Effects of ligand binding on the energy landscape of acyl-CoA-binding protein

TL;DR

This study combines experimental and computational methods to show that ligand binding increases the mechanical stability of acyl-CoA-binding protein by raising the activation free energy for unfolding without altering its unfolding pathway.

Contribution

It provides new insights into how ligand binding stabilizes protein structure mechanically and thermodynamically, with implications for designing stabilizing ligands.

Findings

Ligand binding increases activation free energy for unfolding.

Ligand binding does not change the transition state position.

Unfolding pathway remains largely unaffected by ligand.

Abstract

Binding of ligands is often crucial for function yet the effects of ligand binding on the mechanical stability and energy landscape of proteins are incompletely understood. Here we use a combination of single-molecule optical tweezers and MD simulations to investigate the effect of ligand binding on the energy landscape of acyl-coenzyme A (CoA) binding protein (ACBP). ACBP is a topologically simple and highly conserved four-alpha-helix bundle protein that acts as an intracellular transporter and buffer for fatty-acyl CoA and is active in membrane assembly. We have previously described the behavior of ACBP under tension, revealing a highly extended transition state (TS) located almost halfway between the unfolded and native states. Here, we performed force-ramp and force-jump experiments, in combination with advanced statistical analysis, to show that octanoyl-CoA binding increases the activation free energy for the unfolding reaction of ACBP without affecting the position of the transition state along the reaction coordinate. It follows that ligand binding enhances the mechanical resistance and thermodynamic stability of the protein, without changing its mechanical compliance. Steered molecular dynamics simulations allowed us to rationalize the results in terms of key interactions that octanoyl-CoA establishes with the four alpha-helices of ACBP and showed that the unfolding pathway is marginally affected by the ligand. The results show that ligand-induced mechanical stabilization effects can be complex and may prove useful for the rational design of stabilizing ligands.