NMR Structural And Dynamics Studies Of Hiv-1 Protease

Dental &Craniofacial Research

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Abstract

The mature, active HIV-1 protease is a homodimer that is made up of monomer subunits containing 99 amino acid residues. At least eight FDA-approved anti-protease drugs have been developed. Each of these drugs binds tightly to the dimer active site, thereby inhibiting catalytic activity and preventing propagation of the AIDs virus; however, one unfortunate consequence of targeting a single site on the protease has been the emergence of viral strains which carry multi-drug resistant mutations within their protease molecules. For this reason, there is considerable interest in identifying alternative protease sites that are suitable drug targets. Of particular interest are compounds that bind to the interface of the dimer. Such compounds will block formation of the active protease dimer, but be insensitive to current multidrug-resistant protease variants. In spite of these attractive features, dimerizatioin inhibitors face the formidable challenge of dissociating the tightly bound mature protease homodimer. We have shown that, in contrast with the mature protease, protease constructs, among them those that contain N-terminal extensions (similar to those of the protease precursor, which is embedded within the Gag-Pol polyprotein), have a million-fold larger dissociation constants and are predominantly monomeric at concentrations required for NMR studies. These discoveries suggest that the precursor monomer rather than the mature protease monomer is the target of choice of dimerization inhibitors. We have therefore initiated NMR studies to screen for molecules that interact with models of protease precursor monomer constructs, in order to identify inhibitors of precursor dimerization. Previous structural work that we have done on the protease monomer will aid in the design of dimerization inhibitors. The structual studies have shown that the monomer contains a folded core domain that is common to all monomer constructs that we have examined. The core domain presents preorganized target sites to which small compounds can bind. In addition we have found that mutations in core amino acid residues, which are not in the dimer interface, can destabilize the dimer. These sites are therefore also potential targets for dimer inhibitors. In recent work, we have used a combination of fluorescence and high resolution NMR measurements to follow the urea denaturation profiles of various protease mutants that are predominantly monomeric in solution. Preliminary analysis of this data indicates that either a deletion or an extension of several residues at the N- or C- termini of the mature protease sequence, does not affect the observed denaturation profile of the monomer. This result is consistent with our previous structural studies which show that the N- and C- terminal regions of the mature protease monomer and the monomer precursor (whose N-terminus extends beyond that of the mature protease) are disordered and flexible in solution. In contrast with these results, a Ser-Ala substitution near the active site increased the stability of the monomer against urea denaturation. This observation is thought to result from an enhancement of hydrophobic packing involving the Ala methyl with nearby Leu sidechains. NMR measurements of monomer structural features are being used together with urea denaturation profiles to elucidate structure-stability relationships of various mutants. We have continued studies of the dynamics and interactions of the N- and C-terminal strands that form the primary interface of the protease homodimer. This work is aimed at further understanding the mechanism of protease precursor processing. In the static structural model of the protease, derived from X-ray and NNMR work, terminal residues 1-4 and 96-99 from both monomers form a 4-stranded beta-sheet (the primary dimer interface) to which the exposed autolysis susceptible loop, containing residues 5-9, is connected. However, NMR transverse spin relaxation (R2) dispersion data, hydrogen-deuterium exchange rates and two-dimensional lineshapes provide strong evidence that residues 1-9 of the dimer interface sample two conformations, in dynamic equilibrium. The labile nature of the N-terminus, suggested by the data, is consistent with conclusions described in previous reports on a variety of protease mutants, which revealed that interactions involving the solvent exposed N-terminal strands of the protease are much less important in stabilizing the homodimer than interactions involving the interior C-terminal strands. Furthermore, the proposed dynamic structure rationalizes kinetics data which show that the N-terminal strand folds into the active site of the protease precursor. This conformational switch permits intermolecular cleavage of the immature N-terminus (an early step in Gag-Pol processing). Finally, the flexible N-terminus reveals how the loop containing residues 5-9 becomes fully accessible for autoproteolysis. Analysis of R2 relaxation dispersion profiles can, in principle, provide quantitative information about the chemical shifts and populations of conformations in dynamic equilibrium as well as the rate of the conformational interconversion. Accurate values of these physical parameters provide a basis for characterizing the structural and energetic changes associated with the dynamic process. Recently we have shown that extracting the best values of these parameters and their uncertainties requires careful assessment of experimental errors, both random and systematic, as well as correct application of statistical criteria to ensure that the data are fit with the appropriate statistical model. Current research is focusing upon analysis of errors in the dynamic parameters that result from the common assumption that the sites undergoing exchange have identical intrinsic relaxation rates. We have carried out extensive computer simulations that provide accurate quantities values of such errors for a variety of specific cases. In addition, we used the Carver-Richards equation to obtain general theoretical expressions for the errors. The theoretical equations predict results that are in good agreement with those obtained from the numerical calculations, and provide the spectoscopist with simple expressions for estimating the size of the errors under a wide range of experimental conditions.