Capturing transient states of heterodimeric ABC transporter TM287/288 by time-resolved small-angle X-ray scattering

Structures of the heterodimeric ATP-binding cassette (ABC) transporter TM287/288 have previously been determined in several states indicating large conformational changes during its reaction cycle. However, for a complete description of the cycle, transient states (such as an occluded state) are still missing, as they are difficult to capture with static structural biology techniques. Here, we investigate the kinetics and accompanying structural changes in the course of the reaction cycle of full-length TM287/288 using time-resolved small-angle X-ray scattering initiated by stopped-flow mixing. The use of active-site mutants and state-specific sybodies/nanobodies enabled us to dissect the temporal events involved in the ATP-driven conformational cycle of TM287/288 and reveal a transient occluded state of this ABC transporter.