BPS2026 – Measuring conformational changes of ABCB10 during the basal and substrate-stimulated ATPase cycle using LRET

The ATP-binding cassette (ABC) transporter super-family is comprised of transmembrane proteins which utilize the energy of ATP hydrolysis to facilitate the translocation of substrates across biological membranes. Typically, ABC transporters are expected to follow an alternating access mechanism, where the binding and/or hydrolysis of ATP at the nucleotide binding domains (NBDs), and substrate binding and release at the transmembrane domains (TMDs) are accompanied by transitions between inward- and outward-facing conformations. However, the magnitude and specific triggering events for such conformational changes remain controversial, in part due to the experimental difficulties obtaining structural models of these flexible proteins in conformations that represent catalytically active transporters. We use luminescence resonance energy transfer (LRET) as an alternative approach to measure the conformational equilibrium of the human mitochondrial transporter ABCB10 during active ATP hydrolysis at 37°C in MSP2N2 nanodiscs. ABCB10, like most ABC transporters, displays a basal ATPase activity that is stimulated by substrate; thus, we can compare the conformational changes during the basal and the substrate-stimulated ATP hydrolysis cycle. In the past, we have used this approach to measure distance changes between the NBDs. Here, we are positioning the LRET probes at the top outer loops of the TMDs to estimate the magnitude of their opening when the transporter transitions between inward- and outward-facing conformations. Our TMDs data suggest small separation between the probes during basal ATP hydrolysis and a larger opening in the presence of substrate, likely to promote substrate efflux across the membrane. Interestingly, the detected TMDs opening is not compatible with widely open outward-facing models. In general, our data suggest that ABCB10 functions through small conformational changes and highlights the usefulness of LRET for the study of conformational changes of transmembrane proteins. R01GM145938.