Folding Regulates Autoprocessing of HIV-1 Protease Precursor

Amarnath Chatterjee,P Mridula,Ram Kumar Mishra,Rohit Mittal,Ramakrishna V Hosur

doi:10.1074/jbc.m412603200

Amarnath Chatterjee, P Mridula + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m412603200

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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 degrees 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.

Highlights

Retroviruses including human immunodeficiency virus (HIV),1 use their minimal genetic information by encoding their structural proteins and enzymes as two polyprotein precursors Gag and Gag-Pol [1]
MBP stands for maltose-binding protein of Escherichia coli containing two native cleavage sites, p6pol/PR at the N terminus and PR/reverse transcriptase (RT) at the C terminus.) The first step involves an intramolecular cleavage of the N terminus that is followed by intermolecular cleavage of the C terminus [19, 21]
For the transframe region (TFR)-PR we see only an ϳ11-kDa peak for the PR and a ϳ7-kDa peak for the TFR part; for the TFR-PR-Cnn we see a peak at ϳ18 kDa corresponding to the precursor

Summary

Introduction

Retroviruses including human immunodeficiency virus (HIV), use their minimal genetic information by encoding their structural proteins and enzymes as two polyprotein precursors Gag and Gag-Pol [1]. Pettit et al [20] have recently shown, by co-expressing equivalent amounts of substituted Gag-Pol constructs, that the initial cleavage of the HIV-1 Gag-Pol precursor is intramolecular They showed that competitive active site inhibition by the drug retonavir was 10,000-fold less for the protease embedded in the precursor than for the mature free protease [20]. Functional characterization of the model precursor ⌬TFP-p6pol-PR (⌬TFP is a 5-residue variant of TFP) by examination of the mechanism and the pH rate profile of the autocatalytic reaction to produce mature PR shows that full-length TFR with its native cleavage sites is critical for the regulated autoprocessing of Gag-Pol and for optimal catalytic activity [28]. It has been shown that removal of the p6pol domain from the Gag-Pol polyprotein leads to a significantly higher rate of processing of the Gag-⌬Pol precursor [31]

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