Time-Resolved Mn2+-NO and NO-NO Distance Measurements Reveal That Catalytic Asymmetry Regulates Alternating Access in an ABC Transporter

ATP-binding cassette (ABC) transporters shuttle diverse substrates across biological membranes. Transport is often achieved through a transition between an inward-facing (IF) and an outward-facing (OF) conformation of the transmembrane domains (TMDs). Asymmetric nucleotide-binding sites (NBSs) are present among several ABC subfamilies and their functional role remains elusive. Here we addressed this question using concomitant NO-NO, Mn2+-NO, and Mn2+-Mn2+ pulsed electron-electron double-resonance spectroscopy of TmrAB in a time-resolved manner. This type-IV ABC transporter undergoes a reversible transition in the presence of ATP with a significantly faster forward transition. The impaired degenerate NBS stably binds Mn2+-ATP, and Mn2+ is preferentially released at the active consensus NBS. ATP hydrolysis at the consensus NBS considerably accelerates the reverse transition. Both NBSs fully open during each conformational cycle and the degenerate NBS may regulate the kinetics of this process.

Automated summary

ATP-binding cassette (ABC) transporters are responsible for transporting various substances across biological membranes. The transition between inward-facing (IF) and outward-facing (OF) conformations of transmembrane domains (TMDs) is crucial for the transport process. This study investigated the functional role of asymmetric nucleotide-binding sites (NBSs) in ABC transporters using pulsed electron-electron double-resonance spectroscopy. The results showed that ATP hydrolysis at the consensus NBS accelerates the reverse transition, and the degenerate NBS may regulate the kinetics of this process.