Asymmetry and Conformational Changes of the E. Coli Ribose ABC Transporter

Abstract

The ABC transporter superfamily of proteins is diverse, ubiquitous, and related to a number of genetic diseases as well as multidrug resistance in bacteria and cancer. The ribose transport complex may serve as a model for understanding how this clinically relevant protein family functions. An open question in the field is how ATP hydrolysis is linked to transport, and the stoichiometry of this process. Typical ABC transport systems possess two equivalent sites of ATP hydrolysis, while the ribose transporter possesses an intact active site and a degenerate site. To address how transport is fulfilled by asymmetric ATP hydrolysis, different ribose transport complexes associated with sequential stages of the transport cycle were isolated in the presence of different substrates, and these complexes were studied using Electron Paramagnetic Resonance (EPR) spectroscopy to observe how substrate variation affected their formation and nature. The results show ribose disrupts the interaction of the transmembrane domain (TMD), RbsC, with ribose-binding protein, RbsB. Additionally, apo-RbsB interacts with RbsC to yield the outward-facing conformation of the TMD. In turn, the association of RbsB and RbsC disrupts interactions with the nucleotide-binding domain (NBD), RbsA. This suggests a transport model whereby the apo state of the NBD has a weak interaction with the outward-facing TMD, non-canonical behavior for an ABC system. Mg-ATP loaded RbsA then binds the complex, and is required to return RbsC to an inward-facing state. Subsequent ATP hydrolysis destabilizes the interaction between RbsC and RbsB, completing the transport cycle.