ABC transporters form one of the largest protein superfamilies in all domains of life, catalyzing the movement of diverse substrates across membranes. In this key position, ABC transporters can mediate multidrug resistance in cancer therapy and their dysfunction is linked to various diseases. Here, we describe the 2.7-Å X-ray structure of heterodimeric Thermus thermophilus multidrug resistance proteins A and B (TmrAB), which not only shares structural homology with the antigen translocation complex TAP, but is also able to restore antigen processing in human TAP-deficient cells. TmrAB exhibits a broad peptide specificity and can concentrate substrates several thousandfold, using only one single active ATP-binding site. In our structure, TmrAB adopts an asymmetric inward-facing state, and we show that the C-terminal helices, arranged in a zipper-like fashion, play a crucial role in guiding the conformational changes associated with substrate transport. In conclusion, TmrAB can be regarded as a model system for asymmetric ABC exporters in general, and for TAP in particular. Significance ABC transporters shuttle chemically diverse substances across membranes in an energy-dependent manner. They mediate multidrug resistance in microorganisms and cancer cells and can cause human pathologies when dysfunctional. Although important insights into ABC transporters have been gained in recent years, fundamental questions concerning their mechanism remain open. Here, we identify the protein complex TmrAB as a functional homolog of the antigenic peptide transporter TAP and present its high-resolution structure. The structure adopts an asymmetric conformational state and is characterized by C-terminal zipper helices that are essential for efficient substrate translocation. The structure, together with functional studies, enables us to outline the general conformational dynamics of heterodimeric ABC transporters and to establish TmrAB as a model system for TAP.

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.

Double electron-electron resonance is used here to investigate intermediates of the transport cycle of the Escherichia coli vitamin B12 ATP-binding cassette importer BtuCD-F. Previously, we showed the ATP-induced opening of the cytoplasmic gate I in TM5 helices, later confirmed by the AMP-PNP-bound BtuCD-F crystal structure. Here, other key residues are analyzed in TM10 helices (positions 307 and 322) and in the cytoplasmic gate II, i.e. the loop between TM2 and TM3 (positions 82 and 85). Without BtuF, binding of ATP induces detectable changes at positions 307 and 85 in BtuCD in liposomes. Together with BtuF, ATP triggers the closure of the cytoplasmic gate II in liposomes (reported by both positions 82 and 85). This forms a sealed cavity in the translocation channel in agreement with the AMP-PNP·BtuCD-F x-ray structure. When vitamin B12 and AMP-PNP are simultaneously present, the extent of complex formation is reduced, but the short 82-82 interspin distance detected indicates that the substrate does not affect the closed conformation of this gate. The existence of the BtuCD-F complex under these conditions is verified with spectroscopically orthogonal nitroxide and Gd(III)-based labels. The cytoplasmic gate II remains closed also in the vanadate-trapped state, but it reopens in the ADP-bound state of the complex. Therefore, we suggest that the substrate likely trapped in ATP·BtuCD-F can be released after ATP hydrolysis but before the occluded ADP-bound conformation is reached.