Skip to main content
NCBI home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 2000 Sep;9(9):1631–1641. doi: 10.1110/ps.9.9.1631

Systematic mutational analysis of the active-site threonine of HIV-1 proteinase: rethinking the "fireman's grip" hypothesis.

K Strisovsky 1, U Tessmer 1, J Langner 1, J Konvalinka 1, H G Kräusslich 1
PMCID: PMC2144712  PMID: 11045610

Abstract

Aspartic proteinases share a conserved network of hydrogen bonds (termed "fireman's grip"), which involves the hydroxyl groups of two threonine residues in the active site Asp-Thr-Gly triplets (Thr26 in the case of human immunodeficiency virus type 1 (HIV-1) PR). In the case of retroviral proteinases (PRs), which are active as symmetrical homodimers, these interactions occur at the dimer interface. For a systematic analysis of the "fireman's grip," Thr26 of HIV-1 PR was changed to either Ser, Cys, or Ala. The variant enzymes were tested for cleavage of HIV-1 derived peptide and polyprotein substrates. PR(T26S) and PR(T26C) showed similar or slightly reduced activity compared to wild-type HIV-1 PR, indicating that the sulfhydryl group of cysteine can substitute for the hydroxyl of the conserved threonine in this position. PR(T26A), which lacks the "fireman's grip" interaction, was virtually inactive and was monomeric in solution at conditions where wild-type PR exhibited a monomer-dimer equilibrium. All three mutations had little effect when introduced into only one chain of a linked dimer of HIV-1 PR. In this case, even changing both Thr residues to Ala yielded residual activity suggesting that the "fireman's grip" is not essential for activity but contributes significantly to dimer formation. Taken together, these results indicate that the "fireman's grip" is crucial for stabilization of the retroviral PR dimer and for overall stability of the enzyme.

Full Text

The Full Text of this article is available as a PDF (1.4 MB).

