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

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 298K) 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.

Figure 1.. The two distinct LPS binding sites of MsbA and their molecular interactions.

a) Two views of LPS bound to the interior site or central cavity of MsbA. The protein shown is also bound to the inhibitor G907 (PDB 6BPL). The protein and lipid are shown in cartoon and stick representation, respectively. b) Molecular details of the residues interacting with LPS at the interior site. Bonds are shown as dashed yellow lines along with residue labels. c) Two views of the KDL molecules bound to the two exterior binding sites of MsbA that are symmetrically related (PDB 8DMM). Shown as described in panel A. d) Molecular view of KDL bound to MsbA and shown as described in panel B. The asterisk denotes residues selected for mutant cycle analysis.

Figure 2.. Thermodynamics of KDL binding at the interior site to wild-type and mutant MsbA.

a) Representative deconvoluted native mass spectra of 0.39 μM wild-type MsbA in C10E5 and in the presence of 0.6 μM KDL recorded at different solution temperatures. b) Plot of mole fraction of MsbA (KDL)_0–3 determined from titration of KDL (dots) at 298 K and resulting fit from a sequential ligand binding model (solid line, R² = 0.99). c) van’ t Hoff plot for MsbA(KDL)_1–3 and resulting fit of a nonlinear van’ t Hoff equation. d) Thermodynamics for MsbA and mutants (MsbA^R78A, MsbA^K299A and MsbA^R78A,K299A) binding KDL at 298 K. e) Mutant cycles for MsbA and mutants with (from left to right) ΔΔG (mutant minus wild-type), ΔΔH and Δ(−TΔS) values indicated over the respective arrows. Shown are values at 298K. Reported are the average and standard deviation from repeated measurements (n = 3).

Figure 3.. Triple mutant cycle analysis of the exterior LPS binding site of MsbA.

a) Thermodynamics for MsbA and mutants (MsbA^R188A, MsbA^R238A, MsbA^K243A, MsbA^R188A,R243A, MsbA^R188A,K243A, MsbA^R238A,R243A, and MsbA^{R188A,R238A,K299A}) binding KDL at 298 K. b) Triple mutant cycles for MsbA and mutants with (from left to right) ΔΔG, ΔΔH and Δ(−TΔS) values indicated over the respective arrows. Shown are values at 298K. Reported are the average and standard deviation from repeated measurements (n = 3).

Figure 4.. Mutant cycle of MsbA residues located within the interior and exterior LOS bind sites.

a) Thermodynamic signatures for MsbA and mutants binding KDL at 298 K. b-c) Double mutant cycle analysis for R188 and K299. Shown are results for the first (panel b) and second (panel c) KDL binding to MsbA. Shown from left to right is ΔΔG, ΔΔH and Δ(−TΔS) and the values indicated over the respective arrows at 298K. Reported are the average and standard deviation from repeated measurements (n = 3).

Figure 5.. Double mutant cycles reveal thermodynamic insight into KDL binding vanadate-trapped MsbA.

a) Thermodynamic signatures for MsbA and mutants binding KDL at 298 K. b-c) Double mutant cycle analysis for pairs of R188, R238, and K243 with a total of three combinations. Shown are results for the first (panel b) and second (panel c) KDL binding to MsbA trapped in an open, OF conformation with ADP and vanadate. Within each panel, ΔΔG, ΔΔH and Δ(−TΔS) are shown from left to right and their values at 298K indicated over the respective arrows. Reported are the average and standard deviation from repeated measurements (n = 3).

Figure 6.. The role of solvent in contributing to the molecular recognition of membrane protein-lipid complexes.

The lipid headgroup and binding pocket (basic patch illustrated in blue) on the membrane protein are solvated. The ordered solvent (shown in light blue) is then displaced upon lipid binding the membrane protein leading to solvent reorganization. The displacement of ordered solvent (show in light green) contributes to favorable entropy. This process enables the formation of a high affinity, stable membrane protein-lipid complex.