presence of ADP and Mg2+; and a heretofore unobserved RbsBC complex in the absence of cofactors. The presence of excess ribose also destabilized complex formation between RbsB and RbsC. These observations suggest that RbsABC2 shares functional traits with both type I and type II importers, as well as possessing unique features, and employs a distinct mechanism relative to other ABC transporters.

Background

The ribose transporter is a bacterial ABC importer with a non-canonical organization.

Results

The binding protein complexes with the membrane domain in the absence of substrate, and the ATPase dissociates from the membrane domain during transport.

Conclusion

A distinct model for transport is proposed from in vitro reassembly conditions and EPR data.

Significance

The ribose transporter typifies the diversity of ABC transporter mechanisms.

ABC Transporter
ATPase
Bacterial Metabolism
Electron Paramagnetic Resonance (EPR)
Membrane Transport
Membrane Transporter Reconstitution
Ribose Transport

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*

This work was supported, in whole or in part, by National Institutes of Health Cancer Center Core Grant CA23168, Biophysics Training Grant T32-GM008296, and Public Health Service Grants GM54993 and GM64676.

1

Present address: Beryllium, Bedford, MA 01730.

2

Present address: Dept. of Biochemistry and Molecular Biophysics, Washington University, St. Louis, MO 61130.

3

Present address: Bayer CropScience, 3500 Paramount Parkway, Morrisville, NC 27560.