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J. Biol. Chem., Vol. 283, Issue 19, 13459-13470, May 9, 2008

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Effect of the Active Site D25N Mutation on the Structure, Stability, and Ligand Binding of the Mature HIV-1 Protease^*

Jane M. Sayer, Fengling Liu, Rieko Ishima^¶, Irene T. Weber, and John M. Louis¹

From the Laboratory of Chemical Physics, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-0520, the Department of Biology, Georgia State University, Atlanta, Georgia 30303, and the ^¶Department of Structural Biology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15260

All aspartic proteases, including retroviral proteases, share the triplet DTG critical for the active site geometry and catalytic function. These residues interact closely in the active, dimeric structure of HIV-1 protease (PR). We have systematically assessed the effect of the D25N mutation on the structure and stability of the mature PR monomer and dimer. The D25N mutation (PR_D25N) increases the equilibrium dimer dissociation constant by a factor >100-fold (1.3 ± 0.09 µM) relative to PR. In the absence of inhibitor, NMR studies reveal clear structural differences between PR and PR_D25N in the relatively mobile P1 loop (residues 79-83) and flap regions, and differential scanning calorimetric analyses show that the mutation lowers the stabilities of both the monomer and dimer folds by 5 and 7.3 °C, respectively. Only minimal differences are observed in high resolution crystal structures of PR_D25N complexed to darunavir (DRV), a potent clinical inhibitor, or a non-hydrolyzable substrate analogue, Ac-Thr-Ile-Nle-r-Nle-Gln-Arg-NH₂ (RPB), as compared with PR·DRV and PR·RPB complexes. Although complexation with RPB stabilizes both dimers, the effect on their T_m is smaller for PR_D25N (6.2 °C) than for PR (8.7 °C). The T_m of PR_D25N·DRV increases by only 3 °C relative to free PR_D25N, as compared with a 22 °C increase for PR·DRV, and the mutation increases the ligand dissociation constant of PR_D25N·DRV by a factor of 10⁶ relative to PR·DRV. These results suggest that interactions mediated by the catalytic Asp residues make a major contribution to the tight binding of DRV to PR.

Received for publication, October 12, 2007 , and in revised form, January 11, 2008.

The atomic coordinates and structure factors (codes 3BVA and 3BVB) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was authored, in whole or in part, by National Institutes of Health staff. This work was supported, in whole or in part, by the Intramural Research Program of the NIDDK, National Institutes of Health (NIH), and NIH Grant GM62920. This work was also supported by the Molecular Basis of Disease Program, the Georgia Research Alliance, and the Georgia Cancer Coalition. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S4 and Table S1.

¹ To whom correspondence should be addressed: Laboratory of Chemical Physics, NIDDK, NIH, Bldg. 5, Rm. B2-29, Bethesda, MD 20892-0520. Tel.: 301-594-3122; Fax: 301-480-4001; E-mail: johnl{at}intra.niddk.nih.gov.

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