Selected References

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

  1. Adachi A., Gendelman H. E., Koenig S., Folks T., Willey R., Rabson A., Martin M. A. Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone. J Virol. 1986 Aug;59(2):284–291. doi: 10.1128/jvi.59.2.284-291.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Arad G., Chorev M., Shtorch A., Goldblum A., Kotler M. Point mutation in avian sarcoma leukaemia virus protease which increases its activity but impairs infectious virus production. J Gen Virol. 1995 Aug;76(Pt 8):1917–1925. doi: 10.1099/0022-1317-76-8-1917. [DOI] [PubMed] [Google Scholar]
  3. Bagossi P., Cheng Y. S., Oroszlan S., Tözsér J. Activity of linked HIV-1 proteinase dimers containing mutations in the active site region. Protein Eng. 1996 Nov;9(11):997–1003. doi: 10.1093/protein/9.11.997. [DOI] [PubMed] [Google Scholar]
  4. Bennett R. P., Rhee S., Craven R. C., Hunter E., Wills J. W. Amino acids encoded downstream of gag are not required by Rous sarcoma virus protease during gag-mediated assembly. J Virol. 1991 Jan;65(1):272–280. doi: 10.1128/jvi.65.1.272-280.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bizub D., Weber I. T., Cameron C. E., Leis J. P., Skalka A. M. A range of catalytic efficiencies with avian retroviral protease subunits genetically linked to form single polypeptide chains. J Biol Chem. 1991 Mar 15;266(8):4951–4958. [PubMed] [Google Scholar]
  6. Burstein H., Bizub D., Skalka A. M. Assembly and processing of avian retroviral gag polyproteins containing linked protease dimers. J Virol. 1991 Nov;65(11):6165–6172. doi: 10.1128/jvi.65.11.6165-6172.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cheng Y. S., Yin F. H., Foundling S., Blomstrom D., Kettner C. A. Stability and activity of human immunodeficiency virus protease: comparison of the natural dimer with a homologous, single-chain tethered dimer. Proc Natl Acad Sci U S A. 1990 Dec;87(24):9660–9664. doi: 10.1073/pnas.87.24.9660. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. DiIanni C. L., Davis L. J., Holloway M. K., Herber W. K., Darke P. L., Kohl N. E., Dixon R. A. Characterization of an active single polypeptide form of the human immunodeficiency virus type 1 protease. J Biol Chem. 1990 Oct 5;265(28):17348–17354. [PubMed] [Google Scholar]
  9. Francis S. E., Gluzman I. Y., Oksman A., Knickerbocker A., Mueller R., Bryant M. L., Sherman D. R., Russell D. G., Goldberg D. E. Molecular characterization and inhibition of a Plasmodium falciparum aspartic hemoglobinase. EMBO J. 1994 Jan 15;13(2):306–317. doi: 10.1002/j.1460-2075.1994.tb06263.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Grant S. K., Deckman I. C., Culp J. S., Minnich M. D., Brooks I. S., Hensley P., Debouck C., Meek T. D. Use of protein unfolding studies to determine the conformational and dimeric stabilities of HIV-1 and SIV proteases. Biochemistry. 1992 Oct 6;31(39):9491–9501. doi: 10.1021/bi00154a023. [DOI] [PubMed] [Google Scholar]
  11. Griffiths J. T., Tomchak L. A., Mills J. S., Graves M. C., Cook N. D., Dunn B. M., Kay J. Interactions of substrates and inhibitors with a family of tethered HIV-1 and HIV-2 homo- and heterodimeric proteinases. J Biol Chem. 1994 Feb 18;269(7):4787–4793. [PubMed] [Google Scholar]
  12. Gross I., Hohenberg H., Huckhagel C., Kräusslich H. G. N-Terminal extension of human immunodeficiency virus capsid protein converts the in vitro assembly phenotype from tubular to spherical particles. J Virol. 1998 Jun;72(6):4798–4810. doi: 10.1128/jvi.72.6.4798-4810.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hatfield D. L., Levin J. G., Rein A., Oroszlan S. Translational suppression in retroviral gene expression. Adv Virus Res. 1992;41:193–239. doi: 10.1016/S0065-3527(08)60037-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hill J., Phylip L. H. Bacterial aspartic proteinases. FEBS Lett. 1997 Jun 16;409(3):357–360. doi: 10.1016/s0014-5793(97)00547-4. [DOI] [PubMed] [Google Scholar]
  15. Holladay L. A., Sohpianolpoulos A. J. Nonideal associating systems. I. Documentation of a new method for determining the parameters from sedimentation equilibrium data. J Biol Chem. 1972 Jan 25;247(2):427–439. [PubMed] [Google Scholar]
