Fig 1.
Two Alternate Conformations of Darunavir and the Active Site Mutation V82S in PRS17/DRV Dimer.
A. Fo-Fc omit map contoured at 3σ level shows Ser82 and DRV have two alternate conformations in both subunits. B. Sites of 17 mutations are mapped on PRS17 (pale green cartoon representation) with bound DRV shown as orange sticks. V82S mutation, proximal to the active site, is shown as a green sphere in each subunit. The mutations in the hinge loop cluster are colored as red spheres while the flap mutation cluster is represented as blue spheres. Note that K20R interacts with residues in the hinge loop although it is not contiguous in sequence with this region as described later. The distal mutations perturbing active site aspartates are colored as magenta spheres and remaining mutations are shown as pink spheres. C. Sequence alignment of PRS17 with PR and PR20. Mutations introduced in WT PR to restrict autoproteolysis (Q7K, L33I and L63I) and avoid cysteine-thiol oxidation (C67A and C95A) are indicted by asterisks. Residues identical to PR in PR20 and PRS17 are omitted. The 17 mutations of PRS17 are colored similar to 1B.
Table 1.
Crystallographic Data Collection and Refinement Statistics.
Fig 2.
Hydrogen Bond Interactions of Darunavir with PRS17 and Effect of Leu10 Mutations.
A. Hydrogen bond interactions of DRV with PRS17. PRS17 is in stick representation with green carbons while DRV is shown in ball and stick with white carbons. For the sake of clarity only one conformation of DRV and Ile50 of PRS17/DRV are shown. The hydrogen bond interactions of the second DRV are essentially identical. The hydrogen bonds are shown as broken lines. B. L10I mutation in PRS17 does not break the inter-monomer ion pair between Arg8 and Asp29′ unlike L10F in PR20. Wild-type PR is shown with grey carbons, PRS17 as green carbons and PR20 as salmon carbons. The van der Waals contacts are represented by (-·-) line. The minor conformation of Arg8 in wild-type PR and PR20 and its interactions with Asp29′ are omitted for clarity.
Fig 3.
Conformational Changes in Hinge Loop Mutations E35D, M36I and S37N.
A. Conformational changes in the hinge loop of PRS17/DRV complex in comparison to PR/DRV. B. Comparison of hinge loop between PRS17/DRV and PR20/DRV. C. Hinge loop conformation of PRS17/DRV, PR/DRV and PR20/DRV in subunit B. Wild-type PR, PRS17 and PR20 carbons are shown in grey, green and salmon, respectively.
Fig 4.
Conformational Changes induced by Flap Mutation Cluster of M46L, G48V and I54V.
A. Flap mutation cluster of M46L, G48V and I54V in PRS17/DRV compared to PR/DRV. B. Curling of flap due to flap mutation cluster in inhibitor-free structures of PRS17 (green ribbon), PR (grey ribbon) and PR20 (salmon ribbon). The conformation of flap tip in the circled portion is shown in sticks for inhibitor-free PRS17 (green carbon), PR (grey carbon) and PR20 (salmon carbon). C. Distance in (Å) between the flap tip residue Ile51 and the catalytic Asp25 in inhibitor-free PRS17, PR and PR20. D. Distance (Å) between the flap tip residue Ile51 and Thr80 in the three inhibitor-free structures.
Fig 5.
Effects of Distal mutations A71V, L90M and I93L.
A. L90M mutation in PRS17 (green) induces shortened C-H…O interaction between Met90 and catalytic Asp25 in comparison to wild-type PR (gray). I93L mutation in PRS17 results in loss of van der Waals contact observed between Ile93 and Leu 90 of wild-type PR. B. Distal mutations A71V and I93L in PRS17 are associated with shift in 70’s β strand by ~1 Å and loss of the ion pair between His69 and the carboxylate tail of the second subunit.
Fig 6.
NMR Analysis of Solution Conformation of Inhibitor-free PRS17 D25N.
A. 1H-15N TROSY-HSQC spectrum of the inhibitor-free PRS17D25N recorded in 20 mM sodium phosphate pH 5.7 at 20°C. B. 15N NOE measured under the same conditions for the inhibitor-free PRS17D25N (black) and PR20D25N (green) as a function of the residue number. C. Comparison between the 1DNH RDCs measured for the inhibitor-free PRS17D25N in a dilute solution of squalamine with those predicted from the crystal structures of the inhibitor-free PRS17 (red dots) and PRS17/DRV (gray dots). All the NMR experiments were recorded at 600 MHz.