Fig. 9: GS⁻ binding to and conformational changes of glutathionylated HsGrx5 variants.

a Fraction of bound states with the value for wild-type HsGrx5 (WT) normalized to 100%. Error bars show the standard error of the mean over four replications for each system. Datasets were tested with a two-tailed t test assuming unequal variances; null-hypothesis of equal means could not be rejected with P < 0.05. For WT and HsGrx5^RR P = 0.17; for WT and HsGrx5^loop P = 0.10; for WT and HsGrx5^RR+loop P = 0.08. b For each of the systems of the WT and the variants HsGrx5^RR, HsGrx5^loop, and HsGrx5^RR+loop, bound states were clustered with respect to the structural deviation of the active site and the closest GS⁻ from the solution. The structure representing the most populated cluster is shown in the middle, and important areas of the structure representing the second most populated cluster are shown in the boxes next to it. The covalently bound glutathione moiety at Cys67 is depicted in salmon, the freely diffusing GS⁻ in pink. Residues marked in orange show the mutated residues in the variants; the active site loop is colored red. Residue are numbered always according to the WT sequence. c The occupation density of the diffusing GS⁻ is shown as a blue grid on the structure of each HsGrx5 variant. Bound states were combined across replications for this analysis, and the threshold of the density grids is the same for all variants. Arrows show the additional patterns emerging for the RR and loop variants compared to the WT, respectively. d Partial unfolding of helix 2 (residues marked orange) was observed in one of the replications of HsGrx5 WT (blue), HsGrx5^loop is shown for comparison of the helical conformation (gray). e Residue-wise fraction of secondary structure content averaged over the four MD replications of the HsGrx5 variants and the ScGrx7 WT for helix 2. Only relevant secondary structure types are shown: α-helix in red, turn or bend in blue, and 3₁₀-helix in black. Adjacent residues Cys67/Cys108 and His80/Ser121 have a coil conformation.