Fig. 6: Allosteric modulation of CTSB through dCST3 binding into the discovered pocket.
A Snapshot of major structural clusters as obtained by TIGER2hPE simulations of cathepsin B with different models of the trypsin-processed cystatin C. Binding to an allosteric pocket on CTSB’s surface results in a pronounced opening of the occluding loop. B Average centre-of-mass distance (±SD) between the active site and occluding loop atoms for major cluster states generated throughout different TIGER2hPE simulations. Binding configurations into the allosteric site (four simulations) show significant differences to isolated CST3 (two simulations). Significance was calculated by one-way Anova followed by Dunn–Šidák correction for multiple testing (p < 0.0001 for all binding configurations into the allosteric site (sim5 n = 5935, sim6 n = 8563, sim7 n = 980, sim8 n = 7026) against isolated CST3 (sim1 n = 7378, sim2 n = 5516)). C Resulting fraction of open states using a distance threshold of 16.5 Å. Allosteric modulation by CST3 results in a 3.15-fold increase in active states. D Allosteric sites predicted by AlloSitePro (green, magenta) in superposition with the binding site predicted by TIGER2hPE simulations (cyan) and intersection surface area (purple). Mutation target selected based on simulation data are shown as ball-and-stick. E Activity tests with CTSB wild-type and mutated allosteric site (S169A, Y215F, E221V, D222P, H224L, D333P) under the influence of cystatin C monomer- and dimer-fractions. The wild-type shows a three-fold increase in activity when measured in complex with dimeric cystatin C, similar to the increase in open states. This activating effect is diminished with the allosteric site mutated (WT CTSB vs WT CTSB + CST3 monomer p = 0.0048, mut. CTSB vs mut. CTSB + CST3 monomer p = 0.0009, WT CTSB vs WT CTSB + CST3 dimer p < 0.0001, mut. CTSB vs WT CTSB + CST3 dimer p < 0.0001, WT CTSB + CST3 dimer vs mut. CTSB + CST3 dimer p < 0.0001). F Schematic illustration summarises the effect of CST3 processing for the balance of CTSB and CTSL activity and intracellular trypsinogen activation. Figure 6E shows three independent experiments; significance was calculated by one-way Anova followed by Dunn–Šidák correction for multiple testing. Results are shown as mean ± SD. Significance levels of p < 0.05 are marked by an asterisk. Source data are provided as Source Data file.
