The Protein Journal

, Volume 32, Issue 7, pp 560–567 | Cite as

F99 is Critical for Dimerization and Activation of South African HIV-1 Subtype C Protease

  • Previn Naicker
  • Palesa Seele
  • Heini W. Dirr
  • Yasien Sayed
Article

Abstract

HIV-1 protease (PR) is an obligate homodimer which plays a pivotal role in the maturation and hence propagation of HIV. Although successful developments on PR active site inhibitors have been achieved, the major limiting factor has been the emergence of HIV drug-resistant strains. Disruption of the dimer interface serves as an alternative mechanism to inactivate the enzyme. The terminal residue, F99, was mutated to an alanine to investigate its contribution to dimer stability in the South African HIV-1 subtype C (C-SA) PR. The F99A PR and wild-type C-SA PR were overexpressed and purified. The activities of the PRs and their ability to bind an active site inhibitor, acetyl-pepstatin, were determined in vitro. The F99A PR showed no activity and the inability to bind to the inhibitor. Secondary and quaternary structure analysis were performed and revealed that the F99A PR is monomeric with reduced β-sheet content. The mutation of F99 to alanine disrupted the presumed ‘lock-and-key’ motif at the terminal dimer interface, in turn creating a cavity at the N- and C-terminal antiparallel β-sheet. These findings support the design of inhibitors targeting the C-terminus of the C-SA PR, centered on interactions with the bulky F99.

Keywords

Human immunodeficiency virus Protease F99A Dimer interface Dimerization inhibitors Subtype C 

Abbreviations

AIDS

Acquired immunodeficiency syndrome

C-SA

South African subtype C

DNA

Deoxyribonucleic acid

HIV

Human immunodeficiency virus

PDB

Protein data bank

PR

Protease

Notes

Acknowledgments

This work was supported by the University of the Witwatersrand, South African National Research Foundation (Grant: NRF Thuthuka/REDIBA to Y.S; 60810, 65510 and 68898 to H.W.D) and the South African Research Chairs Initiative of the Department of Science and National Research Foundation (Grant: 64788 to H.W.D).

Conflict of interest

The authors declare that they have no conflict of interest.

