Folding Regulates Autoprocessing of HIV-1 Protease Precursor

Abstract

Autoprocessing of HIV-1 protease (PR) precursors is a crucial step in the generation of the mature protease. Very little is known regarding the molecular mechanism and regulation of this important process in the viral life cycle. In this context we report here the first and complete residue level investigations on the structural and folding characteristics of the 17-kDa precursor TFR-PR-C nn (161 residues) of HIV-1 protease. The precursor shows autoprocessing activity indicating that the solution has a certain population of the folded active dimer. Removal of the 5-residue extension, C nn at the C-terminal of PR enhanced the activity to some extent. However, NMR structural characterization of the precursor containing a mutation, D25N in the PR at pH 5.2 and 32°C under different conditions of partial and complete denaturation by urea, indicate that the precursor has a high tendency to be unfolded. The major population in the ensemble displays some weak folding propensities in both the TFR and the PR regions, and many of these in the PR region are the non-native type. As both D25N mutant and wild-type PR are known to fold efficiently to the same native dimeric form, we infer that TFR cleavage enables removal of the non-native type of preferences in the PR domain to cause constructive folding of the protein. These results indicate that intrinsic structural and folding preferences in the precursor would have important regulatory roles in the autoprocessing reaction and generation of the mature enzyme.

The extremely slow‐exchanging core and acid‐denatured state of green fluorescent protein

“…These experiments have revealed that there can be both native-like and non-native secondary structure present, the exact structure depending upon the protein and experimental conditions. For example, both native and non-native secondary structural preferences were observed in the urea-denatured state of barstar (Bhavesh et al, 2004), in the GdmCldenatured state of HIV-1 protease (Bhavesh et al, 2001;Chatterjee et al, 2005), and in the acid-denatured state of hUBF HMG box 1 (Zhang et al, 2005). In contrast, ␤-type preferences were seen in denatured apomyoglobin which is a highly helical protein (Mohana-Borges et al, 2004).…”

Uncoupling Human Immunodeficiency Virus Type 1 gag and pol Reading Frames: Role of the Transframe Protein p6* in Viral Replication

“…In this regard, sequences adjacent to the PR region have been demonstrated to influence the dimerization efficiency of the Gag-Pol precursors and proper folding of the embedded PR domains, thereby triggering PR autoprocessing (6,10,21,22,23,30,33,42). Accordingly, dimerization defects of PR mutants carrying an inactivated dimer interface were successfully compensated for when the PR was expressed as part of the Gag-Pol precursor (30).…”

“…Proper folding of the embedded homodimeric protease (PR) is an essential prerequisite for full activity following sequential autocleavage from the full-length Gag-Pol polyprotein (summarized in reference 13). Indeed, the p6* residues have been demonstrated to stabilize the PR dimer and dictate the folding propensities of PR precursors, thereby influencing the rate of PR maturation (6,10,21). Interestingly, the autoprocessing of a truncated Gag-PR precursor was clearly accelerated when the p6* sequence was deleted, leading to the proposal that the active site of the PR might be less accessible in the presence of p6* (26).…”

Importance of Protease Cleavage Sites within and Flanking Human Immunodeficiency Virus Type 1 Transframe Protein p6* for Spatiotemporal Regulation of Protease Activation

“…Nuclear magnetic resonance analysis of recombinant p6* protein revealed a widely flexible structure (3,22), and its presence directly upstream of the PR is reminiscent of the propeptides flanking eukaryotic aspartic proteases and has been proven to prevent folding of the PR to its dimeric structure (6). Indeed, we could provide evidence that recombinant p6* protein is a strong competitive inhibitor of PR activity in vitro, and we proposed a model in which the carboxyl-terminal tetrapeptide of p6* following release from the Pol precursor, blocks the substrate binding cleft of the PR, thereby delaying overall processing (37).…”