Identification of Amino Acid Residues in Bone Morphogenetic Protein-1 Important for Procollagen C-proteinase Activity*

Bone morphogenetic protein (BMP)-1, which belongs to the tolloid subgroup of astacin-like zinc metalloproteinases, cleaves the C-propeptides of procollagen at the physiologic site and is, therefore, a procollagen C-proteinase (PCP). Cleavage occurs between a specific alanine or glycine residue (depending on the procollagen chain) and an invariant aspartic acid residue in each of the three chains of procollagen. To learn more about how BMP-1 exhibits PCP activity we mapped the primary structure of BMP-1 onto the x-ray crystal structure of astacin and identified residues in the metalloproteinase domain of BMP-1 for subsequent site-directed mutagenesis studies. Recombinant wild-type and mutant BMP-1 were expressed in COS-7 cells and assayed for PCP activity using type I procollagen as the substrate. We showed that substitution of alanine for Glu⁹⁴, which occurs in the HEXXH zinc-binding motif of BMP-1, abolishes PCP activity. Furthermore, mutation of residues Lys⁸⁷ and Lys¹⁷⁶, which are located in the S1′ pocket of the enzyme and are therefore adjacent to the P1′ residue in the substrate, reduced the proteolytic activity of BMP-1 by ∼50%. A surprising observation was that mutation of Cys⁶⁶ reduced the activity to 20%, suggesting that this residue is crucial for activity. Further experiments showed that Cys⁶⁶ and Cys⁶³, which are located in the tolloid-specific sequence Cys⁶³-Gly⁶⁴-Cys⁶⁵-Cys⁶⁶in the active site, most likely form a disulfide bridge.