Influence of Flanking Sequences on the Dimer Stability of Human Immunodeficiency Virus Type 1 Protease

Wondrak, Ewald M.; Louis, John M.

doi:10.1021/bi960984y

Cited by 51 publications

(68 citation statements)

References 16 publications

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“…15 N and 1 H chem- ical shifts of the terminal residues, e.g. Ile 3 and Ala 95 , of PR T26A /DMP323 are nearly identical to those of the PR/ DMP323, implying very similar terminal ␤-sheet arrangements of PR and PR T26A .…”

Section: Resultsmentioning

confidence: 89%

“…3D), which makes it ideal for studies of the mature PR dimer either free or bound to a native substrate (22). The mutation D25N may also influence the dimer stability of the mature protease (K D ϭ ϳ10 Ϫ6 M), 3 albeit to a much lesser extent than the mutants that form monomers discussed above (also see Table I). The construct PR D25N/Q2C/L97C was used to optimize the protocol for disulfide bond formation (see "Experimental Procedures" for details).…”

Section: Disulfide Bridge Formation Between the N-and C-terminal Stramentioning

confidence: 99%

“…Concluding Remarks-Mature wild-type HIV-1 protease upon its maturation from the Gag-Pol precursor forms a stable dimer exhibiting a subnanomolar dissociation constant (Ͻ5 nM) (3,17). A structure-assisted mutagenesis approach interfaced with experimental assessment of the designed variants by NMR allowed us to define key interactions that influence the monomer-dimer equilibrium (Table I).…”

Section: Disulfide Bridge Formation Between the N-and C-terminal Stramentioning

confidence: 99%

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Revisiting Monomeric HIV-1 Protease

Louis

Ishima

Nesheiwat

et al. 2003

Journal of Biological Chemistry

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Section: Resultsmentioning

confidence: 89%

Section: Disulfide Bridge Formation Between the N-and C-terminal Stramentioning

confidence: 99%

Section: Disulfide Bridge Formation Between the N-and C-terminal Stramentioning

confidence: 99%

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Revisiting Monomeric HIV-1 Protease

Louis

Ishima

Nesheiwat

et al. 2003

Journal of Biological Chemistry

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“…Experiments indicate initial cleavage by a precursor protease still embedded in the GagPol chain occurs through an intramolecular, concentration-independent mechanism with the precursor protease cleaving its own terminus (4) and critically modulated by the N-terminal region (5)(6)(7)(8). Mutations that block N-terminal cleavage result in severe loss of efficiency in catalytic activity.…”

mentioning

confidence: 99%

Kinetic characterization of the critical step in HIV-1 protease maturation

Sadiq

Noé

Fabritiis

2012

Proc. Natl. Acad. Sci. U.S.A.

114

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HIV maturation requires multiple cleavage of long polyprotein chains into functional proteins that include the viral protease itself. Initial cleavage by the protease dimer occurs from within these precursors, and yet only a single protease monomer is embedded in each polyprotein chain. Self-activation has been proposed to start from a partially dimerized protease formed from monomers of different chains binding its own N termini by self-association to the active site, but a complete structural understanding of this critical step in HIV maturation is missing. Here, we captured the critical self-association of immature HIV-1 protease to its extended aminoterminal recognition motif using large-scale molecular dynamics simulations, thus confirming the postulated intramolecular mechanism in atomic detail. We show that self-association to a catalytically viable state requires structural cooperativity of the flexible β-hairpin "flap" regions of the enzyme and that the major transition pathway is first via self-association in the semiopen/open enzyme states, followed by enzyme conformational transition into a catalytically viable closed state. Furthermore, partial N-terminal threading can play a role in self-association, whereas wide opening of the flaps in concert with self-association is not observed. We estimate the association rate constant (k on ) to be on the order of ∼1 × 10 4 s −1 , suggesting that N-terminal self-association is not the rate-limiting step in the process. The shown mechanism also provides an interesting example of molecular conformational transitions along the association pathway.conformational kinetics | Markov state model | high-throughput molecular dynamics H IV, along with all retroviruses, achieves infectious maturation of nascent virus particles through cleavage of polyprotein precursors by the viral protease. In particular, the GagPol chains contain several covalently linked proteins including the protease itself (Fig. 1A). Thus, maturation of the virus is initiated by autocatalysis of viral protease initially embedded in GagPol precursors. HIV-1 protease is functional only in dimeric form (1, 2) because activity of monomeric protease precursor is three orders of magnitude less than the mature dimer (3), and yet only a single monomer is embedded within each precursor. Two individual monomers in different GagPol chains must, therefore, come together to form an embedded dimeric protease, which ultimately cleaves itself into a mature form (Fig. 1B).Experiments indicate initial cleavage by a precursor protease still embedded in the GagPol chain occurs through an intramolecular, concentration-independent mechanism with the precursor protease cleaving its own terminus (4) and critically modulated by the N-terminal region (5-8). Mutations that block N-terminal cleavage result in severe loss of efficiency in catalytic activity. Cleavage at the protease (PR), reverse transcriptase (RT) junction at the C-terminal end of the protease by the precursor protease occurs via an intermolecular, concentrat...

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“…1) by kinetics and NMR revealed that the very low catalytic activity of the protease precursor prior to the cleavage at the N terminus is due to a much higher dimer dissociation constant (K d ) as compared with the mature protease (11). Upon intramolecular cleavage at the N terminus, the protease forms a stable dimer and exhibits a very low K d (Ͻ10 ϫ 10 Ϫ9 M in 50 mM sodium acetate, pH 5, at 25°C (8,13)). This suggests that inhibition of the protease function either by preventing or by disrupting dimer formation prior to its maturation provides an attractive avenue for inhibitor design.…”

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confidence: 99%

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

Ishima

Torchia

Louis

2007

Journal of Biological Chemistry

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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.

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Influence of Flanking Sequences on the Dimer Stability of Human Immunodeficiency Virus Type 1 Protease

Cited by 51 publications

References 16 publications

Revisiting Monomeric HIV-1 Protease

Revisiting Monomeric HIV-1 Protease

Kinetic characterization of the critical step in HIV-1 protease maturation

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

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