  16. Ishima R., Freedberg D. I., Wang Y. X., Louis J. M., Torchia D. A. Flap opening and dimer-interface flexibility in the free and inhibitor-bound HIV protease, and their implications for function. Structure. 1999 Sep 15;7(9):1047–1055. doi: 10.1016/s0969-2126(99)80172-5. [DOI] [PubMed] [Google Scholar]
  17. Klabe R. M., Bacheler L. T., Ala P. J., Erickson-Viitanen S., Meek J. L. Resistance to HIV protease inhibitors: a comparison of enzyme inhibition and antiviral potency. Biochemistry. 1998 Jun 16;37(24):8735–8742. doi: 10.1021/bi972555l. [DOI] [PubMed] [Google Scholar]
  18. Kohl N. E., Emini E. A., Schleif W. A., Davis L. J., Heimbach J. C., Dixon R. A., Scolnick E. M., Sigal I. S. Active human immunodeficiency virus protease is required for viral infectivity. Proc Natl Acad Sci U S A. 1988 Jul;85(13):4686–4690. doi: 10.1073/pnas.85.13.4686. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Konvalinka J., Heuser A. M., Hruskova-Heidingsfeldova O., Vogt V. M., Sedlacek J., Strop P., Kräusslich H. G. Proteolytic processing of particle-associated retroviral polyproteins by homologous and heterologous viral proteinases. Eur J Biochem. 1995 Feb 15;228(1):191–198. doi: 10.1111/j.1432-1033.1995.tb20249.x. [DOI] [PubMed] [Google Scholar]
  20. Konvalinka J., Horejsí M., Andreánsky M., Novek P., Pichová I., Bláha I., Fábry M., Sedlácek J., Foundling S., Strop P. An engineered retroviral proteinase from myeloblastosis associated virus acquires pH dependence and substrate specificity of the HIV-1 proteinase. EMBO J. 1992 Mar;11(3):1141–1144. doi: 10.1002/j.1460-2075.1992.tb05154.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Konvalinka J., Litera J., Weber J., Vondrásek J., Hradílek M., Soucek M., Pichová I., Majer P., Strop P., Sedlácek J. Configurations of diastereomeric hydroxyethylene isosteres strongly affect biological activities of a series of specific inhibitors of human-immunodeficiency-virus proteinase. Eur J Biochem. 1997 Dec 1;250(2):559–566. doi: 10.1111/j.1432-1033.1997.0559a.x. [DOI] [PubMed] [Google Scholar]
  22. Konvalinka J., Litterst M. A., Welker R., Kottler H., Rippmann F., Heuser A. M., Kräusslich H. G. An active-site mutation in the human immunodeficiency virus type 1 proteinase (PR) causes reduced PR activity and loss of PR-mediated cytotoxicity without apparent effect on virus maturation and infectivity. J Virol. 1995 Nov;69(11):7180–7186. doi: 10.1128/jvi.69.11.7180-7186.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kräusslich H. G. Human immunodeficiency virus proteinase dimer as component of the viral polyprotein prevents particle assembly and viral infectivity. Proc Natl Acad Sci U S A. 1991 Apr 15;88(8):3213–3217. doi: 10.1073/pnas.88.8.3213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kunkel T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. doi: 10.1073/pnas.82.2.488. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  26. Lin X., Koelsch G., Wu S., Downs D., Dashti A., Tang J. Human aspartic protease memapsin 2 cleaves the beta-secretase site of beta-amyloid precursor protein. Proc Natl Acad Sci U S A. 2000 Feb 15;97(4):1456–1460. doi: 10.1073/pnas.97.4.1456. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Loeb D. D., Swanstrom R., Everitt L., Manchester M., Stamper S. E., Hutchison C. A., 3rd Complete mutagenesis of the HIV-1 protease. Nature. 1989 Aug 3;340(6232):397–400. doi: 10.1038/340397a0. [DOI] [PubMed] [Google Scholar]
  28. Pearl L. H., Taylor W. R. A structural model for the retroviral proteases. Nature. 1987 Sep 24;329(6137):351–354. doi: 10.1038/329351a0. [DOI] [PubMed] [Google Scholar]
  29. Pearl L., Blundell T. The active site of aspartic proteinases. FEBS Lett. 1984 Aug 20;174(1):96–101. doi: 10.1016/0014-5793(84)81085-6. [DOI] [PubMed] [Google Scholar]
  30. Polgár L., Szeltner Z., Boros I. Substrate-dependent mechanisms in the catalysis of human immunodeficiency virus protease. Biochemistry. 1994 Aug 9;33(31):9351–9357. doi: 10.1021/bi00197a040. [DOI] [PubMed] [Google Scholar]
  31. Rao J. K., Erickson J. W., Wlodawer A. Structural and evolutionary relationships between retroviral and eucaryotic aspartic proteinases. Biochemistry. 1991 May 14;30(19):4663–4671. doi: 10.1021/bi00233a005. [DOI] [PubMed] [Google Scholar]
