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

abstract

  • 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. An extension of the mutant cycle approach is employed to probe basic residues that interact with KDL. We find the molecular recognition of KDL is driven by a positive coupling entropy (as large as -100 kJ/mol at 298 K) that outweighs unfavorable coupling enthalpy. These findings indicate that alterations in solvent reorganization and conformational entropy can contribute significantly to the free energy of protein-lipid association. The results presented herein showcase the advantage of native MS to obtain thermodynamic insight into protein-lipid interactions that would otherwise be intractable using traditional approaches, and this enabling technology will be instrumental in the life sciences and drug discovery.

published proceedings

  • Elife

author list (cited authors)

  • Lyu, J., Zhang, T., Marty, M. T., Clemmer, D., Russell, D. H., & Laganowsky, A.

citation count

  • 1

complete list of authors

  • Lyu, Jixing||Zhang, Tianqi||Marty, Michael T||Clemmer, David||Russell, David H||Laganowsky, Arthur

publication date

  • January 2024

published in