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Disruption of Shape-Complementarity Markers to Create Cytotoxic Variants of Ribonuclease A
- Source :
- Journal of Molecular Biology. 354:41-54
- Publication Year :
- 2005
- Publisher :
- Elsevier BV, 2005.
-
Abstract
- Onconase (ONC), an amphibian member of the bovine pancreatic ribonuclease A (RNase A) superfamily, is in phase III clinical trials as a treatment for malignant mesothelioma. RNase A is a far more efficient catalyst of RNA cleavage than ONC but is not cytotoxic. The innate ability of ONC to evade the cytosolic ribonuclease inhibitor protein (RI) is likely to be a primary reason for its cytotoxicity. In contrast, the non-covalent interaction between RNase A and RI is one of the strongest known, with the RI.RNase A complex having a K(d) value in the femtomolar range. Here, we report on the use of the fast atomic density evaluation (FADE) algorithm to identify regions in the molecular interface of the RI.RNase A complex that exhibit a high degree of geometric complementarity. Guided by these "knobs" and "holes", we designed variants of RNase A that evade RI. The D38R/R39D/N67R/G88R substitution increased the K(d) value of the pRI.RNase A complex by 20 x 10(6)-fold (to 1.4 microM) with little change to catalytic activity or conformational stability. This and two related variants of RNase A were more toxic to human cancer cells than was ONC. Notably, these cytotoxic variants exerted their toxic activity on cancer cells selectively, and more selectively than did ONC. Substitutions that further diminish affinity for RI (which has a cytosolic concentration of 4 microM) are unlikely to produce a substantial increase in cytotoxic activity. These results demonstrate the utility of the FADE algorithm in the examination of protein-protein interfaces and represent a landmark towards the goal of developing chemotherapeutics based on mammalian ribonucleases.
- Subjects :
- Models, Molecular
biology
Cell Survival
Protein Conformation
RNase P
Ribonuclease inhibitor
Computational Biology
Ribonuclease, Pancreatic
Bovine pancreatic ribonuclease
Protein–protein interaction
Amino Acid Substitution
Biochemistry
Structural Biology
Cell Line, Tumor
Cancer cell
Mutagenesis, Site-Directed
biology.protein
Animals
Humans
Cytotoxic T cell
Ribonuclease
Cytotoxicity
Molecular Biology
Algorithms
Protein Binding
Subjects
Details
- ISSN :
- 00222836
- Volume :
- 354
- Database :
- OpenAIRE
- Journal :
- Journal of Molecular Biology
- Accession number :
- edsair.doi.dedup.....b62a99e3b1c124644535b383d61d003a