Submit Data |  Help |  Video Tutorials |  News |  Publications |  FTP Download |  REST API |  Citing RGD |  Contact   

Mutational analysis of predicted interactions between the catalytic and P domains of prohormone convertase 3 (PC3/PC1).

Authors: Ueda, K  Lipkind, GM  Zhou, A  Zhu, X  Kuznetsov, A  Philipson, L  Gardner, P  Zhang, C  Steiner, DF 
Citation: Ueda K, etal., Proc Natl Acad Sci U S A 2003 May 13;100(10):5622-7.
Pubmed: (View Article at PubMed) PMID:12721373
DOI: Full-text: DOI:10.1073/pnas.0631617100

The subtilisin-like prohormone convertases (PCs) contain an essential downstream domain (P domain), which has been predicted to have a beta-barrel structure that interacts with and stabilizes the catalytic domain (CAT). To assess possible sites of hydrophobic interaction, a series of mutant PC3-enhanced GFP constructs were prepared in which selected nonpolar residues on the surface of CAT were substituted by the corresponding polar residues in subtilisin Carlsberg. To investigate the folding potential of the isolated P domain, signal peptide-P domain-enhanced GFP constructs with mutated andor truncated P domains were also made. All mutants were expressed in betaTC3 cells, and their subcellular localization and secretion were determined. The mutants fell into three main groups: (i) Golgisecreted, (ii) ERnonsecreted, and (iii) apoptosis inducing. The destabilizing CAT mutations indicate that the side chains of V292, T328, L351, Q408, H409, V412, and F441 and nonpolar fragments of the side chains of R405 and W413 form a hydrophobic patch on CAT that interacts with the P domain. We also have found that the P domain can fold independently, as indicated by its secretion. Interestingly, T594, which is near the P domain C terminus, was not essential for P domain secretion but is crucial for the stability of intact PC3. T594V produced a stable enzyme, but T594D did not, which suggests that T594 participates in important hydrophobic interactions within PC3. These findings support our conclusion that the catalytic and P domains contribute to the folding and thermodynamic stability of the convertases through reciprocal hydrophobic interactions.

Annotation

Gene Ontology Annotations
Objects Annotated

Additional Information

 
CRRD Object Information
CRRD ID: 734486
Created: 2004-01-15
Species: All species
Last Modified: 2004-08-23
Status: ACTIVE



NHLBI Logo

RGD is funded by grant HL64541 from the National Heart, Lung, and Blood Institute on behalf of the NIH.