Article | Published:

Flexibility and function in HIV-1 protease

Abstract

HIV protease is a homodimeric protein whose activity is essential to viral function. We have investigated the molecular dynamics of the HIV protease, thought to be important for proteinase function, bound to high affinity inhibitors using NMR techniques. Analysis of 15N spin relaxation parameters, of all but 13 backbone amide sites, reveals the presence of significant internal motions of the protein backbone. In particular, the flaps that cover the proteins active site of the protein have terminal loops that undergo large amplitude motions on the ps to ns time scale, while the tips of the flaps undergo a conformational exchange on the μs time scale. This enforces the idea that the flaps of the proteinase are flexible structures that facilitate function by permitting substrate access to and product release from the active site of the enzyme.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1

    Kohl, N.E. et al. Active human immunodeficiency virus protease is required for viral infectivity. Proc. natn. Acad. Sci. U.S.A. 85, 4686–4690 (1988).

  2. 2

    Seelmeier, S., Schmidt, H., Turk, V. & von der Helm, K. Human immunodeficiency virus has an aspartic-type protease that can be inhibited by pepstatin-A. Proc. natn. Acad. Sci. U.S.A. 85, 6612–6616 (1988).

  3. 3

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

  4. 4

    Lam, P.Y.-S., et al. Rational design of potent, bioavailable, nonpeptide cyclic ureas as HIV protease inhibitors. Science 263, 380–384 (1994).

  5. 5

    Grzesiek, S. et al. NMR evidence for the displacement of a conserved interior water molecule in HIV protease by a non-peptide cyclic urea-based inhibitor. J. A. chem. Soc. 116, 1581–1582 (1994).

  6. 6

    Jadhav, P.K. & Woemer, F.J. Synthesis of C2-symmetrical HIV-1 protease inhibitors from D-mannitol. Bioorg. med. Chem. Letts. 2, 353 (1992).

  7. 7

    Harte, W.E. Jr, et al. Domain communication in the dynamics structure of human immunodeficiency virus-1 protease. Proc. natn. Acad. Sci. U.S.A. 87, 8864–8868 (1990).

  8. 8

    Venable, R.M., Brooks, B.R. & Carson, F.W. Theoretical studies of relaxation of a monomeric subunit of HIV-1 protease in water using molecular-dynamics. Proteins Struct. Funct. Genet. 15, 374–384 (1993).

  9. 9

    Abragam, A. The Principles of Nuclear Magnetism (Oxford University Press, Oxford, U.K.; 1961).

  10. 10

    Kay, L.E., Torchia, D.A. & Bax, A. Backbone dynamics of proteins as studied by 15N inverse detected heteronuclear NMR spectroscopy: application to staphylococcal nuclease. Biochemistry 28, 8972–8979 (1989).

  11. 11

    Boyd, J., Hommel, U. & Campbell, I.D. Influence of cross-correlation between dipolar and anisotropic chemical shift relaxation mechanism upon longitudinal relaxation rates of 15N in macromolecules. J. chem. Phys. 175, 477–482 (1990).

  12. 12

    Kay, L.E., Nicholson, L.K., Delaglio, F., Bax, A. & Torchia, D.A. Pulse sequences for removal of the effects of cross correlation between dipolar and chemical-shift anisotropy relaxation mechanisms on the measurement of heteronuclear T1 and T2 values in proteins. J. magn. Reson. 97, 359–375 (1992).

  13. 13

    Palmer, A.G., Skelton, N.J., Chazin, W.J., Wright, P.E. & Rance, M. Suppression of the effects of cross-correlation between dipolar and anisotropic chemical shift relaxation mechanisms in the measurement of spin-spin relaxation rates. Molec. Phys. 75, 699–711 (1992).

  14. 14

    Torchia, D.A., Nicholson, L.K., Cole, H.B.R. & Kay, L.E. Heteronuclear NMR studies of the molecular dynamics of staphylococcal nuclease, in NMR of Proteins (eds Clore, G. M. & Gronenborn, A.M.) 190–219 (Macmillan, London; 1993).

  15. 15

    Wagner, G., Hyberts, S. & Peng, J.W. Study of Protein Dynamics by NMR, in NMR of Proteins (eds Clore, G. M. & Gronenborn, A.M.) 220–257 (Macmillan, London; 1993).

  16. 16

    Lipari, G. & Szabo, A. Model-free approach to the interpretation of nuclear magnetic resonance relaxation in macromolecules. 1. Theory and range of validity. J. Am. chem. Soc. 104, 4546–4559 (1982).

  17. 17

    Lipari, G. & Szabo, A. Model-free approach to the interpretation of nuclear magnetic resonance relaxation in macromolecules. 2. Analysis of experimental results. J. Am. chem. Soc. 104, 4559–4570 (1982).

  18. 18

    Dellwo, M.J. & Wand, A.J. Model-independent and model-dependent analysis of the global and internal dynamics of cyclosporin A. J. Am. chem. Soc. 111, 4571–4578 (1989).

  19. 19

    Clore, G.M. et al. Deviation from the simple two-parameter model-free approach to the interpretation of nitrogen-15 nuclear magnetic relaxation of proteins. J. Am. chem. Soc. 112, 4989–4991 (1990).

