Fig. 3: Structure and activity of evolved pre-SplB variants.
From: Evolution of protease activation and specificity via alpha-2-macroglobulin-mediated covalent capture
a SplB structure (PDB: 2VID) with positions most enriched in directed evolution campaign highlighted in red. Positions previously implicated in SplB activation (black) and the underlying hydrogen bond network are also shown. b Michaelis-Menten kinetics of pre-SplB mutants and mature SplB (n = 3 independent measurements of the same protein purification, mean and SD). c Structural alignment of active SplB (PDB: 2VID) with pre-form SplB variants GS-SplB (PDB: 4K1S), His6-SplB-E3Q/S154R (PDB: 6YV5) and His6-SplB-E2K/E3Q/S154R (PDB: 6YV6). The root-mean-square deviation (RMSD) is indicated for each alignment. Top panel: Overview of the whole structure. Bottom panel: Close-up of the catalytic triad. d Detailed view of the hydrogen bonds formed involving the N-terminus. The residues that were described by Pustelny et al.21 to be important for SplB activation are highlighted. Hydrogen bonds are indicated as green dashes. Source data are provided as a Source Data file for b.
