Revisiting Monomeric HIV-1 Protease

Self Cite

101

An experimental protocol for folding the mature human immunodeficiency virus-1 (HIV-1) protease is presented that facilitates NMR studies at a low protein concentration of ϳ20 M. Under these conditions, NMR spectra show that the mature protease lacking its terminal ␤-sheet residues 1-4 and 96 -99 (PR 5-95 ) exhibits a stable monomer fold spanning the region 10 -90 that is similar to that of the single subunit of the wildtype dimer and the dimer bearing a D25N mutation (PR D25N ). Urea-induced unfolding monitored both by changes in 1 H-15 N heteronuclear single quantum correlation spectra and by protein fluorescence indicates that although PR 5-95 monomer displays a transition profile similar to that of the PR D25N dimer (50% unfolded (U 50 ) ‫؍‬ ϳ1.9 M), extending the protease with 4 residues (SFNF) of its N-terminally flanking sequence in the Gag-Pol precursor ( SFNF PR D25N ) decreases the stability of the fold (U 50 ‫؍‬ ϳ1.5 M). Assigned backbone chemical shifts were used to elucidate differences in the stability of the PR T26A (U 50 ‫؍‬ 2.5 M) and SFNF PR D25N monomers and compared with PR D25N/T26A monomer. Discernible differences in the backbone chemical shifts were observed for N-terminal protease residues 3-6 of SFNF PR D25N that may relate to the increase in the equilibrium dissociation constant (K d ) and the very low catalytic activity of the protease prior to its autoprocessing at its N terminus from the Gag-Pol precursor.

Section: Pr Lacking the Terminal Residues 1-4 And 96 -99 Exhibits A Ssupporting

confidence: 61%

mentioning

confidence: 99%

Mutational and Structural Studies Aimed at Characterizing the Monomer of HIV-1 Protease and Its Precursor

Ishima

Torchia

Louis

2007

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101

“…For this purpose we first prepared a D25N mutant of the precursor, which is inactive as the mutation is at the active site of PR. At the same time it is also known that the D25N mutation does not affect the folding of the protease (37). This mutant precursor is thus stable for weeks together and is ideally suited for structural characterization by NMR.…”

Section: C-terminal Extension At Pr Retards the Autoprocessing Activimentioning

confidence: 91%

Folding Regulates Autoprocessing of HIV-1 Protease Precursor

Chatterjee

Mridula

Mishra

et al. 2005

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.

“…Studies characterizing the HIV protease monomer were not feasible until we recently demonstrated that mutations of the interface residues, such as D29N, R87K, or deletion mutants of the terminal residues 1-4 or 96-99, destabilize the dimer (24,25). While it is apparent that deletion of the terminal residues precludes the formation of the terminal ␤-sheet and disrupts dimerization, these studies also revealed that subtle intermonomer contacts formed by the conserved Asp 29 and Arg 87 residues are essential to the dimerization of the mature protease.…”

Section: Resultsmentioning

confidence: 99%

“…The existence of a folded monomer was recently observed only when unique dimer interface contacts were disrupted via mutations. These mutations increased the dissociation constant of the protease dramatically such that a monomer could be studied by solution NMR at a high protein concentration of up to 1 mM (in monomer) (24,25). A series of mutants PR R87K , PR D29N , PR T26A , PR , and PR has been described to exhibit a monomer fold in the absence of inhibitor.…”

mentioning

confidence: 99%

Solution Structure of the Mature HIV-1 Protease Monomer

Ishima

Torchia

Lynch

et al. 2003

Self Cite

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.