References

  1. 1.
    The Joint Nations Program on HIV/AIDS (UNAIDS) (2012) Global report: UNAIDS report on the global AIDS epidemic 2012. UNAIDS, GenevaGoogle Scholar
  2. 2.
    Walker PR, Pybus OG, Rambaut A, Holmes EC (2005) Comparative population dynamics of HIV-1 subtypes B and C: subtype-specific differences in patterns of epidemic growth. Infect Genet Evol 5(3):199–208CrossRefGoogle Scholar
  3. 3.
    Chakrabarti L, Guyader M, Alizon M, Daniel MD, Desrosiers RC, Tiollais P, Sonigo P (1987) Sequence of simian immunodeficiency virus from macaque and its relationship to other human and simian retroviruses. Nature 328(6130):543–547CrossRefGoogle Scholar
  4. 4.
    Osmanov S, Pattou C, Walker N, Schwardlander B, Esparza J (2000) Estimated global distribution and regional spread of HIV-1 genetic subtypes in the year 2000. J Acquir Immune Defic Syndr 29(2):184–190CrossRefGoogle Scholar
  5. 5.
    Peeters M, Piot P, Vandergroen G (1991) Variability among HIV and SIV strains of African origin. AIDS 5:29–36CrossRefGoogle Scholar
  6. 6.
    Weber IT (1990) Comparison of the crystal structures and intersubunit interactions of human immunodeficiency and rous-sarcoma virus proteases. J Biol Chem 265(18):10492–10496Google Scholar
  7. 7.
    Loeb DD, Hutchison CA, Edgell MH, Farmerie WG, Swarnstrom R (1988) Mutational analysis of human immunodeficiency virus type 1 protease suggests functional homology with aspartic proteinases. J Virol 63(1):111–121Google Scholar
  8. 8.
    Wlodawer A, Miller M, Jaskolski M, Sathyanarayana BK, Baldwin E, Weber IT, Selk LM, Clawson L, Schneider J, Kent SBH (1989) Conserved folding in retroviral proteases: crystal structure of a synthetic hiv-1 protease. Science 245:616–621CrossRefGoogle Scholar
  9. 9.
    Xu D, Lin SL, Nussinov R (1997) Protein binding versus protein folding: the role of hydrophilic bridges in protein associations. J Mol Biol 265(1):68–84CrossRefGoogle Scholar
  10. 10.
    Burgoyne NJ, Jackson RM (2006) Predicting protein interaction sites: binding hot-spots in protein-protein and protein-ligand interfaces. Bioinformatics 22(11):1335–1342CrossRefGoogle Scholar
  11. 11.
    Jones S, Thornton JM (1995) Protein-protein interactions: a review of protein dimer structures. Prog Biophys Mol Biol 63(1):31–65CrossRefGoogle Scholar
  12. 12.
    Ahmed SM, Kruger HG, Govender T, Maguire GEM, Sayed Y, Ibrahim MAA, Naicker P, Soliman MES (2013) Comparison of the molecular dynamics and calculated binding free energies for nine FDA-Approved HIV-1 PR drugs against subtype B and C-SA HIV PR. Chem Biol Drug Des 81(2):208–218CrossRefGoogle Scholar
  13. 13.
    Mosebi S, Morris L, Dirr HW, Sayed Y (2008) Active-site mutations in the South African human immunodeficiency virus type 1 subtype C protease have a significant impact on clinical inhibitor binding: kinetic and thermodynamic study. J Virol 82(22):11476–11479CrossRefGoogle Scholar
  14. 14.
    Velazquez-Campoy A, Vega S, Fleming E, Bacha U, Sayed Y, Dirr HW (2003) Protease inhibition in African subtypes of HIV-1. AIDS Rev 5:165–171Google Scholar
  15. 15.
    Wlodawer A, Gustchina A (2000) Structural and biochemical studies of retroviral proteases. Biochim Biophys Acta 1477(1):16–34CrossRefGoogle Scholar
  16. 16.
    Koh Y, Matsumi S, Das D, Amano M, Davis DA, Li J, Leschenko S, Baldridge A, Shioda T, Yarchoan R, Ghosh AK, Mitsuya H (2007) Potent inhibition of HIV-1 replication by novel non-peptidyl small molecule inhibitors of protease dimerization. J Biol Chem 282(39):28709–28720CrossRefGoogle Scholar
  17. 17.
    Pettit SC, Gulnik S, Everitt L, Kaplan AH (2003) The dimer interfaces of protease and extra-protease domains influence the activation of protease and the specificity of GagPol cleavage. J Virol 77(1):366–374CrossRefGoogle Scholar
  18. 18.
    Mildner AM, Rothrock DJ, Leone JW, Bannow CA, Lull JM, Reardon IM, Sarcich JL, Howe WJ, Tomich CC, Smith CW, Heinrikson RL, Tomasselli AG (1994) The HIV-1 Protease as enzyme and substrate: mutagenesis of autolysis sites and generation of a stable mutant with retained kinetic properties. Biochemistry 33:9405–9413CrossRefGoogle Scholar
