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...