Binding kinetics and conformational changes of the E. coli ribose importer (RbsABC)

Michael James Simon, Purdue University

Abstract

ATP binding cassette (ABC) transporters are essential for all forms of life, facilitating active transport of sugars, peptides, ions, nutrients, and toxins into or out of cells and organelles. The E. coli ribose importer is a multi-subunit ABC transporter composed of three distinct domains. RbsB, the ribose binding domain, recruits ribose in the periplasm and delivers it to the membrane surface where it binds RbsC. RbsC, the transmembrane domain, is a homodimer that allows passage of ribose through the membrane. RbsA, the cytoplasmic ATP binding casstte, is a single polypeptide folded into two ATP binding sites. ATP binding and hydrolysis induces conformational changes in RbsA that are propagated to RbsC and RbsB, driving transport of ribose against a concentration gradient. An assembly/disassembly pathway based on the ATP hydrolysis cycle has been described. In the absence of nucleotide, RbsB is bound to RbsC, upon binding of ATP to RbsA, it associates with RbsC. ATP is then hydrolyzed, phosphate is released, and RbsB dissociates from RbsC. Upon ADP dissociation, RbsA dissociates from RbsC as well. In order to better understand this pathway, we have explored three aspects of ribose transport. First, we have studied the interaction of the substrate, ribose with the different complexes formed during this pathway. Using radio-labeled ribose to observe substrate binding, we found that ribose is released upon association of RbsB with RbsC. Additionally, we have observed that excess ribose induces dissociation of RbsB from RbsC using electron paramagnetic resonance (EPR). From these data we infer that the RbsB-RbsC complex alternates between a transiently associated ribose-bound state and a strongly associated ribose-released state. Second, we have studied the binding kinetics of ribose importer subunits using surface plasmon resonance (SPR). We observed that the free energy of the RbsB-RbsC interaction is sufficient to drive the release of ribose from RbsB, quantitatively verifying interaction data observed previously. Furthermore, we have found that RbsB rapidly associates and dissociates from RbsC, while RbsA associates more weakly with RbsC, but dissociates more slowly. Third, we observed conformational change in RbsB, again using EPR. We have found that RbsB is in a partially open state when associated with RbsC alone or with RbsC bound to cofactor free or ATP bound RbsA. RbsB was then observed to transition to a fully open state when RbsC was bound to RbsA while stabilized by non-hydrolyzable nucleotide analogues mimicking an ATP-Mg2+ bound state. Finally, we observed maximum closure of RbsB when the RbsBC complex was bound to RbsA while stabilized by ADP-Mg2+. Implications of this data in regards to ribose transport and the general mechanisms of ABC transporter operation are discussed.

Degree

Ph.D.

Advisors

Stauffacher, Purdue University.

Subject Area

Biochemistry|Biophysics

COinS