  19. 19.
    Naicker P, Achilonu I, Fanucchi S, Fernandes M, Ibrahim MAA, Dirr HW, Soliman MES, Sayed Y (2013) Structural insights into the South African HIV-1 subtype C protease: impact of hinge region dynamics and flap flexibility in drug resistance. J Biomol Struct Dyn (in press). doi: 10.1080/07391102.07392012.07736774
  20. 20.
    Ido E, Han HP, Kezdy FJ, Tang J (1991) Kinetic-studies of human immunodeficiency virus type-1 protease and its active-site hydrogen-bond mutant A28S. J Biol Chem 266(36):24359–24366Google Scholar
  21. 21.
    Laemmli UK (1970) Cleavage of structural proteins during assembly of head of bacteriophage-T4. Nature 227(5259):680–685CrossRefGoogle Scholar
  22. 22.
    Schagger H (2006) Tricine-SDS-PAGE. Nat Protoc 1(1):16–22CrossRefGoogle Scholar
  23. 23.
    Perkins S (1986) Protein volumes and hydration effects. Eur J Biochem 157:169–180CrossRefGoogle Scholar
  24. 24.
    Woody RW (1995) Circular-dichroism. In: Sauer K (ed) Methods Enzymol, vol 246. Academic Press, New York, pp 34–71Google Scholar
  25. 25.
    Noel AF, Bilsel O, Kundu A, Wu Y, Zitzewitz JA, Matthews CR (2009) The folding free-energy surface of HIV-1 protease: insights into the thermodynamic basis for resistance to inhibitors. J Mol Biol 387(4):1002–1016CrossRefGoogle Scholar
  26. 26.
    Hansen J, Billich S, Schulze T, Sukrow S, Moelling K (1988) Partial purification and substrate analysis of bacterially expressed HIV protease by means of monoclonal antibody. EMBO J 7(6):1785–1791Google Scholar
  27. 27.
    Jones S, Thornton JM (1996) Principles of protein-protein interactions. Proc Natl Acad Sci USA 93(1):13–20CrossRefGoogle Scholar
  28. 28.
    Coman RM, Robbins AH, Goodenow MM, Dunn BM, McKenna R (2008) High-resolution structure of unbound human immunodeficiency virus 1 subtype C protease: implications of flap dynamics and drug resistance. Acta Crystallogr D Biol Crystallogr 64(7):754–763CrossRefGoogle Scholar
  29. 29.
    Manning MC, Illangasekare M, Woody RW (1988) Circular dichroism studies of distorted alpha-helices, twisted beta-sheets, and beta turns. Biophys Chem 31:77–86CrossRefGoogle Scholar
  30. 30.
    Dey S, Pal A, Chakrabarti P, Janin J (2010) The subunit interfaces of weakly associated homodimeric proteins. J Mol Biol 398(1):146–160CrossRefGoogle Scholar
  31. 31.
    Todd MJ, Semo N, Freire E (1998) The structural stability of the HIV-1 protease. J Mol Biol 283(2):475–488CrossRefGoogle Scholar
  32. 32.
    Hostomsky Z, Appelt K, Ogden RC (1989) High-level expression of self-processed HIV-1 protease in Escherichia coli using a synthetic gene. Biochem Biophys Res Commun 161(3):1056–1063CrossRefGoogle Scholar
  33. 33.
    Agniswamy J, Sayer JM, Weber IT, Louis JM (2012) Terminal interface conformations modulate dimer stability prior to amino terminal autoprocessing of HIV-1 protease. Biochemistry 51(5):1041–1050CrossRefGoogle Scholar
  34. 34.
    Babe LM, Rose J, Craik CS (1992) Sythetic interface peptides alter dimeric assembly of the HIV-1 and HIV-2 proteases. Protein Sci 1(10):1244–1253CrossRefGoogle Scholar
  35. 35.
    Ghanta J, Shen CL, Kiessling LL, Murphy RM (1996) A strategy for designing inhibitors of beta-amyloid toxicity. J Biol Chem 271(47):29525–29528CrossRefGoogle Scholar
  36. 36.
    Pinkner JS, Remaut H, Buelens F, Miller E, Aberg V, Pemberton N, Hedenstrom M, Larsson A, Seed P, Waksman G, Hultgren SJ, Almqvist F (2006) Rationally designed small compounds inhibit pilus biogenesis in uropathogenic bacteria. Proc Natl Acad Sci USA 103(47):17897–17902CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Previn Naicker
    • 1
  • Palesa Seele
    • 1
  • Heini W. Dirr
    • 1
  • Yasien Sayed
    • 1
  1. 1.Protein Structure-Function Research Unit, School of Molecular and Cell BiologyUniversity of the WitwatersrandJohannesburgSouth Africa

Personalised recommendations