Dynamic properties of chemically synthesized Gly51 analogues of HIV-1 protease. (A) “Snapshots” of the conformational states (open, semiopen, and closed overlaid in B) of HIV-1 protease chemical analogues labeled with nitroxide-spin label, measured at 55 K using pulse-EPR spectroscopy. Populations of conformers are depicted for Gly51, L-Ala51, D-Ala51 and Aib51 homodimers in green, blue, red, and black, respectively. In the case of the D-Ala51 homodimer analogue, the semiopen conformer is overpopulated. (C) ¹⁵N-HSQC spectra overlaid for L-Ala51, D-Ala51, and Aib51 homodimers with the same color coding as in A. Peaks for the wild-type HIV-1 protease (in green) for corresponding residues were reconstructed from a previous study (21). Order parameters S², obtained by model-free analysis of R₁, R₂ and ¹H-¹⁵N NOE values, versus residue number are depicted as Inset. (D) CPMG ¹⁵N relaxation dispersion data for catalytic residue Asp25 in three HIV-1 protease symmetric homodimers obtained at two magnetic fields (600 and 900 MHz). Remarkably, the chemical exchange rates for the three analogue enzyme molecules are drastically different, and are correlated with the dynamic properties of the flaps and with the catalytic rates for proteolysis (Table 1; entries 5, 7, and 8).

Active-site structures revealed by MD simulations, and a scheme for the mechanism of HIV-1 protease catalysis taking into account the dynamics of the flaps. (A) An asymmetric structure of catalytic Asp25 and Asp25′ and the nucleophilic water molecule dominated in the MD trajectory for the asymmetric [L-Ala51; D-Ala51′] chemical analogue of HIV-1 protease with the L-domain having a protonated Asp25 and the D-domain having a charged Asp25′ side chain. (B) Symmetric structure for catalytic residues and nucleophilic water was observed as the most populous state in the MD simulation for the symmetric [L-Ala51; L-Ala51′] and [D-Ala51; D-Ala51′] chemical analogues. (C) Scheme for the mechanism of HIV-1 protease catalysis. Asymmetric conformers are preorganized for catalysis. Conformational isomerizations are depicted by blue arrows, and electron rearrangements are depicted by red arrows. TS1 and TS2 are earlier and later transition states, respectively, and E.I is the enzyme complex with the tetrahedral intermediate.

Structural features of HIV-1 protease. (A) Homodimeric catalytically active form of HIV-1 protease (2 × 99 amino acids) complexed with the peptidomimetic reduced isostere-containing MVT-101 inhibitor. (B) The β-turn structures (residues 37–61) from each monomer, known as “flaps,” donate hydrogen bonds to the substrate (or inhibitor) through a structural water (water 301) molecule (in green). (C) Flaps with β-turn type I (Upper Left) and β-turn type II (Lower Right) conformations depicted separately for clarity. Residue Gly51 has D-amino acid conformation in the β-turn type I structure and L-amino acid conformation in the β-turn type II structure. (D) Flap X-ray structures in chemically synthesized [L-Ala51; D-Ala51′] covalent dimer HIV-1 protease molecule; 2F_o - F_c electron density contoured in blue at 1.5σ level for the 1.6-Å resolution crystal structure [Protein Data Bank (PDB) ID code 3FSM; see SI Appendix].

X-ray structures of HIV-1 protease and its ester chemical analogues 9 and 10 complexed with substrate-based inhibitors. (A and B) In complexes of enzymes 9 and 10 with MVT-101 inhibitor, the electron density for structural water 301 molecule is significantly diffused—most strikingly in the enzyme 10 complex (see B), where we could not locate clear density for a water molecule at the flaps/inhibitor interface. (C and D) Interestingly, with the mechanistically based hydrated ketomethylene inhibitor KVS-1 (9), water 301 is well-populated in structures of enzymes 9 and 10. Moreover, in the complex of enzyme 10, there is second structural water molecule. [In all structures, side chains are deleted for clarity except the residues of interest. The 2F_o - F_celectron density map was contoured at 1.0 σ level (in magenta) for selected flap residues and water molecules, and at a level of 3σ (in green) for residues Asp25 and Asp25′. Distances between distal oxygens of two catalytic Asp25 and Asp25′ and PDB codes are specified for each structure].

“Activity–flexibility” relationship for a series of chemical analogues of HIV-1 protease. The “activity” axis contains relative values of k_cat and k_cat/K_m for enzyme analogues 1–8 (see Table 1) normalized to those of the wild-type enzyme 1. The “flexibility” axis is built based on the assumption that the flap structure of the enzyme molecule containing Gly at position 51 is most flexible, followed by molecules containing L-Ala, D-Ala, and Aib (α-aminoisobutyric acid) replacing Gly. Most of the data follow a general trend where higher flexibility leads to higher activity. The exception is enzyme analogue 2 with one flap containing L-Ala51 and another D-Ala51′.