Fig. 1: Cryo-EM reconstruction of PAPP-A2 at 3.13 Å resolution.

a Schematic domain organization of PAPP-A2. Numbering shown here and discussed in the text refers to mature PAPP-A2 after removal of the signal secretion and pro-peptide sequences. Domains corresponding to observed density are color-coded while unresolved domains are shown in white. b Cryo-EM map density of PAPP-A2 monomer with domains colored according to Fig. 1a. c Alignment of PAPP-A2 (colored domains) with one copy of PAPP-A_BP5 (PDB 7ufg). PAPP-A is shown in transparent gray and olive cartoon and one copy of the anchor peptide is shown as orange cartoon. d PAPP-A2 active site. PAPP-A2 MP domain residues are shown as green sticks. Zinc and water are in gray and red spheres, respectively. Zinc coordination bonds are shown as yellow dashes and hydrogen bonds are black dashes. Aligned PAPP-A residues (from PDB 7ufg) are shown as plum sticks and IGFBP5 anchor peptide is shown as transparent orange cartoon with sticks. Residues for both PAPP-A and IGFBP5 are labeled in parentheses to differentiate them from PAPP-A2 residues. e Cleavage assays for wildtype and active site PAPP-A2 mutants using IGFBP5 as the substrate. For assays containing IGFBP5 as the substrate shown here and in the remainder of the report, unless indicated otherwise, a concentration of 500 nM was used and reactions were incubated for 4 h at 37 °C (please see METHODS section for further details). Data shown in graphs here and in the rest of the report, unless indicated otherwise, represent a concentration of 30 nM PAPP-A2 in assays. Protein quality and representative, primary data are shown in Supplementary Fig. 4a and Supplementary Fig. 4b, respectively. Additional data for all experiments is included in Supplementary Data 2. f Cleavage assays for wild type and active site PAPP-A2 mutants using IGFBP3 as a substrate. Representative, primary data is shown in Supplementary Fig. 4c. Assays shown here and elsewhere in the manuscript used 500 nM of IGFBP3. IGFBP3 was observed to be cleaved less efficiently than IGFBP5. Therefore, for assays containing IGFBP3 as a substrate shown here and in the remainder of the report, reactions were incubated for 18 h at 37 °C. g Cleavage assays for wild type and anchor peptide binding deficient mutant PAPP-A2 or PAPP-A using IGFBP5 as a substrate. Protein quality is shown in Supplementary Fig. 5a and Supplementary Fig. 5b, and representative data is in Supplementary Fig. 5c. In assay results shown in Fig. 1.e–g, error bars represent the standard deviation of experiments done in triplicate.