Fig. 4: The catalytic domain (CD) of the PAPP-A subunit.

a Cartoon representation of the isolated CD (red) with the ZnM loop (gray) and the two inserted LNR modules (LNR1-2, green) colored separately. This color scheme is used throughout the figure. The active site cleft, containing the Zn2+ ion coordinated by three histidine residues (H562, H566, H572), is facing right and located between the LNR modules and the ZnM loop. b Enlarged view of the active site cleft of the CD shown in two orientations. The methionine residue of the Met-turn (M636) is indicated. In the position of the glutamic acid residue (E563), which serves to polarize a water molecule involved in nucleophilic attack at the scissile peptide bond, is shown a glutamine residue, present in the recombinant protein (E563Q) used in this study. The ZnM loop is anchored to the core of the CD by two disulfide bonds (C327-C587 and C612-C643). One disulfide bond (C583-C622) and an unpaired cysteine residue (C600) is present within the ZnM loop. The position of a Ca2+ ion and coordinating residues (E589, D598, T603) of the ZnM loop are shown. Insets show map density around the active site Zn2+ ion (top) and the Ca2+ ion of the ZnM loop (bottom). The map (MAP1) was contoured at 4.0σ. c Dimeric PAPP-A and the 2:2 PAPP-A·STC2 complex are both able to cleave a 26-residue peptide derived from the PAPP-A substrate, IGFBP-4 (L(118)QKHFAKIRDRSTSGGKMKVNGAPRE(143), cleavage occurs at MK). The assay is based on intramolecular quenched fluorescence. Average values with error bars (SD) are plotted. Kinetic parameters with SD for both reactions are shown. n = 3 (PAPP-A) or 4 (PAPP-A·STC2) independent experiments. Source data are provided as a Source data file.