Molecular Dynamics Study of HIV-Protease
V. Hornak, A. Okur, R.C. Rizzo, and C. Simmerling

Due to its central role in processing viral polypeptide precursors, HIV-1 protease (HIV-PR) continues to be one of the primary targets of anti-AIDS drug discovery. The introduction of HIV-1 protease (HIV-PR) inhibitors has led to a dramatic increase in patient survival; however, these gains are threatened by the emergence of multi-drug resistant strains. Design of inhibitors that overcome resistance would be greatly facilitated by deeper insight into the mechanistic events associated with binding of substrates and inhibitors, as well as an understanding of the effects of resistance mutations on the structure and dynamic behavior of HIV-PR. An extensive set of X-ray crystal structures of HIV-1 protease has been solved [1], revealing a C2 symmetric homodimer with a large substrate binding pocket covered by two glycine rich β-hairpins, or flaps [2-4]. Consistent structural differences are present between the bound and free states of the protein. In all of the liganded forms, the flaps are pulled in towards the bottom of the active site (“closed” form), while the structures for the unbound enzyme all adopt a “semi-open” conformation with the flaps shifted away from the catalytic site, but still substantially closed over the active site and in contact with each other. Although large-scale flap opening is presumably required for normal substrate access to the active site, no crystallographic structures representing such an open configuration have been reported and thus the mechanism of inhibitor entry and binding remains unknown.

In contrast to the static view provided by crystallography, molecular dynamics (MD) simulations can provide valuable insight into time-dependent structural variation. We performed unrestrained, all-atom molecular dynamics simulations of HIV-PR that sampled large conformational changes of the active site flaps [5]. The unliganded protease underwent spontaneous and reproducible conversions between the “closed” and “semi-open” forms observed in crystal structures of inhibitor-bound and unliganded protease, respectively. Simulations in the presence of a cyclic urea inhibitor yield stable closed flaps. Furthermore, we observed several events in which the flaps of the unliganded protease opened to a much greater degree than observed in crystal structures and subsequently returned to the semi-open state. Our data strongly support the hypothesis that the unliganded protease predominantly populates the semi-open conformation, with closed and fully open structures being a minor component of the overall ensemble. The results also provide a model for the flap opening and closing that is considered to be essential to enzyme function.

Further computational studies investigated whether the model for the open state based on our simulations was capable of binding to inhibitors and adopting the closed form. We manually placed an inhibitor in the proximity of the flaps (see Figure 1). During subsequent simulations, the inhibitor reproducibly induced spontaneous conversion to the closed form as seen in all inhibitor-bound HIV-PR crystal structures, with root mean square deviation (RMSD) of ~1 Å from the crystal structure of the complex for the inhibitor and each flap despite initial RMSD values of 6 - 11 Å [6]. The results demonstrate that all-atom simulations have the ability to significantly improve poorly docked ligand conformations and reproduce large-scale receptor conformational changes that occur upon binding.
 

References

• [1] Vondrasek, J. and Wlodawer, A. Proteins-Structure Function and Genetics 49: 429-431 (2002).
• [2] Navia, M.A., Fitzgerald, P.M.D., McKeever, B.M., Leu, C.T., Heimbach, J.C., Herber, W.K., Sigal, I.S., Darke, P.L., and Springer, J.P. Nature 337: 615-620 (1989).
• [3] Wlodawer, A., Miller, M., Jaskolski, M., Sathyanarayana, B.K., Baldwin, E., Weber,I.T., Selk, L.M., Clawson, L., Schneider, J., and Kent, S.B.H. Science 245: 616-621 (1989).
• [4] Lapatto, R., Blundell, T., Hemmings, A., Overington, J., Wilderspin, A., Wood, S., Merson, J.R., Whittle, P.J., Danley, D.E., Geoghegan, K.F., Hawrylik, S.J., Lee, S.E., Scheld, K.G., and Hobart, P.M. Nature 342: 299-302 (1989).
• [5] Lapatto, R., Blundell, T., Hemmings, A., Overington, J., Wilderspin, A., Wood, S., Merson, J.R., Whittle, P.J., Danley, D.E., Geoghegan, K.F., Hawrylik, S.J., Lee, S.E., Scheld, K.G., and Hobart, P.M. Nature 342: 299-302 (1989).
• [6] ] Hornak, V., Okur, A., Rizzo, R. and Simmerling, C. HIV-1 protease flaps spontaneously close to the correct structure in simulations following manual placement of an inhibitor into the open state. J. Am. Chem. Soc. 128: 2812 (2006).

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