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.
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.ABC transporter | conformational dynamics | membrane proteins | peptide transport | transporter associated with antigen processing
Polyacrylamide gel electrophoresis (PAGE) and immunoblotting (Western blotting) are the most common methods in life science. In conjunction with these methods, the polyhistidine-tag has proven to be a superb fusion tag for protein purification as well as specific protein detection by immunoblotting, which led to a vast amount of commercially available antibodies. Nevertheless, antibody batch-to-batch variations and nonspecific binding complicate the laborious procedure. The interaction principle applied for His-tagged protein purification by metal-affinity chromatography using N-nitrilotriacetic acid (NTA) was employed to develop small high-affinity lock-and-key molecules coupled to a fluorophore. These multivalent NTA probes allow specific detection of His-tagged proteins by fluorescence. Here, we report on HisQuick-PAGE as a fast and versatile immunoblot alternative, using such high-affinity fluorescent super-chelator probes. The procedure allows direct, fast, and ultra-sensitive in-gel detection and analysis of soluble proteins as well as intact membrane protein complexes and macromolecular ribonucleoprotein particles.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.