Skip to main content
NCBI home page
The EMBO Journal logoLink to The EMBO Journal
. 1992 Mar;11(3):1141–1144. doi: 10.1002/j.1460-2075.1992.tb05154.x

An engineered retroviral proteinase from myeloblastosis associated virus acquires pH dependence and substrate specificity of the HIV-1 proteinase.

J Konvalinka 1, M Horejsí 1, M Andreánsky 1, P Novek 1, I Pichová 1, I Bláha 1, M Fábry 1, J Sedlácek 1, S Foundling 1, P Strop 1
PMCID: PMC556556  PMID: 1547777

Abstract

In an attempt to understand the structural reasons for differences in specificity and activity of proteinases from two retroviruses encoded by human immunodeficiency virus (HIV) and myeloblastosis associated virus (MAV), we mutated five key residues predicted to form part of the enzyme subsites S1, S2 and S3 in the substrate binding cleft of the wild-type MAV proteinase wMAV PR. These were changed to the residues occupying a similar or identical position in the HIV-1 enzyme. The resultant mutated MAV proteinase (mMAV PR) exhibits increased enzymatic activity, altered substrate specificity, a substantially changed pH activity profile and a higher pH stability close to that observed in the HIV-1 PR. This dramatic alteration of MAV PR activity achieved by site-directed mutagenesis suggests that we have identified the amino acid residues contributing substantially to the differences between MAV and HIV-1 proteinases.

Full text

PDF
1141

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Jaskólski M., Miller M., Rao J. K., Leis J., Wlodawer A. Structure of the aspartic protease from Rous sarcoma retrovirus refined at 2-A resolution. Biochemistry. 1990 Jun 26;29(25):5889–5898. doi: 10.1021/bi00477a002. [DOI] [PubMed] [Google Scholar]
  2. Kay J., Dunn B. M. Viral proteinases: weakness in strength. Biochim Biophys Acta. 1990 Jan 30;1048(1):1–18. doi: 10.1016/0167-4781(90)90015-t. [DOI] [PubMed] [Google Scholar]
  3. Konvalinka J., Blaha I., Skrabana R., Sedlacek J., Pichova I., Kapralek F., Kostka V., Strop P. Subsite specificity of the proteinase from myeloblastosis associated virus. FEBS Lett. 1991 Apr 22;282(1):73–76. doi: 10.1016/0014-5793(91)80447-b. [DOI] [PubMed] [Google Scholar]
  4. Konvalinka J., Strop P., Velek J., Cerna V., Kostka V., Phylip L. H., Richards A. D., Dunn B. M., Kay J. Sub-site preferences of the aspartic proteinase from the human immunodeficiency virus, HIV-1. FEBS Lett. 1990 Jul 30;268(1):35–38. doi: 10.1016/0014-5793(90)80966-m. [DOI] [PubMed] [Google Scholar]
  5. Kotler M., Danho W., Katz R. A., Leis J., Skalka A. M. Avian retroviral protease and cellular aspartic proteases are distinguished by activities on peptide substrates. J Biol Chem. 1989 Feb 25;264(6):3428–3435. [PubMed] [Google Scholar]
  6. Kräusslich H. G., Wimmer E. Viral proteinases. Annu Rev Biochem. 1988;57:701–754. doi: 10.1146/annurev.bi.57.070188.003413. [DOI] [PubMed] [Google Scholar]
  7. Lapatto R., Blundell T., Hemmings A., Overington J., Wilderspin A., Wood S., Merson J. R., Whittle P. J., Danley D. E., Geoghegan K. F. X-ray analysis of HIV-1 proteinase at 2.7 A resolution confirms structural homology among retroviral enzymes. Nature. 1989 Nov 16;342(6247):299–302. doi: 10.1038/342299a0. [DOI] [PubMed] [Google Scholar]
  8. Miller M., Schneider J., Sathyanarayana B. K., Toth M. V., Marshall G. R., Clawson L., Selk L., Kent S. B., Wlodawer A. Structure of complex of synthetic HIV-1 protease with a substrate-based inhibitor at 2.3 A resolution. Science. 1989 Dec 1;246(4934):1149–1152. doi: 10.1126/science.2686029. [DOI] [PubMed] [Google Scholar]
  9. Roberts N. A., Martin J. A., Kinchington D., Broadhurst A. V., Craig J. C., Duncan I. B., Galpin S. A., Handa B. K., Kay J., Kröhn A. Rational design of peptide-based HIV proteinase inhibitors. Science. 1990 Apr 20;248(4953):358–361. doi: 10.1126/science.2183354. [DOI] [PubMed] [Google Scholar]
  10. Schechter I., Berger A. On the size of the active site in proteases. I. Papain. Biochem Biophys Res Commun. 1967 Apr 20;27(2):157–162. doi: 10.1016/s0006-291x(67)80055-x. [DOI] [PubMed] [Google Scholar]
  11. Sedlácek J., Strop P., Kaprálek F., Pecenka V., Kostka V., Trávnícek M., Ríman J. Processed enzymatically active protease (p15gag) of avian retrovirus obtained in an E. coli system expressing a recombinant precursor (Pr25lac-delta gag). FEBS Lett. 1988 Sep 12;237(1-2):187–190. doi: 10.1016/0014-5793(88)80198-4. [DOI] [PubMed] [Google Scholar]
  12. Strop P., Konvalinka J., Stys D., Pavlickova L., Blaha I., Velek J., Travnicek M., Kostka V., Sedlacek J. Specificity studies on retroviral proteinase from myeloblastosis-associated virus. Biochemistry. 1991 Apr 9;30(14):3437–3443. doi: 10.1021/bi00228a013. [DOI] [PubMed] [Google Scholar]
  13. Wlodawer A., Miller M., Jaskólski M., Sathyanarayana B. K., Baldwin E., Weber I. T., Selk L. M., Clawson L., Schneider J., Kent S. B. Conserved folding in retroviral proteases: crystal structure of a synthetic HIV-1 protease. Science. 1989 Aug 11;245(4918):616–621. doi: 10.1126/science.2548279. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES