Structural elements of PC2 required for interaction with its helper protein 7B2.
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.
Zhu
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Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans, Louisiana 70112, USA.
Muller
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Mains
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Lindberg
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D000595:Amino Acid Sequence; D002460:Cell Line; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D051859:Neuroendocrine Secretory Protein 7B2; D010907:Pituitary Hormones / Q000378:metabolism*; D045706:Proprotein Convertase 2; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D013381:Subtilisins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism
D009419:Nerve Tissue Proteins; D051859:Neuroendocrine Secretory Protein 7B2; D010907:Pituitary Hormones; D014443:Tyrosine; D013381:Subtilisins; D045706:Proprotein Convertase 2