Interflap Distances in HIV-1 Protease Determined by Pulsed EPR Measurements

Galiano, Luis; Bonora, Marco; Fanucci, Gail E.

doi:10.1021/ja073684k

Cited by 53 publications

(113 citation statements)

References 19 publications

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“…Pulsed EPR experiment distance measurements for double labeled GM2AP constructs were performed on a Bruker E580 spectrometer with an MD-5 resonator with the four-pulse DEER sequence as described previously. 32−35 DEER data were analyzed with DEERAnalysis2011 software, available online at , and in-house Matlab DSim as described previously. 36−38 Because the average distances determined from pulsed experiments for double labeled sites in GM2AP were found to be near 20 Å, low temperature CW EPR experiments were also performed for distance determination.…”

Section: Methodsmentioning

confidence: 99%

Characterizing Solution Surface Loop Conformational Flexibility of the GM2 Activator Protein

et al. 2014

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GM2AP has a β-cup topology with numerous X-ray structures showing multiple conformations for some of the surface loops, revealing conformational flexibility that may be related to function, where function is defined as either membrane binding associated with ligand binding and extraction or interaction with other proteins. Here, site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy and molecular dynamic (MD) simulations are used to characterize the mobility and conformational flexibility of various structural regions of GM2AP. A series of 10 single cysteine amino acid substitutions were generated, and the constructs were chemically modified with the methanethiosulfonate spin label. Continuous wave (CW) EPR line shapes were obtained and subsequently simulated using the microscopic order macroscopic disorder (MOMD) program. Line shapes for sites that have multiple conformations in the X-ray structures required two spectral components, whereas spectra of the remaining sites were adequately fit with single-component parameters. For spin labeled sites L126C and I66C, spectra were acquired as a function of temperature, and simulations provided for the determination of thermodynamic parameters associated with conformational change. Binding to GM2 ligand did not alter the conformational flexibility of the loops, as evaluated by EPR and NMR spectroscopies. These results confirm that the conformational flexibility observed in the surface loops of GM2AP crystals is present in solution and that the exchange is slow on the EPR time scale (>ns). Furthermore, MD simulation results are presented and agree well with the conformational heterogeneity revealed by SDSL.

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Section: Methodsmentioning

confidence: 99%

Characterizing Solution Surface Loop Conformational Flexibility of the GM2 Activator Protein

et al. 2014

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“…Our SDSL DEER investigations on HIV-1PR clearly demonstrate the ability of this method to characterize conformational sampling ensembles and ensemble populations. 15, 43, 74–77, 88 …”

Section: Introductionmentioning

confidence: 99%

Pulsed EPR characterization of HIV-1 protease conformational sampling and inhibitor-induced population shifts

Liu

Casey

Blackburn

et al. 2016

Phys. Chem. Chem. Phys.

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The conformational landscape of HIV-1 protease (PR) can be experimentally characterized by pulsed-EPR double electron-electron resonance (DEER). For this characterization, nitroxide spin labels are attached to an engineered cysteine residue in the flap region of HIV-1 PR. DEER distance measurements from spin-labels contained within each flap of the homodimer provide a detailed description of the conformational sampling of apo-enzyme as well as induced conformational shifts as a function inhibitor binding. The distance distribution profiles are further interpreted in terms of a conformational ensemble scheme that consists of four unique states termed “curled/tucked”, “closed”, “semi-open” and “wide-open” conformations. Reported here are the DEER results for a drug-resistant variant clinical isolate sequence, V6, in the presence of FDA approved protease inhibitors (PIs) as well as a non-hydrolyzable substrate mimic, CaP2. Results are interpreted in the context of the current understanding of the relationship between conformational sampling, drug resistance, and kinetic efficiency of HIV-1PR as derived from previous DEER and kinetic data for a series of HIV-1PR constructs that contain drug-pressure selected mutations or natural polymorphisms. Specifically, these collective results support the notion that inhibitor-induced closure of the flaps correlates with inhibitor efficiency and drug resistance. This body of work also suggests DEER as a tool for studying conformational sampling in flexible enzymes as it relates to function.

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“…Flaps have been shown to regulate entry to the active site of proteases by providing access for substrate and/or inhibitors binding (15,18). Flap opening and closure in HIV protease (16) have been well studied using molecular dynamics (16,(19)(20)(21)(22)(23)(24)(25)(26). In general, different parameters have been proposed to describe the flap motion in the case of the HIV protease (20).…”

Section: Introductionmentioning

confidence: 99%

Investigation of flap flexibility of β-secretase using molecular dynamic simulations

Kumalo

Bhakat

Soliman

2015

Journal of Biomolecular Structure and Dynamics

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Flap motif and its dynamics were extensively reported in aspartate proteases, e.g. HIV proteases and plasmepsins. Herein, we report the first account of flap dynamics amongst different conformations of β-secretase using molecular dynamics simulation. Various parameters were proposed and a selected few were picked which could appropriately describe the flap motion. Three systems were studied, namely Free (BACEFree) and two ligand-bound conformations, which belonged to space groups P6122 (BACEBound1) and C2221 (BACEBound2), respectively and four parameters (distance between the flaps tip residue, Thr72 and Ser325, d1; dihedral angle, ϕ (Thr72-Asp32-Asp228-Ser325); TriCα angles, θ1 (Thr72-Asp32-Ser325), and θ2 (Thr72-Asp228-Ser325)) were proposed to understand the change in dynamics of flap domain and the extent of flap opening and closing. Analysis of, θ2, d1, θ1 and ϕ confirmed that the BACEFree adopted semi-open, open and closed conformations with slight twisting during flap opening. However, BACEBound1 (P6122) showed an adaptation to open conformation due to lack of hydrogen bond interaction between the ligand and flap tip residue. A slight flap twisting, ϕ (lateral twisting) was observed for BACEBound1 during flap opening which correlates with the opening of BACEFree. Contradictory to the BACEBound1, the BACEBound2 locked the flap in a closed conformation throughout the simulation due to formation of a stable hydrogen bond interaction between the flap tip residue and ligand. Analyses of all three systems highlight that d1, θ2 and ϕ can be precisely used to describe the extent of flap opening and closing concurrently with snapshots along the molecular dynamics trajectory across several conformations of β-secretase.

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Interflap Distances in HIV-1 Protease Determined by Pulsed EPR Measurements

Cited by 53 publications

References 19 publications

Characterizing Solution Surface Loop Conformational Flexibility of the GM2 Activator Protein

Characterizing Solution Surface Loop Conformational Flexibility of the GM2 Activator Protein

Pulsed EPR characterization of HIV-1 protease conformational sampling and inhibitor-induced population shifts

Investigation of flap flexibility of β-secretase using molecular dynamic simulations

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