Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Visualizing transient events in amino-terminal autoprocessing of HIV-1 protease

Nature volume 455, pages 693–696 (2008)Cite this article

This article has been updated

Abstract

HIV-1 protease processes the Gag and Gag-Pol polyproteins into mature structural and functional proteins, including itself, and is therefore indispensable for viral maturation1,2. The mature protease is active only as a dimer3,4,5 with each subunit contributing catalytic residues6. The full-length transframe region protease precursor appears to be monomeric yet undergoes maturation via intramolecular cleavage of a putative precursor dimer5,7,8,9,10,11, concomitant with the appearance of mature-like catalytic activity7,9. How such intramolecular cleavage can occur when the amino and carboxy termini of the mature protease are part of an intersubunit β-sheet located distal from the active site is unclear. Here we visualize the early events in N-terminal autoprocessing using an inactive mini-precursor with a four-residue N-terminal extension that mimics the transframe region protease precursor5,12. Using paramagnetic relaxation enhancement, a technique that is exquisitely sensitive to the presence of minor species13,14,15,16, we show that the mini-precursor forms highly transient, lowly populated (3–5%) dimeric encounter complexes that involve the mature dimer interface but occupy a wide range of subunit orientations relative to the mature dimer. Furthermore, the occupancy of the mature dimer configuration constitutes a very small fraction of the self-associated species (accounting for the very low enzymatic activity of the protease precursor), and the N-terminal extension makes transient intra- and intersubunit contacts with the substrate binding site and is therefore available for autocleavage when the correct dimer orientation is sampled within the encounter complex ensemble.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Intermolecular PRE profiles.
Figure 2: Ensemble simulated annealing and the protease mini-precursor encounter complex ensemble.
Figure 3: PRE profiles with spin labels attached at the N- and C termini of the SFNF PR(D25N) mini-precursor.

Similar content being viewed by others

Change history

  • 14 November 2008

    In the online-only extended Methods, a sentence in the second paragraph of the 'NMR experiments' section has been corrected.

References

  1. Louis, J. M., Weber, I. T., Tozser, J., Clore, G. M. & Gronenborn, A. M. HIV-1 protease: maturation, enzyme specificity, and drug resistance. Adv. Pharmacol. 49, 111–146 (2000)

    Article  CAS  Google Scholar 

  2. Louis, J. M., Ishima, R., Torchia, D. A. & Weber, I. T. HIV-1 protease: structure, dynamics and inhibition. Adv. Pharmacol. 55, 261–298 (2007)

    Article  CAS  Google Scholar 

  3. Wlodawer, A. & Erikson, J. Structure-based inhibitors of HIV-1 protease. Annu. Rev. Biochem. 62, 543–585 (1993)

    Article  CAS  Google Scholar 

  4. Wlodawer, A. & Vondrasek, J. Inhibitors of HIV-1 protease: a major success of structure-assisted drug design. Annu. Rev. Biophys. Biomol. Struct. 27, 249–284 (1998)

    Article  CAS  Google Scholar 

  5. Ishima, R., Torchia, D. A., Lynch, S. M., Gronenborn, A. M. & Louis, J. M. Solution structure of the mature HIV-1 protease monomer: insight into the tertiary fold and stability of a precursor. J. Biol. Chem. 278, 43311–43319 (2003)

    Article  CAS  Google Scholar 

  6. Miller, M. et al. Conserved folding in retroviral proteases: crystal structure of a synthetic HIV-1 protease. Science 246, 1149–1152 (1989)

    Article  ADS  CAS  Google Scholar 

  7. Louis, J. M., Nashed, N. T., Parris, K. D., Kimmel, A. R. & Jerina, D. M. Kinetics and mechanism of autoprocessing of human immunodeficiency virus type 1 protease from an analog of the Gag-Pol polyprotein. Proc. Natl Acad. Sci. USA 91, 7970–7974 (1994)

    Article  ADS  CAS  Google Scholar 

  8. Co, E. et al. Proteolytic processing mechanisms of a miniprecursor of the aspartic protease of human immunodeficiency virus type 1. Biochemistry 33, 1248–1254 (1994)

    Article  CAS  Google Scholar 

  9. Louis, J. M., Wondrak, E. M., Kimmel, A. R., Wingfield, P. T. & Nashed, N. T. Proteolytic processing of HIV-1 protease precursor, kinetics and mechanism. J. Biol. Chem. 274, 23437–23442 (1999)

    Article  CAS  Google Scholar 

  10. Louis, J. M., Clore, G. M. & Gronenborn, A. M. Autoprocessing of HIV-1 protease is tightly coupled to protein folding. Nature Struct. Biol. 6, 868–874 (1999)

