Double and triple thermodynamic mutant cycles reveal the basis for specific MsbA-lipid interactions.

Structural and functional studies of the ATP-binding cassette transporter MsbA have revealed two distinct lipopolysaccharide (LPS) binding sites: one located in the central cavity and the other at a membrane-facing, exterior site. Although these binding sites are known to be important for MsbA function, the thermodynamic basis for these specific MsbA-LPS interactions is not well understood. Here, we use native mass spectrometry to determine the thermodynamics of MsbA interacting with the LPS-precursor 3-deoxy-D- manno -oct-2-ulosonic acid (Kdo) 2 -lipid A (KDL). The binding of KDL is solely driven by entropy, despite the transporter adopting an inward-facing conformation or trapped in an outward-facing conformation with adenosine 5'-diphosphate and vanadate. Double and single mutant cycles reveal that pairwise residues engage KDL with a positive coupling energy, which stems from positive coupling entropy (as large as -100 kJ/mol at 298K) outweighing unfavorable coupling enthalpy. Our results provide new insight into how pairwise interactions can thermodynamically contribute to specific, high-affinity lipid binding in membrane proteins, which may have implications in the design of small molecules targeting specific lipid-protein interactions.