Rapid structural fluctuations of the free HIV protease flaps in solution: Relationship to crystal structures and comparison with predictions of dynamics calculations

Freedberg, Darón I.; Ishima, Rieko; Jacob, Jaison; Wang, Yun‐Xing; Kustanovich, I. M.; Louis, John M.; Torchia, Dennis A.

doi:10.1110/ps.33202

Cited by 202 publications

(248 citation statements)

References 48 publications

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“…This agrees well with previous calculations and experimental measurements of the relaxation rate of fast-flap conformational opening, which exhibits a small kinetic barrier (k flap ≈ 0.1 ns −1 ) (10,15,33,34). Even though self-association via both flap opening and lateral threading was observed, it is difficult to attribute any event solely to either one or the other mechanism.…”

Section: Resultssupporting

confidence: 91%

“…Access to this site is modulated by a pair of flexible β-hairpin flaps (Fig. 1C) that must first open to allow entry and then close (10)(11)(12)(13)(14)(15) to make substrate cleavage viable through a structurally conserved recognition pattern (16)(17)(18)(19). Recent advances in paramagnetic relaxation enhancement (PRE) (20) reveal transient N-terminal contacts within the active site, but a complete structural characterization of this transient process in full atomic detail remains an outstanding challenge and is the aim of this work.…”

mentioning

confidence: 99%

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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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Section: Resultssupporting

confidence: 91%

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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“…Fig. 8 shows plots of S 2 values determined for apo-inactive constructs Bi, PR5i, and AEi The results of Bi are similar to those obtained previously for a related subtype B construct called PMPR (19,21,31,59,64). Consistent with earlier findings, S 2 values plotted as a function of residue number (Fig.…”

supporting

confidence: 81%

“…The flaps must open to allow substrate entry and likely the release of catalytic product (19,20). Molecular dynamics (MD) simulations have shown nominally three states in this process as follows: namely closed, semiopen, and wide open conformations (20,21).…”

Section: Hiv-1 Protease (Hiv-1 Pr)mentioning

confidence: 99%

The Role of Select Subtype Polymorphisms on HIV-1 Protease Conformational Sampling and Dynamics

Huang

Britto

Kear-Scott

et al. 2014

Journal of Biological Chemistry

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Background: HIV-1 protease is an essential enzyme for HIV maturation. Results: Select and naturally occurring polymorphisms alter the conformational sampling and backbone dynamics of HIV-1 protease. Conclusion: These mutations lead to an alternative flap ensemble that we suspect is a curled flap orientation. Significance: The mechanism of distal mutations on drug resistance is unclear, but altered dynamics and conformational equilibria likely play key roles.

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“…The NOE experiments were repeated three times to calculate the average and SD of the NOE values. The overall rotational correlation time for backbone amide motion was determined using the protocol described previously (41).…”

Section: Methodsmentioning

confidence: 99%

Crystal and solution structures of an odorant-binding protein from the southern house mosquito complexed with an oviposition pheromone

Mao

Xu²,

Xü

et al. 2010

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

120

132

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Culex mosquitoes introduce the pathogens responsible for filariasis, West Nile virus, St. Louis encephalitis, and other diseases into humans. Currently, traps baited with oviposition semiochemicals play an important role in detection efforts and could provide an environmentally friendly approach to controlling their populations. The odorant binding proteins (OBPs) in the female's antenna play a crucial, if yet imperfectly understood, role in sensing oviposition cues. Here, we report the X-ray crystallography and NMR 3D structures of OBP1 for Culex quinquefasciatus (CquiOBP1) bound to an oviposition pheromone (5R,6S)-6-acetoxy-5-hexadecanolide (MOP). In both studies, CquiOBP1 had the same overall six-helix structure seen in other insect OBPs, but a detailed analysis revealed an important previously undescribed feature. There are two models for OBPmediated signal transduction: (i) direct release of the pheromone from an internal binding pocket in a pH-dependent fashion and (ii) detection of a pheromone-induced conformational change in the OBP·pheromone complex. Although CquiOBP1 binds MOP in a pHdependent fashion, it lacks the C terminus required for the pHdependent release model. This study shows that CquiOBP binds MOP in an unprecedented fashion using both a small central cavity for the lactone head group and a long hydrophobic channel for its tail.AaegOBP1 | AgamOBP1 | CquiOBP1 | NMR | X-ray crystallography

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Rapid structural fluctuations of the free HIV protease flaps in solution: Relationship to crystal structures and comparison with predictions of dynamics calculations

Cited by 202 publications

References 48 publications

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

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

The Role of Select Subtype Polymorphisms on HIV-1 Protease Conformational Sampling and Dynamics

Crystal and solution structures of an odorant-binding protein from the southern house mosquito complexed with an oviposition pheromone

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