Bacterial binding protein-dependent ATP binding cassette (ABC) transporters facilitate uptake of essential nutrients. The crystal structure of Escherichia coli BtuF, the protein that binds vitamin B 12 and delivers it to the periplasmic surface of the ABC transporter BtuCD, reveals a bi-lobed fold resembling that of the ferrichrome binding protein FhuD. B 12 is bound in the ''base-on'' conformation in a deep cleft formed at the interface between the two lobes of BtuF. A stable complex between BtuF and BtuCD (with the stoichiometry BtuC 2D2F) is demonstrated to form in vitro and was modeled using the individual crystal structures. Two surface glutamates from BtuF may interact with arginine residues on the periplasmic surface of the BtuCD transporter. These glutamate and arginine residues are conserved among binding proteins and ABC transporters mediating iron and B 12 uptake, suggesting that they may have a role in docking and the transmission of conformational changes.A TP binding cassette (ABC) transporters are a ubiquitous family of importer and exporter proteins that invariably consist of two membrane-spanning domains, which form a translocation pathway, and two cytoplasmic ABC domains, which power the transport reaction through binding and hydrolysis of ATP (1). Although most eukaryotic ABC transporters export hydrophobic molecules from the cytoplasm (2), the majority of bacterial ABC transporters import essential nutrients that are delivered to them by specific binding proteins (1,3,4). These proteins bind their substrates selectively and with high affinity, which is thought to ensure the specificity of the transport reaction (3). The association of a substrate-loaded binding protein with its cognate transporter has been shown to stimulate ATP hydrolysis by the cytoplasmic ABC domains (5). The binding protein remains docked to the cognate transporter until one or both of the hydrolysis products are released, as shown by experiments that used vanadate to trap an intermediate close to the transition state (6). This finding suggested that the binding protein, associated with the transporter during substrate translocation, may prevent the escape of substrate into the periplasmic space.The structures of many different binding proteins have been solved, revealing a common architecture: two domains, each consisting of a central ␤-sheet and surrounding ␣-helices, with the substrate binding site located in a cleft between them (7). Recently, the crystal structure of the binding protein-dependent ABC transporter, BtuCD, which facilitates import of vitamin B 12 into Escherichia coli, was determined at 3.2-Å resolution (8). We have now solved the crystal structure of E. coli BtuF, the cognate periplasmic binding protein for BtuCD (9, 10) at 2.0-Å resolution. In addition, we could form a stable complex between BtuF and BtuCD in vitro. These results provide general insights into the interaction of binding proteins with their cognate ABC transporters. Materials and MethodsPurification and Crystallization. The btuf gene (p...

BtuCD is an ATP binding cassette (ABC) transporter that facilitates uptake of vitamin B(12) into the cytoplasm of Escherichia coli. The crystal structures of BtuCD and its cognate periplasmic binding protein BtuF have been recently determined. We have now explored BtuCD-F function in vitro, both in proteoliposomes and in various detergents. BtuCD reconstituted into proteoliposomes has a significant basal ATP hydrolysis rate that is stimulated by addition of BtuF and inhibited by sodium ortho-vanadate. When using different detergents to solubilize BtuCD, the basal ATP hydrolysis rate, the ability of BtuF to stimulate hydrolysis, and the extent to which sodium ortho-vanadate inhibits ATP hydrolysis all vary significantly. Reconstituted BtuCD can mediate transport of vitamin B(12) against a concentration gradient when coupled to ATP hydrolysis by BtuD in the liposome lumen and BtuF outside the liposomes. These in vitro studies establish the functional competence of the BtuCD and BtuF preparations used in the crystallographic analyses for both ATPase and transport activities. Furthermore, the tight binding of BtuF to BtuCD under the conditions studied suggests that the binding protein may not dissociate from the transporter during the catalytic cycle, which may be relevant to the mechanisms of other ABC transporter systems.