Abstract
The structures of the eukaryotic subtilisin protease family members can be divided into four distinct domains as follows: the proregion, the catalytic domain, the P domain, and the carboxyl-terminal region. Although these enzymes are evolutionarily related, only prohormone convertase 2 (PC2) requires 7B2 for activation. To examine the potential contribution of each domain of PC2 to PC2-7B2 interactions, we performed sequential deletions, site-directed mutagenesis, and domain swapping to replace individual domains or particular amino acids of pro-PC2 with the corresponding segments/amino acids of pro-PC1. These chimeras and mutant enzyme molecules were then expressed in AtT-20 cells and analyzed for 7B2 binding, maturation ability, and enzymatic activity. The results revealed that 1) the PC2 proregion is required but is not sufficient to confer 7B2 binding; 2) the P domain is required for the stabilization of PC2 structure and is not exchangeable with the P domain of PC1; and 3) the carboxyl-terminal domain is not involved in 7B2 binding. Site-directed mutagenesis of pro-PC2 further showed that a single residue replacement in the catalytic domain, Tyr-194 --> Asp, prevented pro-PC2 from binding 7B2 and blocked activation. This residue is present within a loop rich in aromatic amino acids which appears to be on the surface of the molecule as extrapolated from the crystal structure of subtilisin. This loop may represent the primary recognition site for 7B2 within the catalytic domain.
Highlights
The processing of many peptide hormones and other protein precursors is mediated by a family of subtilisin-like serine endoproteases that act through cleavage at paired or multiple basic residues
We first examined the role of the PC2 proregion in the binding of 7B2 by a co-immunoprecipitation study using FurP:PC2 and PC2 containing a furin proregion (PC2-P):PC1 [18]
AtT-20/PC2/21-kDa 7B2, AtT20/furin rather than the PC2 proregion (Fur-P):PC2/21-kDa 7B2, and AtT-20/PC2-P:PC1/21-kDa 7B2 cells were labeled with [35S]Met and -Cys ProMix for 20 min, and cell extracts were immunoprecipitated under non-denaturing conditions using antisera LSU18 and LSU3 to examine potential co-immunoprecipitation of PC2 domains with 7B2
Summary
The processing of many peptide hormones and other protein precursors is mediated by a family of subtilisin-like serine endoproteases that act through cleavage at paired or multiple basic residues. To examine the potential contribution of these PC2-specific sequences to 7B2 binding (as well as the contributions of the proregion, the catalytic region, the P domain, and the carboxylterminal tail of pro-PC2), we used sequential deletion and domain swapping as well as site-directed mutagenesis to replace individual domains or particular amino acids of pro-PC2 with the corresponding segments or individual amino acids of pro-PC1. These chimeras and mutant enzyme molecules were expressed in neuroendocrine cells and analyzed for 7B2 binding, for maturation, and for enzymatic activity. We further identified a single amino acid in the catalytic domain, Tyr-194, as critical to PC2–7B2 binding
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