    Article  CAS  Google Scholar 

  11. Pettit, S. C., Everitt, L. E., Choudhury, S., Dunn, B. M. & Kaplan, A. H. Initial cleavage of the human immunodeficiency virus type 1 GagPol precursor by its activated protease occurs by an intramolecular mechanism. J. Virol. 78, 8477–8485 (2004)

    Article  CAS  Google Scholar 

  12. Ishima, R., Torchia, D. A. & Louis, J. M. Mutational and structural studies aimed at characterizing the monomer of HIV-1 protease and its precursor. J. Biol. Chem. 282, 17190–17199 (2007)

    Article  CAS  Google Scholar 

  13. Iwahara, J. & Clore, G. M. Detecting transient intermediates in macromolecular binding by paramagnetic NMR. Nature 440, 1227–1230 (2006)

    Article  ADS  CAS  Google Scholar 

  14. Tang, C., Iwahara, J. & Clore, G. M. Visualization of transient encounter complexes in protein-protein association. Nature 444, 383–386 (2006)

    Article  ADS  CAS  Google Scholar 

  15. Volkov, A. N., Worall, J. A., Holtzmann, E. & Ubbink, M. Solution structure and dynamics of the complex between cytochrome c and cytochrome c peroxidase determined by paramagnetic NMR. Proc. Natl Acad. Sci. USA 103, 18945–18950 (2006)

    Article  ADS  CAS  Google Scholar 

  16. Tang, C., Schwieters, C. D. & Clore, G. M. Open-to-closed transition in apo maltose-binding protein observed by paramagnetic NMR. Nature 449, 1078–1082 (2007)

    Article  ADS  CAS  Google Scholar 

  17. Tessmer, U. & Kräusslich, H.-G. Cleavage of human immunodeficiency virus type 1 proteinase from the N-terminally adjacent p6* protein is essential for efficient Gag polyprotein processing and viral infectivity. J. Virol. 72, 3459–3463 (1998)

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Ludwig, C., Leiherer, A. & Wagner, G. Importance of protease cleavage sites within and flanking human immunodeficiency virus type 1 transframe protein p6* for spatiotemporal regulation of protease activation. J. Virol. 82, 4573–4584 (2008)

    Article  CAS  Google Scholar 

  19. Wondrak, E. M., Nashed, N. T., Haber, M. T., Jerina, D. M. & Louis, J. M. A transient precursor of the HIV-1 protease: isolation, characterization and kinetics of maturation. J. Biol. Chem. 271, 4477–4481 (1996)

    Article  CAS  Google Scholar 

  20. Cherry, E. et al. Characterization of human immunodeficiency virus type-1 (HIV-1) particles that express protease-reverse transcriptase fusion proteins. J. Mol. Biol. 284, 43–56 (1998)

    Article  CAS  Google Scholar 

  21. Iwahara, J., Tang, C. & Clore, G. M. Practical aspects of 1H transverse paramagnetic relaxation enhancement measurements on macromolecules. J. Magn. Reson. 184, 185–195 (2007)

    Article  ADS  CAS  Google Scholar 

  22. Schwieters, C. D., Kuszewski, J. & Clore, G. M. Using Xplor-NIH for NMR molecular structure determination. Prog. Nucl. Magn. Reson. Spectrosc. 48, 47–62 (2006)

    Article  CAS  Google Scholar 

  23. Iwahara, J., Schwieters, C. D. & Clore, G. M. Ensemble approach for NMR structure refinement against 1H paramagnetic relaxation enhancement data arising from a flexible paramagnetic group attached to a macromolecule. J. Am. Chem. Soc. 126, 5879–5896 (2004)

    Article  CAS  Google Scholar 

  24. Spinelli, S., Liu, Q. Z., Alzari, P. M., Hirel, P. H. & Poljak, R. J. The three-dimensional structure of the aspartyl protease from the HIV-1 isolate BRU. Biochimie 73, 1391–1396 (1991)

    Article  CAS  Google Scholar 

  25. Schwieters, C. D. & Clore, G. M. Reweighted atomic densities to represent ensembles of NMR structures. J. Biomol. NMR 23, 221–225 (2002)

    Article  CAS  Google Scholar 

  26. Wondrak, E. M. & Louis, J. M. Influence of flanking sequences on the dimer stability of human immunodeficiency virus type 1 protease. Biochemistry 35, 12957–12962 (1996)

    Article  CAS  Google Scholar 

  27. Mahalingam, B. et al. Crystal structures of HIV protease V82A and L90M mutants reveal changes in the indinavir-binding site. Eur. J. Biochem. 271, 1516–1524 (2004)

    Article  CAS  Google Scholar 

  28. Altieri, A. S., Hinton, D. P. & Byrd, R. A. Association of biomolecular systems via pulsed field gradient NMR self-diffusion measurements. J. Am. Chem. Soc. 117, 7566–7567 (1995)

