ATP Binding Cassette (ABC) transporters shuttle diverse substrates across biological membranes. Transport is achieved through an ATP-driven closure of the two nucleotide binding sites (NBSs), which induces a transition between an inward-facing (IF) and an outward-facing (OF) conformation of the connected transmembrane domains (TMDs)1,2. While this IF-to-OF transition is rather well understood, the sequence of ATP hydrolysis and release during the reverse transition (OF-to-IF) remains elusive. This is particularly relevant for heterodimeric ABC transporters with asymmetric NBSs. These transporters possess an ATPase active consensus NBS (c-NBS) and a degenerate NBS (d-NBS) with little or no ATPase activity3. Asymmetric NBSs are present among several ABC subfamilies and their functional relevance is not well understood.
Here, we addressed these open questions using DEER/PELDOR spectroscopy of the heterodimeric ABC exporter TmrAB. We replaced the Mg2+ ions with paramagnetic Mn2+ ions to simultaneously monitor ATP-Mn2+ binding and the conformation of the NBSs. In addition, a nitroxide spin pair was introduced at the TMDs to monitor its coupled motion with the NBSs. Our Mn2+-NO DEER/PELDOR results reveal stable ATP binding at the d-NBS, whereas at the c-NBS, ATP binding is seen only upon vanadate trapping. In the non-functional E-to-Q variant, ATP binding is observed at both NBSs, suggesting that ATP is immediately hydrolyzed at the functional c-NBS. Concomitant observation of the TMDs (NO-NO PELDOR) and the NBSs (Mn2+-NO PELDOR) shows a significant fraction of transporters in the OF conformation, while the c-NBS has lost Mn2+. Further time-resolved measurements suggest that ATP hydrolysis at the active c-NBS triggers the reverse (OF-to-IF) transition, whereas opening of the impaired d-NBS limits the overall kinetics of this process. This regulatory function of the d-NBS may be modulated by the presence of substrate and or ADP.