Fig. 1. The hexapeptide H5 achieves high reactivity and selectivity for GzmB by accessing a unique binding pocket.

a Fluorogenic hexapeptide H5 and fluorescence quantum yields of intact and cleaved probe. b Tetra- and hexapeptide sequences, fluorescence increases and t₅₀ values upon incubation (25 μM) with hGzmB (20 nM). Data as means ± SEM (n = 3). c Time-course fluorescence of H5 (25 μM, green, 510 nm) and Ac-IEPD-AMC (25 μM, blue, 450 nm) after incubation with hGzmB (20 nM) at 37 °C. Probe H5 alone (25 μM, black, 510 nm). Data as means ± SEM (n = 5). d Cleavage rate of probe H5 by hGzmB (20 nM) as a function of substrate concentration. Data as individual replicates and kinetic values determined using the Michaelis-Menten equation (n = 3). e Fluorescence changes of probe H5 (25 µM) after incubation with proteases (20 nM) at 37 °C for 60 min. Data as means ± SEM (n = 3). For active GzmB vs pro-GzmB, p < 0.0001. For active GzmB vs GzmB+ inhibitor, p = 0.0003. For active GzmB vs GzmA, p < 0.0001. f Representative binding mode of IEPD-Dabcyl in T1 (left) and IEPDAL-Dabcyl in H5 (right) from the MD simulations. g Detailed interactions at P1’ and P2’ sites for the probe H5 with overlaid structures from 3 independent runs. P-values from two-tailed t-tests. Source data (b–e) provided as a Source Data file.