Trp42 rotamers report reduced flexibility when the inhibitor acetyl-pepstatin is bound to HIV-1 protease

B. Ullrich, M. Laberge, F. Tölgyesi, J. Fidy, Z. Szeltner, L. Polgár

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

The Q7K/L33I/L63I HIV-1 protease mutant was expressed in Escherichia coli and the effect of binding a substrate-analog inhibitor, acetyl-pepstatin, was investigated by fluorescence spectroscopy and molecular dynamics. The dimeric enzyme has four intrinsic tryptophans, located at positions 6 and 42 in each monomer. Fluorescence spectra and acrylamide quenching experiments show two differently accessible Trp populations in the apoenzyme with kq1 = 6.85 × l09 M-1 s-1 and kq2 = 1.88 × 109 M-1 s-1, that merge into one in the complex with kq = 1.78 × 109 M-1 s-1. 500 ps trajectory analysis of Trp X1/X2 rotameric interconversions suggest a model to account for the observed Trp fluorescence. In the simulations, Trp6/Trp6B rotameric interconversions do not occur on this timescale for both HIV forms. In the apoenzyme simulations, however, both Trp42s and Trp42Bs are flipping between X1/X2 states; in the complexed form, no such interconverions occur. A detailed investigation of the local Trp environments sampled during the molecular dynamics simulation suggests that one of the apoenzyme Trp42B rotameric interconversions would allow indole-quencher contact, such as with nearby Tyr59. This could account for the short lifetime component. The model thus interprets the experimental data on the basis of the conformational fluctuations of Trp42s alone. It suggests that the rotameric interconversions of these Trps, located relatively far from the active site and at the very start of the flap region, becomes restrained when the apoenzyme binds the inhibitor. The model is thus consistent with associating components of the fluorescence decay in HIV-1 protease to ground state conformational heterogeneity.

Original languageEnglish
Pages (from-to)2232-2245
Number of pages14
JournalProtein Science
Volume9
Issue number11
Publication statusPublished - 2000

Fingerprint

Apoenzymes
Fluorescence
Molecular Dynamics Simulation
Molecular dynamics
Acrylamide
Fluorescence Spectrometry
Fluorescence spectroscopy
Tryptophan
Escherichia coli
Ground state
Quenching
Catalytic Domain
Monomers
Trajectories
HIV
Streptomyces pepsin inhibitor
Human immunodeficiency virus 1 p16 protease
Computer simulation
Substrates
Enzymes

Keywords

  • 500 ps molecular dynamics
  • Acrylamide quenching
  • Dihedral trajectories
  • Dynamics influenced by inhibitor
  • Ground state heterogeneity
  • Trp fluorescence
  • Trp rotamers

ASJC Scopus subject areas

  • Biochemistry

Cite this

Trp42 rotamers report reduced flexibility when the inhibitor acetyl-pepstatin is bound to HIV-1 protease. / Ullrich, B.; Laberge, M.; Tölgyesi, F.; Fidy, J.; Szeltner, Z.; Polgár, L.

In: Protein Science, Vol. 9, No. 11, 2000, p. 2232-2245.

Research output: Contribution to journalArticle

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abstract = "The Q7K/L33I/L63I HIV-1 protease mutant was expressed in Escherichia coli and the effect of binding a substrate-analog inhibitor, acetyl-pepstatin, was investigated by fluorescence spectroscopy and molecular dynamics. The dimeric enzyme has four intrinsic tryptophans, located at positions 6 and 42 in each monomer. Fluorescence spectra and acrylamide quenching experiments show two differently accessible Trp populations in the apoenzyme with kq1 = 6.85 × l09 M-1 s-1 and kq2 = 1.88 × 109 M-1 s-1, that merge into one in the complex with kq = 1.78 × 109 M-1 s-1. 500 ps trajectory analysis of Trp X1/X2 rotameric interconversions suggest a model to account for the observed Trp fluorescence. In the simulations, Trp6/Trp6B rotameric interconversions do not occur on this timescale for both HIV forms. In the apoenzyme simulations, however, both Trp42s and Trp42Bs are flipping between X1/X2 states; in the complexed form, no such interconverions occur. A detailed investigation of the local Trp environments sampled during the molecular dynamics simulation suggests that one of the apoenzyme Trp42B rotameric interconversions would allow indole-quencher contact, such as with nearby Tyr59. This could account for the short lifetime component. The model thus interprets the experimental data on the basis of the conformational fluctuations of Trp42s alone. It suggests that the rotameric interconversions of these Trps, located relatively far from the active site and at the very start of the flap region, becomes restrained when the apoenzyme binds the inhibitor. The model is thus consistent with associating components of the fluorescence decay in HIV-1 protease to ground state conformational heterogeneity.",
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AU - Ullrich, B.

