Role of invariant Thr80 in human immunodeficiency virus type 1 protease structure, function, and viral infectivity

Abstract

Sequence variability associated with human immunodeficiency virus type 1 (HIV-1) is useful for inferring structural and/or functional constraints at specific residues within the viral protease. Positions that are invariant even in the presence of drug selection define critically important residues for protease function. While the importance of conserved active-site residues is easily understood, the role of other invariant residues is not. This work focuses on invariant Thr80 at the apex of the P1 loop of HIV-1, HIV-2, and simian immunodeficiency virus protease. In a previous study, we postulated, on the basis of a molecular dynamics simulation of the unliganded protease, that Thr80 may play a role in the mobility of the flaps of protease. In the present study, both experimental and computational methods were used to study the role of Thr80 in HIV protease. Three protease variants (T80V, T80N, and T80S) were examined for changes in structure, dynamics, enzymatic activity, affinity for protease inhibitors, and viral infectivity. While all three variants were structurally similar to the wild type, only T80S was functionally similar. Both T80V and T80N had decreased the affinity for saquinavir. T80V significantly decreased the ability of the enzyme to cleave a peptide substrate but maintained infectivity, while T80N abolished both activity and viral infectivity. Additionally, T80N decreased the conformational flexibility of the flap region, as observed by simulations of molecular dynamics. Taken together, these data indicate that HIV-1 protease functions best when residue 80 is a small polar residue and that mutations to other amino acids significantly impair enzyme function, possibly by affecting the flexibility of the flap domain.

FIG. 1.

HIV-1 protease colored by sequence conservation (58). Residues colored red are invariant in both untreated and treated populations of patients. Those colored yellow are invariant only in the untreated population. Invariant glycine residues are colored blue. The side chains of the catalytic aspartic acids (Asp25), Trp6, Trp42, and Thr80 are displayed. (a) Unliganded protease structure (PDB code 1HHP). (b) Protease structure following 9-ns MD simulation.

FIG. 2.

General structural features of WT and variant proteases (WT, black; T80V, red; T80N, yellow; T80S, green). (a) Far-UV CD spectra of WT and variant proteases. (b) Tryptophan fluorescence spectra of WT and variant proteases, unbound and bound to SQV. The unliganded WT protease is shown as a solid line, and the unliganded variant proteases are shown as filled symbols. The WT protease bound to SQV is shown as a dotted line, and the variant proteases are shown as empty symbols. AU, arbitrary units.

FIG. 3.

Mutation at residue 80 affects protease activity. (a) Enzymatic activity. The increase in fluorescence over time observed when 10 μl of a 2 μM protease solution was mixed with 190 μl of 2 μM fluorescent substrate is shown. Both the WT (black) and T80S (green) completely cleaved the fluorescent substrate within 10 min. The initial burst in fluorescence occurs during the 20 s required for mixing. T80V (red) required 10 min to reach the same fluorescence that the WT and the T80S variant achieved in the first 20 s. The T80N (yellow) variant had virtually undetectable increases in fluorescence. AU, arbitrary units. (b) Western analysis of mutant virus particles. Lane 1, pNLCH used as the WT; lane 2, T80S mutant; lane 3, another clone of the T80S mutant; lane 4, T80V mutant; lane 5, T80N mutant; lane 6, another clone of the T80N mutant; lane 7, D25A active-site mutant of the protease used as a control for a negative processing phenotype. Molecular size markers were included in the gel, and their migration positions (sizes are in kilodaltons) are shown on the right.

FIG. 4.

Difference distance plots of SQV_T80N versus SQV_T80S. Relative changes in the distance between two alpha-carbon atoms in the SQV_T80S structure versus the SQV_T80N structure are shown. Green, blue, and red contours distinguish the ranges −1.0 to −0.5 Å, 0.5 to 1.0 Å, and 1 Å and above, respectively.

FIG. 5.

P1 loops of the crystal structures of SQV_T80N and SQV_T80S superimposed. Side chain oxygen atoms are in red, and side chain nitrogen atoms are in blue. Water molecules within 5 Å of residue 80 are shown as van der Waals spheres. (a) Asn80 (SQV_T80N, yellow; SQV, orange) and Ser80 (SQV_T80S, green; SQV, cyan) adopt very different conformations in the P1 loop. (b) Asn80′ and Ser80′ adopt similar conformations, and the P1 loops in this monomer are not significantly different between the two structures. The modeling program MIDAS (16) was used to superimpose the two protease structures, SQV_T80N and SQV_T80S, on the basis of the highly conserved terminal region (positions 1 to 9 and 86 to 99).

FIG. 6.

Differences in van der Waals contacts between the protease and SQV in the SQV_T80S and SQV_T80N crystal structures. Distances between these two structures that changed by more than 0.2 Å were considered significant. Side chain oxygen atoms are in red, and side chain nitrogen atoms are in blue. (a) van der Waals spheres are used to show contacts between protease atoms and SQV atoms that decreased by more than 0.2 Å in SQV_T80S relative to SQV_T80N. (b) van der Waals spheres are used to show contacts between protease atoms and SQV atoms that decreased by more than 0.2 Å in SQV_T80N relative to SQV_T80S.

FIG. 7.

Snapshots from the four MD simulations at 0.0, 1.5, 3.0, and 4.5 ns (WT, black; T80V, red; T80S, green; T80N, yellow). The flap region in the WT, T80V, and T80S simulations unfolded into the solvent within 4.5 ns. In the T80N simulations, the flap region was less mobile.