Cryo-electron microscopy (cryo-EM) has the capacity to capture molecular machines in action 1 – 3 . ATP-binding cassette (ABC) exporters are highly dynamic membrane proteins that extrude a wide range of substances from the cytosol 4 – 6 and thereby contribute to essential cellular processes, adaptive immunity, and multidrug resistance 7 , 8 . Despite their vital importance, the coupling of nucleotide binding, hydrolysis, and release to the conformational dynamics remains poorly resolved, especially for heterodimeric/asymmetric ABC exporters that abound in humans. Here, we present eight high-resolution cryo-EM structures that delineate the full functional cycle of an asymmetric ABC exporter in lipid environment. Cryo-EM analysis under active turnover conditions reveals distinct inward-facing (IF) conformations, one of them with bound peptide substrate, and previously undescribed asymmetric post-hydrolysis states with dimerized nucleotide-binding domains (NBDs) and a closed extracellular gate. Capturing an outward-facing (OF) open conformation requires a slow-down in ATP hydrolysis, indicating the transient nature of this state vulnerable to substrate re-entry. ATP-bound pre-hydrolysis and vanadate-trapped states are conformationally equivalent and both comprise co-existing OF conformations with open and closed extracelluar gates. In contrast, the post-hydrolysis states from the turnover experiment exhibit asymmetric ADP/ATP occlusion after phosphate release from the canonical site and display a progressive separation of the nucleotide-binding domains and unlocking of the intracellular gate. Our findings reveal that phosphate release, not ATP hydrolysis, triggers the return of the exporter to the IF conformation. By mapping the conformational landscape during active turnover, aided by mutational and chemical modulation of kinetic rates to trap the key intermediates, we resolved fundamental and so-far hidden steps of the substrate translocation cycle of asymmetric ABC transporters.

In this protocol, we describe a procedure for incorporating ATP-binding cassette (ABC) transporters into large unilamellar vesicles (LUVs) and assays to determine ligand binding and solute translocation by these membrane-reconstituted systems. The reconstitution technique as described has been optimized for ABC transporters but can be readily adapted for other types of transport systems. Purified transporters are inserted into detergent-destabilized preformed liposomes and detergent is subsequently removed by adsorption onto polystyrene beads. Next, Mg-ATP or an ATP-regenerating system is incorporated into the vesicle lumen by one or more cycles of freezing-thawing, followed by extrusion through polycarbonate filters to obtain unilamellar vesicles. Binding and translocation of substrates are measured using isotope-labeled ligands and rapid filtration to separate the proteoliposomes from the surrounding medium. Quantitative information is obtained about dissociation constants (K(d)) for ligand binding, number of binding-sites, transport affinities (K(m)), rates of transport, and the activities of transporter molecules with opposite orientations in the membrane. The full protocol can be completed within 4-5 d.

Members of the SLC11 (NRAMP) family transport iron and other transition-metal ions across cellular membranes. These membrane proteins are present in all kingdoms of life with a high degree of sequence conservation. To gain insight into the determinants of ion selectivity, we have determined the crystal structure of Staphylococcus capitis DMT (ScaDMT), a close prokaryotic homolog of the family. ScaDMT shows a familiar architecture that was previously identified in the amino acid permease LeuT. The protein adopts an inward-facing conformation with a substrate-binding site located in the center of the transporter. This site is composed of conserved residues, which coordinate Mn2+, Fe2+ and Cd2+ but not Ca2+. Mutations of interacting residues affect ion binding and transport in both ScaDMT and human DMT1. Our study thus reveals a conserved mechanism for transition-metal ion selectivity within the SLC11 family.

Methods for the cloning of large numbers of open reading frames into expression vectors are of critical importance for challenging structural biology projects. Here we describe a system termed fragment exchange (FX) cloning that facilitates the high-throughput generation of expression constructs. The method is based on a class IIS restriction enzyme and negative selection markers. FX cloning combines attractive features of established recombination- and ligation-independent cloning methods: It allows the straightforward transfer of an open reading frame into a variety of expression vectors and is highly efficient and very economic in its use. In addition, FX cloning avoids the common but undesirable feature of significantly extending target open reading frames with cloning related sequences, as it leaves a minimal seam of only a single extra amino acid to either side of the protein. The method has proven to be very robust and suitable for all common pro- and eukaryotic expression systems. It considerably speeds up the generation of expression constructs compared to traditional methods and thus facilitates a broader expression screening.