Fig. 3: ML model and functional analysis of the IGFBP5 anchor peptide binding site in PAPP-A2.
From: Cryo-EM structure of human PAPP-A2 and mechanism of substrate recognition
a ML-PAPP-A2/IGFBP5 model. PAPP-A2 domains are color-coded and IGFBP5 is shown in orange. Some regions of IGFBP5 are shown as transparent cartoon to make the anchor peptide (boxed) more visible. Confidence scores are shown in Supplementary Fig. 7a. A full, zoomed-in view of the binding site is shown in Supplementary Fig. 7c. b Zoomed-in view of the anchor peptide binding site in the M1 domain (cyan) of the ML-PAPP-A2/IGFBP5 model. c Zoomed-in view of the anchor-peptide binding site in the M1 domain (cyan) from the PAPP-ABP5 (PDB 7ufg). d Cleavage comparison of wildtype and mutant PAPP-A2 and PAPP-A proteins in the M1 domain anchor peptide binding site. Error bars represent the standard deviation of experiments done in triplicate. Protein quality for wildtype and mutant PAPP-A2, and a representative cleavage assay is shown in Supplementary Fig. 9a and Supplementary Fig. 9b, respectively. Protein quality for wildtype and mutant PAPP-A, and a representative cleavage assay is shown in Supplementary Fig. 9c and Supplementary Fig. 9d, respectively. e Zoomed-in view of the ML-PAPP-A2/IGFBP5 model around anchor peptide residues 136-138. The MP domain is in green and the M1 domain is in cyan. Black, dashed lines represent hydrogen bonds. f Zoomed-in view of PAPP-ABP5 (PDB 7ufg) around anchor peptide residues 136-138. The red dashes indicate that residue R137 is too far away to interact with the MP domain. g Cleavage comparison of PAPP-A2 and PAPP-A on wild type and R136D/R137D/K138D IGFBP5 mutant substrate. Error bars represent the standard deviation of experiments done in triplicate. IGFBP5 substrate protein quality is in Supplemental Fig. 8d. The graphed data is representative of cleavage efficiency at a concentration of 8 nM for both PAPP-A and PAPP-A2. No additional cleavage was observed for IGFBP5 R136D/R137D/K138D at higher concentrations of PAPP-A2 (Supplementary Fig. 10a), but PAPP-A cleaved the substrate completely at concentrations above 8 nM (Supplementary Fig. 10b).