    Article  CAS  Google Scholar 

  29. Chou, J. J., Baber, J. L. & Bax, A. Characterization of phospholipid mixed micelles by translational diffusion. J. Biomol. NMR 29, 299–308 (2004)

    Article  CAS  Google Scholar 

  30. Grzesiek, S. & Bax, A. The importance of not saturating H2O in protein NMR: application to sensitivity enhancement and NOE measurements. J. Am. Chem. Soc. 115, 12593–12594 (1993)

    Article  CAS  Google Scholar 

  31. Clore, G. M. & Gronenborn, A. M. Two-, three- and four-dimensional NMR methods for obtaining larger and more precise three-dimensional structures of proteins in solution. Annu. Rev. Biophys. Biophys. Chem. 20, 29–63 (1991)

    Article  CAS  Google Scholar 

  32. Clore, G. M. & Gronenborn, A. M. Multidimensional heteronuclear nuclear magnetic resonance of proteins. Methods Enzymol. 239, 349–363 (1994)

    Article  CAS  Google Scholar 

  33. Grzesiek, S., Stahl, S. J., Wingfield, P. T. & Bax, A. The CD4 determinant of downregulation by HIV-1 Nef directly binds to Nef: mapping of the Nef binding surface by NMR. Biochemistry 35, 10256–10261 (1996)

    Article  CAS  Google Scholar 

  34. Wishart, D. S. & Sykes, B. D. The 13C chemical-shift index: a simple method for the identification of protein secondary structure using 13C chemical-shift data. J. Biomol. NMR 4, 171–180 (1994)

    Article  CAS  Google Scholar 

  35. Shen, Y. et al. Consistent blind protein structure generation from NMR chemical shift data. Proc. Natl Acad. Sci. USA 105, 4685–4690 (2008)

    Article  ADS  CAS  Google Scholar 

  36. Katoh, E. et al. A solution NMR study of the binding kinetics and internal dynamics of an HIV-1 protease-substrate complex. Protein Sci. 12, 1376–1385 (2003)

    Article  CAS  Google Scholar 

  37. Clore, G. M. & Kuszewski, J. χ1 rotamer populations and angles of mobile surface side chains are accurately predicted by a torsion angle database potential of mean force. J. Am. Chem. Soc. 124, 2866–2867 (2002)

    Article  CAS  Google Scholar 

  38. Kuszewski, J., Gronenborn, A. M. & Clore, G. M. Improving the packing and accuracy of NMR structures with a pseudopotential for the radius of gyration. J. Am. Chem. Soc. 121, 2337–2338 (1999)

    Article  CAS  Google Scholar 

  39. Tang, C., Ghirlando, R. & Clore, G. M. Visualization of transient ultra-weak protein self-association in solution using paramagnetic relaxation enhancement. J. Am. Chem. Soc. 130, 4048–4056 (2008)

    Article  CAS  Google Scholar 

  40. Kim, Y. C., Tang, C., Clore, G. M. & Hummer, G. Replica exchange simulations of transient encounter complexes in protein-protein association. Proc. Natl Acad. Sci. USA 105, 12855–12860 (2008)

    Article  ADS  CAS  Google Scholar 

Download references

Acknowledgements

We thank R. Ishima for providing initial backbone assignments for the SFNFPR(D25N) protease construct; C. Schwieters for many discussions; Y. Sheng for help with the CS-Rosetta calculations; Y. Kim for providing the code for structure clustering and d.r.m.s. calculations; and J. Sayer for MALDI measurements. This work was supported by funds from the Intramural Program of the NIH, NIDDK and the AIDS Targeted Antiviral program of the Office of the Director of the NIH (to G.M.C.).

Author information

Author notes

  1. Chun Tang

    Present address: Present address: Department of Biochemistry, University of Missouri, Columbia, Missouri 65211, USA.,

Authors and Affiliations

  1. Laboratory of Chemical Physics, Building 5, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-0520, USA ,

    Chun Tang, John M. Louis, Annie Aniana, Jeong-Yong Suh & G. Marius Clore

Authors
  1. Chun Tang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. John M. Louis
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Annie Aniana
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jeong-Yong Suh
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. G. Marius Clore
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to G. Marius Clore.

Supplementary information

Supplementary Information

This file contains Supplementary Figures and Legends S1-S3 and Supplementary Table S1 In the original Supplementary Fig. 1d, the values of the translational diffusion coefficients (Ds) for the monomeric precursor and mature HIV-1 protease dimer were inadvertently transposed; these have now been corrected. (PDF 287 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Tang, C., Louis, J., Aniana, A. et al. Visualizing transient events in amino-terminal autoprocessing of HIV-1 protease. Nature 455, 693–696 (2008). https://doi.org/10.1038/nature07342

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature07342

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing