Binding mode of the activity‐modulating C‐terminal segment of <scp>NS</scp>2B to <scp>NS</scp>3 in the dengue virus <scp>NS</scp>2B–<scp>NS</scp>3 protease

Cruz, Laura de la; Chen, Wan-Na; Graham, Bimbil; Otting, Gottfried

doi:10.1111/febs.12729

Cited by 59 publications

(107 citation statements)

References 68 publications

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“…This observation further substantiates the hypothesis that product release is the rate-limiting step in the catalytic cycle. This interpretation is further supported by structural data1738. In the first reported ligand-bound DENV crystal structure, two different complexes were observed in the asymmetric unit, one with the full tetrapeptide inhibitor (bz-Nle-KRR-H) and one with a degraded peptide, which was modelled as a di-Arg degraded product17.…”

Section: Discussionmentioning

confidence: 68%

“…5), even after 1000 s (data not shown), indicating that structural changes induced by substrate binding appear to be irreversible on the time scale employed for the catalytic measurements. Previous data have shown that cleavage products can remain bound to the active site in NS3pro1738, possibly supported by stabilising electrostatic interactions. It therefore seems likely that the hydrolysis products may remain bound to the protease until displaced by the next incoming substrate molecule.…”

Section: Resultsmentioning

confidence: 85%

“…Available crystallographic data suggest that the β-hairpin of NS2B H may be relatively flexible and directed towards the solvent, forming no interactions with NS3pro5. The observation of extreme broadening of NMR resonances supports structural instability of the ligand-free complex and the potential for the β-hairpin to adopt varied conformations20383940. Alternatively, NMR studies utilising 15 N-relaxation rates, paramagnetic relaxation enhancement (PREs) and pseudo-contact shifts (PCS) in the unlinked protease complex have indicated that the crystallised ‘open’ conformation of DENV protease is instead a result of destabilisation or degradation of the complex caused by high pH and ionic strength2038.…”

Section: Discussionmentioning

confidence: 94%

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Product release is rate-limiting for catalytic processing by the Dengue virus protease

Shannon

Pedroso

Chappell

et al. 2016

Sci Rep

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Dengue Virus (DENV) is the most prevalent global arbovirus, yet despite an increasing burden to health care there are currently no therapeutics available to treat infection. A potential target for antiviral drugs is the two-component viral protease NS2B-NS3pro, which is essential for viral replication. Interactions between the two components have been investigated here by probing the effect on the rate of enzyme catalysis of key mutations in a mobile loop within NS2B that is located at the interface of the two components. Steady-state kinetic assays indicated that the mutations greatly affect catalytic turnover. However, single turnover and fluorescence experiments have revealed that the mutations predominantly affect product release rather than substrate binding. Fluorescence analysis also indicated that the addition of substrate triggers a near-irreversible change in the enzyme conformation that activates the catalytic centre. Based on this mechanistic insight, we propose that residues within the mobile loop of NS2B control product release and present a new target for design of potent Dengue NS2B-NS3 protease inhibitors.

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Section: Discussionmentioning

confidence: 68%

Section: Resultsmentioning

confidence: 85%

Section: Discussionmentioning

confidence: 94%

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Product release is rate-limiting for catalytic processing by the Dengue virus protease

Shannon

Pedroso

Chappell

et al. 2016

Sci Rep

View full text Add to dashboard Cite

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“…The diversity of conformations of the NS2B C-terminal region indicated that the NS2B C-terminus is highly flexible, and that the conformations seen in the crystal structures may only represent one or a few of many conformations of NS2B in solution. Recently, using unlinked NS2B-NS3 constructs, a few NMR studies indicated that the DENV NS2B-NS3 protease mainly displayed the active “closed” conformation regardless of inhibitor binding [62–65]. However, NMR studies also indicated that (1) the C-terminal portion of NS2B was still found to be flexible; (2) conformational changes were also observed and could be induced upon buffer manipulations; and (3) active-site inhibitor further stabilized the “closed” conformation [36, 63–65].…”

Section: Discussionmentioning

confidence: 99%

A conformational switch high-throughput screening assay and allosteric inhibition of the flavivirus NS2B-NS3 protease

et al. 2017

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The flavivirus genome encodes a single polyprotein precursor requiring multiple cleavages by host and viral proteases in order to produce the individual proteins that constitute an infectious virion. Previous studies have revealed that the NS2B cofactor of the viral NS2B-NS3 heterocomplex protease displays a conformational dynamic between active and inactive states. Here, we developed a conformational switch assay based on split luciferase complementation (SLC) to monitor the conformational change of NS2B and to characterize candidate allosteric inhibitors. Binding of an active-site inhibitor to the protease resulted in a conformational change of NS2B and led to significant SLC enhancement. Mutagenesis of key residues at an allosteric site abolished this induced conformational change and SLC enhancement. We also performed a virtual screen of NCI library compounds to identify allosteric inhibitors, followed by in vitro biochemical screening of the resultant candidates. Only three of these compounds, NSC135618, 260594, and 146771, significantly inhibited the protease of Dengue virus 2 (DENV2) in vitro, with IC50 values of 1.8 μM, 11.4 μM, and 4.8 μM, respectively. Among the three compounds, only NSC135618 significantly suppressed the SLC enhancement triggered by binding of active-site inhibitor in a dose-dependent manner, indicating that it inhibits the conformational change of NS2B. Results from virus titer reduction assays revealed that NSC135618 is a broad spectrum flavivirus protease inhibitor, and can significantly reduce titers of DENV2, Zika virus (ZIKV), West Nile virus (WNV), and Yellow fever virus (YFV) on A549 cells in vivo, with EC50 values in low micromolar range. In contrast, the cytotoxicity of NSC135618 is only moderate with CC50 of 48.8 μM on A549 cells. Moreover, NSC135618 inhibited ZIKV in human placental and neural progenitor cells relevant to ZIKV pathogenesis. Results from binding, kinetics, Western blot, mass spectrometry and mutagenesis experiments unambiguously demonstrated an allosteric mechanism for inhibition of the viral protease by NSC135618.

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“…Amazingly, unlike other proteases with a chymotrypsin-like fold, the flavivirus proteases including dengue protease, requires a stretch of approximately 40 amino acids from the cytosolic domain of NS2B for catalytic activity, thus called two-component protease [6][7][8]. Intriguingly, while the protease domains adopt highly similar structures in all crystal structures determined to date, the NS2B cofactors have been found to assume two distinctive structures, namely open (inactive) and closed (active) conformations by X-ray crystallography and NMR spectroscopy [7][8][9][10][11][12].…”

Section: Introductionmentioning

confidence: 99%

NMR and MD Studies Reveal That the Isolated Dengue NS3 Protease Is an Intrinsically Disordered Chymotrypsin Fold Which Absolutely Requests NS2B for Correct Folding and Functional Dynamics

2015

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Dengue genome encodes a two component protease complex (NS2B-NS3pro) essential for the viral maturation/infectivity, thus representing a key drug target. Previously, due to its “complete insolubility”, the isolated NS3pro could not be experimentally studied and it remains elusive what structure it adopts without NS2B and why NS2B is indispensable. Here as facilitated by our previous discovery, the isolated NS3pro has been surprisingly deciphered by NMR to be the first intrinsically-disordered chymotrypsin-like fold, which exists in a loosely-packed state with non-native long-range interactions as revealed by paramagnetic relaxation enhancement (PRE). The disordered NS3pro appears to be needed for binding a human host factor to trigger the membrane remodeling. Moreover, we have in vitro refolded the NS3pro in complex with either NS2B (48–100) or the full-length NS2B (1–130) anchored into the LMPC micelle, and the two complexes have similar activities but different dynamics. We also performed molecular dynamics (MD) simulations and the results revealed that NS2B shows the highest structural fluctuations in the complex, thus providing the dynamic basis for the observation on its conformational exchange between open and closed states. Remarkably, the NS2B cofactor plays a central role in maintaining the correlated motion network required for the catalysis as we previously decoded for the SARS 3CL protease. Indeed, a truncated NS2B (48–100;Δ77–84) with the flexible loop deleted is able to trap the NS2B-NS3pro complex in a highly dynamic and catalytically-impotent state. Taken together, our study implies potential strategies to perturb the NS2B-NS3pro interface for design of inhibitors for treating dengue infection.

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Binding mode of the activity‐modulating C‐terminal segment of NS2B to NS3 in the dengue virus NS2B–NS3 protease

Cited by 59 publications

References 68 publications

Product release is rate-limiting for catalytic processing by the Dengue virus protease

Product release is rate-limiting for catalytic processing by the Dengue virus protease

A conformational switch high-throughput screening assay and allosteric inhibition of the flavivirus NS2B-NS3 protease

NMR and MD Studies Reveal That the Isolated Dengue NS3 Protease Is an Intrinsically Disordered Chymotrypsin Fold Which Absolutely Requests NS2B for Correct Folding and Functional Dynamics

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