Materials and methodsThe sequences for the different transporters were subcloned into various pET vectors (Novagen), which attached poly---histidine tags at the amino---or carboxy---terminus. The constructs were expressed in E. coli BL21 (DE3) (Stratagene) in a 50---liter fermenter at 37°C and were induced using 1 mM IPTG. BtuC and BtuD were coexpressed from a single plasmid, with the BtuC subunit containing an NH 2 ---terminal decahistidine tag. BtuCD protein was solubilized in 1% dodecyl---N,N---dimethylamineoxide (LDAO) and purified using Ni---NTA metal affinity chromatography (Qiagen), followed by gel filtration. Crystals were grown by mixing BtuCD protein (20 mg/ml) with an equal volume of reservoir solution (100 mM Tris pH 8.0, 300 mM magnesium nitrate, 21% polyethylene glycol 2000, 0.8% 2---methyl---2,4---pentanediol in D 2 O) in sitting drops. Crystals grew to a final size of 0.1 0.15 0.5 mm 3 in a week, and were frozen in liquid N 2 before data collection. Cells expressing selenomethionine BtuCD protein were grown in M9 medium supplemented with seleno---D,L---methionine. Purification and crystallization of the selenomethionine protein was as described above for the native protein. Data sets were collected at the Advanced Photon Source (APS) or at the Stanford Synchrotron Radiation Laboratory (SSRL).

Mechanosensitive ion channels play a critical role in transducing physical stresses at the cell membrane into an electrochemical response. The MscL family of large-conductance mechanosensitive channels is widely distributed among prokaryotes and may participate in the regulation of osmotic pressure changes within the cell. In an effort to better understand the structural basis for the function of these channels, the structure of the MscL homolog from Mycobacterium tuberculosis was determined by x-ray crystallography to 3.5 angstroms resolution. This channel is organized as a homopentamer, with each subunit containing two transmembrane α helices and a third cytoplasmic α helix. From the extracellular side, a water-filled opening approximately 18 angstroms in diameter leads into a pore lined with hydrophilic residues which narrows at the cytoplasmic side to an occluded hydrophobic apex that may act as the channel gate. This structure may serve as a model for other mechanosensitive channels, as well as the broader class of pentameric ligand-gated ion channels exemplified by the nicotinic acetylcholine receptor.

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...