Fig. 6: Allosteric activation of monomeric HTRA1 by peptidic modulators.
From: Rational correction of pathogenic conformational defects in HTRA1
a Model of activation of trimeric and monomeric HTRA1. Left panel: in wt HTRA1, the substrate bound to the active site interacts with loop L3, followed by an inter-protomer loop L3-LD interaction. This interaction mediates the proper positioning of loops L1 and L2, and of the catalytic site. Right panel: in monomeric mutant HTRA1, loop L3-LD interaction is disrupted. VDAC2 acts as a surrogate of the missing loop L3 from an adjacent protomer leading to the proper positioning of loop LD and thus of loops L1 and L2. Therefore, in active monomeric HTRA1, the activation domain resembles that of classic monomeric serine proteases61,62. b Binding of VDAC2 to HTRA1 wt trimers or to R274Q monomers analyzed by native MS. Graphs depict the occupancy (average ± SD of 4 independent datasets). c Oligomeric states of HTRA1 wt and R274Q in the absence (Ctrl) or presence of VDAC2 analyzed by NMR DOSY (mean ± SD of 6 technical replicates). d Normalized cleavage rates of peptidic fluorescence-quenched substrate (2 µM) by HTRA1 wt (30 nM) and R274Q (300 nM) plotted vs. VDAC2 concentration (mean ± SD of 3 independent datasets with technical duplicates). Data were fitted to the hyperbolic weak-binding equation to obtain Vmax and Kd for peptide binding; brackets: SE of the fit. e Model of the binding mode of VDAC2 at the N-terminal tip of HTRA1-R274Q. Backbones of HTRA1 (orange) and VDAC2 (magenta), sidechains of the catalytic triad’s residues shown with sticks (licorice style). Green: hydrophilic residues; gray: hydrophobic residues. Surface representation is used for HTRA1 (transparent surface). Inset: close-up view of selected HTRA1’s residues nearby VDAC2. Sidechains of HTRA1’s residues depicted with sticks (licorice style) and colored in yellow except oxygen (red) and nitrogen (blue). Sidechains of VDAC2 residues are shown with balls and sticks (CPK style) and colored in light blue except oxygen (red) and nitrogen (blue). f Representative structures of the 10 largest clusters of the advanced sampling simulations (three replicas combined). Numbers: relative population of each cluster (percent of the simulation’s frames).
