Conformational selection drives substrate-ATP coupling in the type VI ABC transporter

Abstract

The mechanism of substrate-ATP coupling is central to the function of ABC transporters. Substrates bind to the specialized transmembrane domains (TMDs), whereas ATP binding and hydrolysis occur in the conserved intracellular nucleotide-binding domains (NBDs). Efficient substrate transport requires coupling between these processes, in which the intracellular coupling helices play an essential role. Here, we probed both coupling helices of the type VI ABC transporter LptB2FG(C), the core complex responsible for intermembrane lipopolysaccharide (LPS) transport in Gram-negative bacteria, using site-specific 19F labeling and ultra-fast MAS NMR. We show that both coupling helices exist in an equilibrium among three major conformational states. Progression through the coupling cycle occurs via conformational selection, as LPS and nucleotide binding shift the conformational equilibrium, providing evidence for bidirectional communication between the TMDs and NBDs. Furthermore, the conformational distributions of the two coupling helices are asymmetric. Finally, complex formation with LptC, a unique feature of type VI ABC transporters, symmetrizes the conformational landscape of the coupling helices and facilitates transitions between conformational states, thereby enhancing the efficiency of coupling ATP hydrolysis to LPS transport. Together, our findings establish the coupling helices as dynamic allosteric elements that integrate nucleotide and substrate binding through conformational selection, providing a mechanistic framework for substrate-ATP coupling in ABC transporters.