Accessibility explains preferred thiol-disulfide isomerization in a protein domain

Abstract

Disulfide bonds are key stabilizing and yet potentially labile cross-links in proteins. While spontaneous disulfide rearrangement through thiol-disulfide exchange is increasingly recognized to play an important physiological role, its molecular determinants are still largely unknown. Here, we used a novel hybrid Monte Carlo and Molecular Dynamics scheme to elucidate the molecular principles of thiol-disulfide exchange in proteins, for a mutated immunoglobulin domain as a model system. Unexpectedly, using simple proximity as the criterion for thiol-disulfide exchange, our method correctly predicts the experimentally observed regiospecificity and selectivity of the cysteine-rich protein. While redox reactivity has been examined primarily on the level of transition states and activation barriers, our results argue for accessibility of the disulfide by the attacking thiol given the highly dynamic and sterically demanding protein as a major bottleneck of thiol-disulfide exchange. This scenario may be similarly at play in other proteins with or without an evolutionarily designed active site.

Figure 1

(a) Scheme of acid-base catalyzed thiol-disulfide exchange reaction proceeding through a classical S_N2 trigonal bipyramidal transition state. (b) Schematic representation of force-clamp MD simulations during which the I27* domain unfolded under constant force application. The five cysteine residues contained in I27* are highlighted (carbon alpha and carbon beta atoms: purple spheres and sulfur atom: yellow sphere). The disulfide bond between 24Cys and 55Cys prevented the protein from fully stretching and stabilized a closed disorder loop. This loop confined two of the three free cysteines (32Cys and 47Cys) in proximity to the 24Cys–55Cys disulfide bond. The third free cysteine, 63Cys, at the C-terminal part of the protein moved away from the disulfide bond upon stretching.

Figure 2

I27* unfolds at 480pN in the majority of force-clamp MD simulations. (a) End-to-end distances as a function of time for all 100 simulations. Each horizontal panel comprises 20 simulations, 100 ns each, separated by vertical lines, resulting in 10 µs of cumulative simulation time. (b) Cumulative frequency graph representing the time of unfolding for 73 out of the 100 force-clamp MD simulations for which unfolding was observed.

Figure 3

Accessibility of the disulfide bond by the free thiols in I27* strongly varies. Distributions of distances, d_S-S, between the sulfur of one of the free cysteines (32Cys, 47Cys or 67Cys) and the sulfur of one of the disulfide bonded cysteines (24Cys or 55Cys). To guide the eye, the distance monitored is highlighted with the red line on the right scheme of each panel. Inset: close-up for distances lower than 0.5 nm.

Figure 4

Attack of the disulfide bond by 32Cys does not compromise the wide conformational space available to the loop. (a) Projection of loop dynamics along the first two eigenvectors obtained from principle component analysis (PCA) colored according to the distance between 32Cys (left) or 47Cys (right) and the center-of-mass of the disulfide bond. (b) Typical conformations representing the width of conformational space along the two major eigenvectors.

Figure 5

Thiol-disulfide exchange in I27* is regioselective for steric reasons. (a) Ratio of 55Cys (N₅₅) and 24Cys (N₂₄) swap counts, calculated with the distance criterion (green) and with the energy-based Metropolis criterion (magenta). The standard error was obtained by bootstrapping. The experimental ratios (gray) were taken from Alegre-Cebollada et al.. Data for a distance cut-off of 0.5 nm is shown (see SI, Figs S3 and S4 for other distances). Counts of swaps based on distance criterion were obtained from 100 simulations, while those based on the Metropolis criterion from a subset of 20 simulations. (b) Solvent accessible surface area (SASA) of conformations allowing a 32–>24 and a 32–>55 S_N2 reaction, and of all conformations. *** denotes p < 0.001. Bottom: a representative conformation for a 32–>24 (blue) and 32–>55 (red) reaction.