Journal Article

Subnanometre-resolution electron cryomicroscopy structure of a heterodimeric ABC exporter

Tomasiak, Thomas M., Stiller, Sebastian B., Kim, JungMin, Winter, Michael B., Cheng, Yifan, Tampé, Robert, Wu, Shenping, Robles-Colmanares, Yaneth, Stroud, Robert M., Craik, Charles S., Mergel, Claudia

2015

Overview

The subnanometre-resolution electron cryomicroscopy structure of TmrAB, a heterodimeric ABC transport protein, in a nucleotide-free, inward-facing conformation, is determined. An ABC transporter at 8.2-Å resolution The ATP-binding cassette (ABC) transporter is implicated in a number of human diseases and is an important drug target. It is a small hetero-oligomeric protein presenting a challenge to structural biologists. Here Yifan Cheng and colleagues report the 8.2 Å resolution electron cryomicroscopy structure of TmrAB, a 135 kDa heterodimeric ABC transport protein, in a nucleotide-free, inward-facing conformation. The structure shows that the cytoplasmic nucleotide-binding domains of this ABC transporter are in contact with each other. Comparison with the structures of other ABC transporters in various states suggest that the cytoplasmic nucleotide-binding domains slide and rotate during the transition from the inward-facing to the outward-facing conformation. ATP-binding cassette (ABC) transporters translocate substrates across cell membranes, using energy harnessed from ATP binding and hydrolysis at their nucleotide-binding domains 1 , 2 . ABC exporters are present both in prokaryotes and eukaryotes, with examples implicated in multidrug resistance of pathogens and cancer cells, as well as in many human diseases 3 , 4 . TmrAB is a heterodimeric ABC exporter from the thermophilic Gram-negative eubacterium Thermus thermophilus ; it is homologous to various multidrug transporters and contains one degenerate site with a non-catalytic residue next to the Walker B motif 5 . Here we report a subnanometre-resolution structure of detergent-solubilized TmrAB in a nucleotide-free, inward-facing conformation by single-particle electron cryomicroscopy. The reconstructions clearly resolve characteristic features of ABC transporters, including helices in the transmembrane domain and nucleotide-binding domains. A cavity in the transmembrane domain is accessible laterally from the cytoplasmic side of the membrane as well as from the cytoplasm, indicating that the transporter lies in an inward-facing open conformation. The two nucleotide-binding domains remain in contact via their carboxy-terminal helices. Furthermore, comparison between our structure and the crystal structures of other ABC transporters suggests a possible trajectory of conformational changes that involves a sliding and rotating motion between the two nucleotide-binding domains during the transition from the inward-facing to outward-facing conformations.

• Publisher: Nature Publishing Group UK,Nature Publishing Group
• Subject: 101/28 / 631/535/1258/1259 / 631/92/577 / 82/1 / Analysis / Antigens / Antigens - chemistry / Antigens - immunology / ATP / ATP-Binding Cassette Transporters - chemistry / ATP-Binding Cassette Transporters - immunology / ATP-Binding Cassette Transporters - ultrastructure / Binding Sites / Carrier proteins / Cryoelectron Microscopy / Crystallography, X-Ray / Humanities and Social Sciences / Hydrolysis / letter / Membranes / Microscopy / Models, Molecular / multidisciplinary / Nucleotides - metabolism / Physiological aspects / Protein Multimerization / Protein Structure, Quaternary / Protein Structure, Tertiary / Proteins / Rotation / Science / Structure / Symmetry / Thermus thermophilus - chemistry