Solution Structure of the Mature HIV-1 Protease Monomer

Abstract

We present the first solution structure of the HIV-1 protease monomer spanning the region Phe 1 -Ala 95(PR 1-95 ). Except for the terminal regions (residues 1-10 and 91-95) that are disordered, the tertiary fold of the remainder of the protease is essentially identical to that of the individual subunit of the dimer. In the monomer, the side chains of buried residues stabilizing the active site interface in the dimer, such as Asp 25 , Asp 29 , and Arg 87 , are now exposed to solvent. The flap dynamics in the monomer are similar to that of the free protease dimer. We also show that the protease domain of an optimized precursor flanked by 56 amino acids of the N-terminal transframe region is predominantly monomeric, exhibiting a tertiary fold that is quite similar to that of PR 1-95 structure. This explains the very low catalytic activity observed for the protease prior to its maturation at its N terminus as compared with the mature protease, which is an active stable dimer under identical conditions. Adding as few as 2 amino acids to the N terminus of the mature protease significantly increases its dissociation into monomers. Knowledge of the protease monomer structure and critical features of its dimerization may aid in the screening and design of compounds that target the protease prior to its maturation from the Gag-Pol precursor.

One Site Mutation Disrupts Dimer Formation in Human DPP-IV Proteins

“…Since two of the three triad residues (Asp 708 and His 740 ) are located on the C-terminal loop and close to the actual dimerization interface (17), the optimal alignment of the triad needed for catalysis, the conformation of the substrate binding pocket or/and the position of an oxyanion hole might be affected upon monomer formation. The studies on dimeric HCMV and HIV proteases have revealed that upon the introduction of the deletion/mutation at the dimer interface, the active site configuration is changed and a loop involved in oxyanion hole stabilization is distorted (39,44). The failure of the DPP-IV propeller loop to hold the dimer together upon the introduction of the mutation on the C-terminal loop emphasizes the importance of the C-terminal loop in dimer formation and maintenance.…”

Mode localization in the cooperative dynamics of protein recognition

“…These C-terminal residues stabilize the dimer by forming antiparallel β-strands with the N-terminal residues 1 -5. [37] Without this stabilizing element, both the NMR relaxation rates and the ensemble of NMR conformers [9,10] are consistent with an N-terminal tail that is highly flexible. Important conserved regions are the active site residues 22 -34, the flap loop residues 47 -52 which control access to the active site, and a region containing an α-helix and part of the hydrophobic core residues 74 -87.…”

“…[10] Figure 3 shows the mode dependent localized fluctuations as a function of the amino acid position inside the protein primary sequence. In its active dimeric form aspartyl protease catalyzes the cleaving of the peptide chain for the separation of the protein products of the HIV genome.…”

“…The extent of the energetic barriers involved indicates if a new dynamically active region emerges upon binding that is likely involved in the following step in a reaction pathway, or if entropically-relevant multiple states emerge that are distributed along the protein for a straightforward entropy-enthalpy compensation mechanism. As an example we study both monomer and dimerized HIV protease, [9,10] and free Insulin Growth Factor II Receptor (IGF2R) domain 11 and the IGF2R:IGF2 complex. [11] Other theoretical models based upon effective harmonic descriptions, which calculate sequence dependent protein flexibility, are Normal Mode Analysis (NMA), and general Elastic Network Models (ENM) including the Gaussian Network Model (GNM).…”

Predicting protein dynamics from structural ensembles

“…These proteins were N-TIMP-1 (1D2B) [58], a de Novo α-helix bundle protein s836 (2JUA) [59], Cellular retinol-binding protein I CPB1 (1MX7) [60], Kinase-associated protein phosphatase KAPP (1MZK) [61,62], Insulin Growth Factor 2 Receptor IFG2R domain 11 (2M6T) [63], Ubiquitin (1UBQ) [64,65], and HIV Protease monomer (1Q9P). [66,67] The input parameters to the LE4PD equation change from protein to protein: the structural parameters such as bond length, monomer friction, hydrodynamic radius, and the pairwise bond correlations are determined from the structural ensemble, while the thermodynamic parameters such as solvent viscosity, and temperature, are defined by the experimental conditions. The viscosity was set to account for temperature dependence and content of deuterated water.…”