The structure of Escherichia coli BtuF and binding to its cognate ATP binding cassette transporter

Abstract

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...

Interactions between TonB from Escherichia coli and the Periplasmic Protein FhuD

“…A hydrogen bond with the siderophore is also formed with FhuD residues Asn-215 and Ser-219 through an intermediate water molecule. The overall fold of FhuD is similar to that of BtuF (14,15), the periplasmic cobalamin-binding protein of E. coli. Periplasmic metal-binding proteins TroA (16) and PsaA (17) are also structurally related to FhuD.…”

The Role of FhuD2 in Iron(III)-Hydroxamate Transport in Staphylococcus aureus

“…, and E. coli (EC), and BtuF from E. coli. The eight proteins were aligned using ClustalX (28), and then the alignment was manually adjusted based on a superposition of the crystal structures of E. coli FhuD (4) and BtuF (6). Shaded boxes indicate residues that are conserved in at least six of the eight proteins.…”

“…For this type of modeling, an accurate sequence alignment is critical. Acidic residues that are conserved in this binding protein superfamily, and which are thought to be important for interaction with the integral membrane complex (6), are located at positions 97 and 231 in the FhuD2 sequence, and these conserved positions were used to "anchor" the alignment shown in Fig. 1.…”

“…The structures of different hydroxamate siderophores are accommodated in the binding pocket by subtle rearrangements in the positions of the side chains. In a recent study of the high resolution structure of BtuF, Borths et al (6) highlight two highly conserved glutamate residues that are located on the surface of each of the two lobes of the protein. When they aligned the BtuF structure above the structure of BtuCD, the ABC transporter, it was found that the BtuF glutamates align with the position of positively charged pockets of arginines present on the periplasmic surface of BtuCD.…”

“…This has led to the suggestion that interprotein Glu-Arg salt bridges might form important docking contacts between the binding protein and the ABC transporter. Notably, both the glutamates and the arginines are conserved in a number of iron(III)-siderophore transport systems in different bacteria (6).…”