Monitoring Inhibitor-Induced Conformational Population Shifts in HIV-1 Protease by Pulsed EPR Spectroscopy

Blackburn, Mandy E.; Veloro, Angelo M.; Fanucci, Gail E.

doi:10.1021/bi901201q

Cited by 40 publications

(126 citation statements)

References 27 publications

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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 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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supporting

confidence: 81%

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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“…In the respective works they used several different inhibitors and found a higher populational density for the closed state, in most cases of the complexed enzyme and semi-open conformation for the apo state. Blackburn et al, 2009 had previously reported, additionally, the interflap distance for the HIV-PR complexed with a peptidomimetic molecule (Ca-p2), which was also found to be mainly in the closed state.…”

Section: Standard Molecular Dynamics Induces Conformational Sampling mentioning

confidence: 86%

“…In the current study, the conformational dynamics of the flaps in HIV-PR were investigated using the distance between the Lys55 side chains nitrogen atom. This distance was previously referred to by electron paramagnetic resonance (EPR) spectroscopy experiments (Blackburn et al, 2009;Huang et al, 2014Huang et al, , 2012Torbeev et al, 2009) to show the conformational states of the flap structures. Huang et al and Torbeev et al have measured the interflap distance using inhibitor-bound and apo HIV-PR.…”

Section: Standard Molecular Dynamics Induces Conformational Sampling mentioning

confidence: 99%

Unraveling HIV protease flaps dynamics by Constant pH Molecular Dynamics simulations

Soares

Tôrres

Silva

et al. 2016

Journal of Structural Biology

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a b s t r a c tThe active site of HIV protease (HIV-PR) is covered by two flaps. These flaps are known to be essential for the catalytic activity of the HIV-PR, but their exact conformations at the different stages of the enzymatic pathway remain subject to debate. Understanding the correct functional dynamics of the flaps might aid the development of new HIV-PR inhibitors. It is known that, the HIV-PR catalytic efficiency is pHdependent, likely due to the influence of processes such as charge transfer and protonation/deprotonation of ionizable residues. Several Molecular Dynamics (MD) simulations have reported information about the HIV-PR flaps. However, in MD simulations the protonation of a residue is fixed and thus it is not possible to study the correlation between conformation and protonation state. To address this shortcoming, this work attempts to capture, through Constant pH Molecular Dynamics (CpHMD), the conformations of the apo, substrate-bound and inhibitor-bound HIV-PR, which differ drastically in their flap arrangements. The results show that the HIV-PR flaps conformations are defined by the protonation of the catalytic residues Asp25/Asp25 0 and that these residues are sensitive to pH changes. This study suggests that the catalytic aspartates can modulate the opening of the active site and substrate binding.

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“…The ELDOR frequency (pump position) was assigned to the maximum of the nitroxide absorption spectrum, with the observe position (chosen by selection of the static magnetic field) being placed at a 24 Gauss higher magnetic field strength on the field swept absorption spectrum. Temperature was maintained at 65 K during acquisition, which lasted 4-24 h. The background-corrected DEER decay was analyzed using the Tikhonov regularization method provided in the software DeerAnalysis2013.2 (37) to determine the distribution of distances encoded in the spectrum, followed by Gaussian analysis of the distribution assuming a model of discrete conformational states (38). In all cases the dominant distance distributions reported by Tikhonov regularization were well fit by two Gaussians (SI Appendix, Figs.…”

Section: Methodsmentioning

confidence: 99%

Site-directed spectroscopy of cardiac myosin-binding protein C reveals effects of phosphorylation on protein structural dynamics

Colson

Thompson

Espinoza-Fonseca

et al. 2016

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

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We have used the site-directed spectroscopies of time-resolved fluorescence resonance energy transfer (TR-FRET) and double electronelectron resonance (DEER), combined with complementary molecular dynamics (MD) simulations, to resolve the structure and dynamics of cardiac myosin-binding protein C (cMyBP-C), focusing on the N-terminal region. The results have implications for the role of this protein in myocardial contraction, with particular relevance to β-adrenergic signaling, heart failure, and hypertrophic cardiomyopathy. N-terminal cMyBP-C domains C0-C2 (C0C2) contain binding regions for potential interactions with both thick and thin filaments. Phosphorylation by PKA in the MyBP-C motif regulates these binding interactions. Our spectroscopic assays detect distances between pairs of site-directed probes on cMyBP-C. We engineered intramolecular pairs of labeling sites within cMyBP-C to measure, with high resolution, the distance and disorder in the protein's flexible regions using TR-FRET and DEER. Phosphorylation reduced the level of molecular disorder and the distribution of C0C2 intramolecular distances became more compact, with probes flanking either the motif between C1 and C2 or the Pro/Ala-rich linker (PAL) between C0 and C1. Further insight was obtained from microsecond MD simulations, which revealed a large structural change in the disordered motif region in which phosphorylation unmasks the surface of a series of residues on a stable α-helix within the motif with high potential as a protein-protein interaction site. These experimental and computational findings elucidate structural transitions in the flexible and dynamic portions of cMyBP-C, providing previously unidentified molecular insight into the modulatory role of this protein in cardiac muscle contractility. muscle | protein kinase A | fluorescence resonance energy transfer | DEER | molecular dynamics simulation

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Monitoring Inhibitor-Induced Conformational Population Shifts in HIV-1 Protease by Pulsed EPR Spectroscopy

Cited by 40 publications

References 27 publications

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

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

Unraveling HIV protease flaps dynamics by Constant pH Molecular Dynamics simulations

Site-directed spectroscopy of cardiac myosin-binding protein C reveals effects of phosphorylation on protein structural dynamics

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