Subnanometre-resolution electron cryomicroscopy structure of a heterodimeric ABC exporter

Abstract

ATP-binding cassette (ABC) transporters translocate substrates across cell membranes, using energy harnessed from ATP binding and hydrolysis at their nucleotide binding domains (NBDs)1,2. ABC exporters are present in both prokaryotes and eukaryotes with examples implicated in multidrug resistance of pathogens and cancer cells, as well as in many human diseases3,4. TmrAB is a heterodimeric ABC exporter from the thermophilic Gram-negative eubacterium Thermus thermophilus homologous to various multidrug transporters and containing one degenerate site with a non-catalytic residue next to the Walker B motif5. Here we report a subnanometer resolution structure of detergent-solubilized TmrAB in a nucleotide-free, inward-facing conformation by single particle electron cryomicroscopy (cryo-EM). The reconstructions clearly resolved characteristic features of ABC transporters, including helices in the transmembrane domain (TMD) and NBDs. A cavity in the TMD 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 NBDs remain in contact via their C-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 NBDs during the transition from the inward facing to outward facing conformations.

Molecular Dynamics Flexible Fitting Simulations Identify New Models of the Closed State of the Cystic Fibrosis Transmembrane Conductance Regulator Protein

“…[15][16]32 Efforts towards the development of such maps take advantage of impressive improvements in EM technology and the concomitant recording of medium to high resolution EM maps of CFTR and its homologues. [15][16][60][61] Moreover, recording maps in the presence and absence of known CFTR modulators could inform on their binding sites. A combination of MDFF and docking simulations could therefore be used to elucidate the binding modes of these modulators and to suggest ways to improve them, paving the way to structure-based drug design.…”

The Effects of Electron Beam Exposure Time on Transmission Electron Microscopy Imaging of Negatively Stained Biological Samples

“…Previous studies (Henderson, 1995) using theoretical considerations demonstrated that unstained molecules larger than approximately 100 kDa can be reconstructed to provide structural details. This molecular weight limit does not apply to negative staining TEM of proteins as well as cryo-EM (Kim et al, 2015); therefore, the use of correct instrumentation and optimized analytical parameters can maximize the quality of A B C 1 sec 2 sec 4 sec Fig. 4.…”

Structural and Functional Basis for Lipid Synergy on the Activity of the Antibacterial Peptide ABC Transporter McjD

“…For example, the ABC exporter TmrAB, from Thermus thermophilus, is associated with phosphatidylglycerol (PG) and lipid A, as revealed by mass spectrometry (8). However, electron density in the low resolution electron cryo-microscopy single particle reconstruction envelope of TmrAB was assigned to the detergent micelle (9). In the low resolution crystal structure of the eukaryotic P-glycoprotein, no lipids were observed (10,11), but non-denaturing mass spectrometry identified tightly associated lipids and a clear dependence of ligand and lipid binding (12).…”