BtuCD is an adenosine triphosphate-binding cassette (ABC) transporter that translocates vitamin B12 from the periplasmic binding protein BtuF into the cytoplasm of Escherichia coli. The 2.6 angstrom crystal structure of a complex BtuCD-F reveals substantial conformational changes as compared with the previously reported structures of BtuCD and BtuF. The lobes of BtuF are spread apart, and B12 is displaced from the binding pocket. The transmembrane BtuC subunits reveal two distinct conformations, and the translocation pathway is closed to both sides of the membrane. Electron paramagnetic resonance spectra of spin-labeled cysteine mutants reconstituted in proteoliposomes are consistent with the conformation of BtuCD-F that was observed in the crystal structure. A comparison with BtuCD and the homologous HI1470/71 protein suggests that the structure of BtuCD-F may reflect a posttranslocation intermediate.

Transport across cellular membranes is an essential process that is catalyzed by diverse membrane transport proteins. The turnover rates of certain transporters are inhibited by their substrates in a process termed trans-inhibition, whose structural basis is poorly understood. We present the crystal structure of a molybdate/tungstate ABC transporter (ModBC) from Methanosarcina acetivorans in a trans-inhibited state. The regulatory domains of the nucleotide-binding subunits are in close contact and provide two oxyanion binding pockets at the shared interface. By specifically binding to these pockets, molybdate or tungstate prevent adenosine triphosphatase activity and lock the transporter in an inward-facing conformation, with the catalytic motifs of the nucleotide-binding domains separated. This allosteric effect prevents the transporter from switching between the inward-facing and the outward-facing states, thus interfering with the alternating access and release mechanism.

HmuUV is a bacterial ATP-binding cassette (ABC) transporter that catalyzes heme uptake into the cytoplasm of the gram-negative pathogen Yersinia pestis. We report the crystal structure of HmuUV at 3.0 Å resolution in a nucleotide-free state, which features a heme translocation pathway in an outward-facing conformation, poised to accept a heme from the cognate periplasmic binding protein HmuT. A new assay allowed us to determine in vitro rates of HmuUV-catalyzed heme transport into proteoliposomes and to establish the role of conserved residues in the translocation pathway of HmuUV and at the interface with HmuT. Differences in architecture relative to the related vitamin B(12) transporter BtuCD suggest an adaptation of HmuUV for its smaller substrate. Our study also suggests that type II ABC importers, which include bacterial iron-siderophore, heme and cobalamin transporters, have a coupling mechanism distinct from that of other ABC transporters.

ATP-binding cassette (ABC) transporters are ubiquitous integral membrane proteins that translocate substrates across cell membranes. The alternating access of their transmembrane domains to opposite sides of the membrane powered by the closure and reopening of the nucleotide binding domains is proposed to drive the translocation events. Despite clear structural similarities, evidence for considerable mechanistic diversity starts to accumulate within the importers subfamily. We present here a detailed study of the gating mechanism of a type II ABC importer, the BtuCD-F vitamin B 12 importer from Escherichia coli, elucidated by EPR spectroscopy. Distance changes at key positions in the translocation gates in the nucleotide-free, ATP-and ADP-bound conformations of the transporter were measured in detergent micelles and liposomes. The translocation gates of the BtuCD-F complex undergo conformational changes in line with a "two-state" alternating access model. We provide the first direct evidence that binding of ATP drives the gates to an inward-facing conformation, in contrast to type I importers specific for maltose, molybdate, or methionine. Following ATP hydrolysis, the translocation gates restore to an apo-like conformation. In the presence of ATP, an excess of vitamin B 12 promotes the reopening of the gates toward the periplasm and the dislodgment of BtuF from the transporter. The EPR data allow a productive translocation cycle of the vitamin B 12 transporter to be modeled. ATP-binding cassette (ABC)2 transporters couple the energy of ATP hydrolysis to the translocation of substrates across biological membranes. They constitute the largest transmembrane protein family present in all branches of life and mediate the active transport of various substances such as sugars, amino acids, peptides, vitamins, iron siderophores, opines, metals, etc. across the membrane (1-3). Biochemical evidence supports a "two-state, alternating access" mechanistic model for both ABC exporters and importers (4). In this model, an ATP-bound conformation of the transmembrane domains facing the extracellular side of the membrane is converted to an inward-facing conformation via ATP hydrolysis in the nucleotide-binding domains (NBDs). This conformational transition ensures net substrate uptake by the importers or net expulsion by the exporters. Among the canonical ABC importers, which are characterized by the presence of a soluble periplasmic substrate binding protein, two structurally different types exist, namely type I (e.g. maltose, molybdate, and methionine systems) and type II (e.g. vitamin B 12 , heme, and metal systems) (3). For type I ABC importers the details of the alternating access mechanism have been confirmed by a large body of experimental evidence, including the crystal structures showing the maltose transporter in different states during the nucleotide cycle (5-8). The transmembrane domains (TMDs) of type I importers, featuring 10 -14 helices, alternate from an ATP-bound outward-facing conformation where the substrate b...