  20. 20

    Barbato, G., Ikura, M., Kay, L.E., Pastor, R. & Bax, A. Backbone dynamics of calmodulin studied by 15N relaxation using inverse detected two-dimensional NMR spectroscopy: the central helix is flexible. Biochemistry 31, 5269–5278 (1992).

  21. 21

    Kordel, J., Skelton, N.J., Akke, M., Palmer, A.G. & Chazin, W.J. Backbone dynamics of calcium-loaded calbindin D9k studies by two-dimensional proton detected 15N NMR spectroscopy. Biochemistry 31, 4856–4559 (1992).

  22. 22

    Nicholson, L.K. et al. Dynamics of methyl groups in proteins as studied by proton-detected 13C NMR spectroscopy. Application to the leucine residues of Staphylococcal Nuclease. Biochemistry 31, 5253–5263 (1992).

  23. 23

    Constantine, K.L. et al. Relaxation study of the backbone dynamics of human profilin by two-dimensional 1H-15N NMR. FEBS Letts 336, 457–461 (1993).

  24. 24

    Farrow, N.A. et al. Backbone dynamics of a free and a phosphopeptide-complexed Src homology 2 domain studied by 15N NMR relaxation. Biochemistry 33, 5984–6003 (1994).

  25. 25

    Farrar, T.C. & Becker, E.D. Pulse and Fourier Transform NMR 1–115 (Academic Press, New York, 1997).

  26. 26

    Szyperski, T., Luginbuhl, P., Otting, G., Guntert, P. & Wuethrich, K. . protein dynamics studied by rotating frame 15N spin relaxation times. J. Biomol. NMR 3, 151–164 (1993).

  27. 27

    Yamazaki, T. et al. Secondary structure and signal assignments of human-immunodeficiency-virus-1 protease complexed to a novel, structure-based inhibitor. Eur. J. Biochem. 219, 707–712 (1994).

  28. 28

    Clore, G.M., Driscoll, PC., Wingfield, P.T .& Gronenborn, A. Analysis of the backbone dynamics of interleukin-1β using two-dimensional inverse detected heteronuclear 15N-1H NMR spectroscopy. Biochemistry 29, 7387–7401 (1990).

  29. 29

    Stone, M.J. et al. The backbone dynamics of the Bacillus subtilis glucose permease IIA domain determined from 15N NMR relaxation measurements. Biochemistry 31, 4393–4406 (1992).

  30. 30

    Loeb, D.D. et al. Complete mutagenesis of the HIV-1 Protease. Nature 340, 397–400 (1989).

  31. 31

    Yamazaki, T. et al. NMR and X-ray evidence that the HIV protease catalytic aspartyl groups are protonated in the complex formed by the protease and a non-peptide cyclic urea-based inhibitor. J. Am. chem. Soc. 116, 1994 (1994).

  32. 32

    Bai, Y, Milne, J.S., Mayne, L. & Englander, S.W. Primary structure effects on peptide group hydrogen exchange. Prot., Struct. Funct. Genet. 17, 75–86 (1993).

  33. 33

    Rose, J.R., Salto, R. & Craik, C.S. Regulation of autoproteolysis of the HIV-1 and HIV-2 proteases with engineered amino acid substitutions. J. biol. Chem. 268, 11939–11945 (1993).

  34. 34

    Cheng, Y.-S.E. et al. High-level synthsis of recombinant HIV-1 protease and the recovery of active enzyme from inclusion bodies. Gene 87, 243–248 (1990).

  35. 35

    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).

  36. 36

    Peng, J.W., Thanabal, V. & Wagner, G. 2D heteronuclear NMR measurements of spin-lattice relaxation times in the rotating frame of X nuclei in heteronuclear HX spin systems. J. magn. Reson. 95, 421–427 (1991).

  37. 37

    Palmer, A.G., Wright, P.E. & Ranee, M. Measurement of relaxation time constants for methyl groups by proton-detected heteronuclear NMR spectroscopy. Chem. Phys. Letts. 185, 41–46 (1991).

  38. 38

    Press, W.H., Flannery, B.P., Teukolsky, S.A. & Vetterling, W.T. Numerical Recipes in C (Cambridge University Press, Cambridge, U.K., 1988).

  39. 39

    Venable, R.M. & Pastor, R.W. Frictional models for stochastic simulations of proteins. Biopolymers 27, 1001–1014 (1988).

  40. 40

    Woessner, D.E. Nuclear spin relaxation in ellipsoids undergoing rotational brownian motion. J. chem. Phys. 37, 647–654 (1962).

  41. 41

    Palmer, A.G., Ranee, M. & Wright, P.E. Intramolecular motions of a zinc finger DNA-binding domain from Xfin characterized by proton-detected natural abundance 13C heteronuclear NMR spectroscopy. J. Am. chem. Soc. 113, 4371–4380 (1991).

  42. 42

    Kraulis, P. Molscript - a program to produce both detailed and schematic plots of protein structures. J. appl. Crystallogr. 24, 946–950 (1991).

Download references

Author information

Rights and permissions

Reprints and Permissions

About this article

Further reading