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AU - Szeltner, Z.

AU - Polgár, L.

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N2 - The Q7K/L33I/L63I HIV-1 protease mutant was expressed in Escherichia coli and the effect of binding a substrate-analog inhibitor, acetyl-pepstatin, was investigated by fluorescence spectroscopy and molecular dynamics. The dimeric enzyme has four intrinsic tryptophans, located at positions 6 and 42 in each monomer. Fluorescence spectra and acrylamide quenching experiments show two differently accessible Trp populations in the apoenzyme with kq1 = 6.85 × l09 M-1 s-1 and kq2 = 1.88 × 109 M-1 s-1, that merge into one in the complex with kq = 1.78 × 109 M-1 s-1. 500 ps trajectory analysis of Trp X1/X2 rotameric interconversions suggest a model to account for the observed Trp fluorescence. In the simulations, Trp6/Trp6B rotameric interconversions do not occur on this timescale for both HIV forms. In the apoenzyme simulations, however, both Trp42s and Trp42Bs are flipping between X1/X2 states; in the complexed form, no such interconverions occur. A detailed investigation of the local Trp environments sampled during the molecular dynamics simulation suggests that one of the apoenzyme Trp42B rotameric interconversions would allow indole-quencher contact, such as with nearby Tyr59. This could account for the short lifetime component. The model thus interprets the experimental data on the basis of the conformational fluctuations of Trp42s alone. It suggests that the rotameric interconversions of these Trps, located relatively far from the active site and at the very start of the flap region, becomes restrained when the apoenzyme binds the inhibitor. The model is thus consistent with associating components of the fluorescence decay in HIV-1 protease to ground state conformational heterogeneity.

AB - The Q7K/L33I/L63I HIV-1 protease mutant was expressed in Escherichia coli and the effect of binding a substrate-analog inhibitor, acetyl-pepstatin, was investigated by fluorescence spectroscopy and molecular dynamics. The dimeric enzyme has four intrinsic tryptophans, located at positions 6 and 42 in each monomer. Fluorescence spectra and acrylamide quenching experiments show two differently accessible Trp populations in the apoenzyme with kq1 = 6.85 × l09 M-1 s-1 and kq2 = 1.88 × 109 M-1 s-1, that merge into one in the complex with kq = 1.78 × 109 M-1 s-1. 500 ps trajectory analysis of Trp X1/X2 rotameric interconversions suggest a model to account for the observed Trp fluorescence. In the simulations, Trp6/Trp6B rotameric interconversions do not occur on this timescale for both HIV forms. In the apoenzyme simulations, however, both Trp42s and Trp42Bs are flipping between X1/X2 states; in the complexed form, no such interconverions occur. A detailed investigation of the local Trp environments sampled during the molecular dynamics simulation suggests that one of the apoenzyme Trp42B rotameric interconversions would allow indole-quencher contact, such as with nearby Tyr59. This could account for the short lifetime component. The model thus interprets the experimental data on the basis of the conformational fluctuations of Trp42s alone. It suggests that the rotameric interconversions of these Trps, located relatively far from the active site and at the very start of the flap region, becomes restrained when the apoenzyme binds the inhibitor. The model is thus consistent with associating components of the fluorescence decay in HIV-1 protease to ground state conformational heterogeneity.

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