In Vitro Reassembly of the Ribose ATP-binding Cassette Transporter Reveals a Distinct Set of Transport Complexes

Abstract

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, RbsC 2 ; and a periplasmic substrate binding protein, RbsB. To investigate the transport mechanism of the complex RbsABC 2 , we probed intersubunit interactions by varying the presence of the substrate ribose and the hydrolysis cofactors, ATP/ADP and Mg 2+ . We were able to purify a full complex, RbsABC 2 , in the presence of stable, transition state mimics (ATP, Mg 2+ , and VO 4 ); a RbsAC complex in the presence of ADP and Mg 2+ ; 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 RbsABC 2 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.