  32. Richards A. D., Phylip L. H., Farmerie W. G., Scarborough P. E., Alvarez A., Dunn B. M., Hirel P. H., Konvalinka J., Strop P., Pavlickova L. Sensitive, soluble chromogenic substrates for HIV-1 proteinase. J Biol Chem. 1990 May 15;265(14):7733–7736. [PubMed] [Google Scholar]
  33. 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]
  34. Rosé J. R., Babé L. M., Craik C. S. Defining the level of human immunodeficiency virus type 1 (HIV-1) protease activity required for HIV-1 particle maturation and infectivity. J Virol. 1995 May;69(5):2751–2758. doi: 10.1128/jvi.69.5.2751-2758.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Royer M., Cerutti M., Gay B., Hong S. S., Devauchelle G., Boulanger P. Functional domains of HIV-1 gag-polyprotein expressed in baculovirus-infected cells. Virology. 1991 Sep;184(1):417–422. doi: 10.1016/0042-6822(91)90861-5. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
  38. Shire S. J., Holladay L. A., Rinderknecht E. Self-association of human and porcine relaxin as assessed by analytical ultracentrifugation and circular dichroism. Biochemistry. 1991 Aug 6;30(31):7703–7711. doi: 10.1021/bi00245a006. [DOI] [PubMed] [Google Scholar]
  39. Spector T. Refinement of the coomassie blue method of protein quantitation. A simple and linear spectrophotometric assay for less than or equal to 0.5 to 50 microgram of protein. Anal Biochem. 1978 May;86(1):142–146. doi: 10.1016/0003-2697(78)90327-5. [DOI] [PubMed] [Google Scholar]
  40. Studier F. W., Rosenberg A. H., Dunn J. J., Dubendorff J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990;185:60–89. doi: 10.1016/0076-6879(90)85008-c. [DOI] [PubMed] [Google Scholar]
  41. Szeltner Z., Polgár L. Conformational stability and catalytic activity of HIV-1 protease are both enhanced at high salt concentration. J Biol Chem. 1996 Mar 8;271(10):5458–5463. doi: 10.1074/jbc.271.10.5458. [DOI] [PubMed] [Google Scholar]
  42. Todd M. J., Semo N., Freire E. The structural stability of the HIV-1 protease. J Mol Biol. 1998 Oct 23;283(2):475–488. doi: 10.1006/jmbi.1998.2090. [DOI] [PubMed] [Google Scholar]
  43. Tözsér J., Yin F. H., Cheng Y. S., Bagossi P., Weber I. T., Harrison R. W., Oroszlan S. Activity of tethered human immunodeficiency virus 1 protease containing mutations in the flap region of one subunit. Eur J Biochem. 1997 Feb 15;244(1):235–241. doi: 10.1111/j.1432-1033.1997.00235.x. [DOI] [PubMed] [Google Scholar]
  44. Vogt V. M., Eisenman R., Diggelmann H. Generation of avian myeloblastosis virus structural proteins by proteolytic cleavage of a precursor polypeptide. J Mol Biol. 1975 Aug 15;96(3):471–493. doi: 10.1016/0022-2836(75)90174-6. [DOI] [PubMed] [Google Scholar]
  45. Vogt V. M., Eisenman R. Identification of a large polypeptide precursor of avian oncornavirus proteins. Proc Natl Acad Sci U S A. 1973 Jun;70(6):1734–1738. doi: 10.1073/pnas.70.6.1734. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Wallqvist A., Smythers G. W., Covell D. G. A cooperative folding unit in HIV-1 protease. Implications for protein stability and occurrence of drug-induced mutations. Protein Eng. 1998 Nov;11(11):999–1005. doi: 10.1093/protein/11.11.999. [DOI] [PubMed] [Google Scholar]
  47. Weber I. T. Comparison of the crystal structures and intersubunit interactions of human immunodeficiency and Rous sarcoma virus proteases. J Biol Chem. 1990 Jun 25;265(18):10492–10496. [PubMed] [Google Scholar]
  48. 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]
  49. Xie D., Gulnik S., Gustchina E., Yu B., Shao W., Qoronfleh W., Nathan A., Erickson J. W. Drug resistance mutations can effect dimer stability of HIV-1 protease at neutral pH. Protein Sci. 1999 Aug;8(8):1702–1707. doi: 10.1110/ps.8.8.1702. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. YPHANTIS D. A. EQUILIBRIUM ULTRACENTRIFUGATION OF DILUTE SOLUTIONS. Biochemistry. 1964 Mar;3:297–317. doi: 10.1021/bi00891a003. [DOI] [PubMed] [Google Scholar]
  51. Zamyatnin A. A. Protein volume in solution. Prog Biophys Mol Biol. 1972;24:107–123. doi: 10.1016/0079-6107(72)90005-3. [DOI] [PubMed] [Google Scholar]
  52. von der Helm K. Retroviral proteases: structure, function and inhibition from a non-anticipated viral enzyme to the target of a most promising HIV therapy. Biol Chem. 1996 Dec;377(12):765–774. [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES