X-ray crystallographic analyses of RbsN1/2A and RbsR will be performed. RbsN1/2A is a complete ATP-binding cassette (ABC) representing half of RbsA, the ATPase of the binding protein mediated ribose membrane transport system of E. coli. It is homologous to the ABC's of the cystic fibrosis-related transmembrane conductance regulator and to the multiple drug resistance (P-glycoprotein) proteins in humans. Crystals which we have obtained diffract to at least 2.3 Angstrom. RbsR is the repressor that regulates expression of the ribose transporter proteins. It is a member of the family of repressors that include LacI and PurR. It has been crystallized in the presence of its dsDNA operator sequence. Crystals have been obtained under two different conditions, the first of which diffract to at least 3.8 Angstrom. The crystals of RbsN1/2A represent the first crystals suitable for x-ray analysis of an ABC. The crystals of RbsR will yield information which will address the question of how repressors which are closely related by sequence can bind DNA by opposite mechanisms, RbsR and Lacl as apo -repressors and PurR as the holo-repressor. We also propose to continue work on the purification, stabilization, and crystallization of the other components of the ribose transporter. These are RbsA (the complete ATP-binding protein), RbsC (the component which is largely membrane-embedded), and RbsA-C (a chimeric protein containing all of RbsA joined to RbsC by a flexible factor Xa-cleavable bridge). Our preliminary work has succeeded in developing protocols which yield milegram quantities of each of these proteins in at least 95% purity. This proposal addresses a stability problem with RbsA and RbsA portion of RbsA-C, and it also addresses the requirement for appropriate detergents in which to isolate and manipulate the RbsC (and its component of RbsA-C) for crystal screens and in vitro reconstitution studies. We also propose to reconstitute transport of ribose in vitro in phospholipid vesicles. In vitro assays will be highly desirable for mechanistic studies once structures of the proteins are obtained, and they will permit determination of the stoichiometry of the constituents of the transporter and the mole ratios of ATP hydrolyzed to ribose transported. The ribose transporter is related to a large family of membrane transporters f all kingdoms of living organisms. There is no detailed structural information on any of these transporters, and the ribose transporter is the first member of the family which has yielded suitable crystals (to our knowledge) and adequate quantities of pure constituents to pursue atomic resolution structural analyses.
| Clifton, Matthew C; Simon, Michael J; Erramilli, Satchal K et al. (2015) In vitro reassembly of the ribose ATP-binding cassette transporter reveals a distinct set of transport complexes. J Biol Chem 290:5555-65 |
| Stewart, Jeffrey B; Hermodson, Mark A (2003) Topology of RbsC, the membrane component of the Escherichia coli ribose transporter. J Bacteriol 185:5234-9 |
