Conformational Dynamics of the Dengue Virus Protease Revealed by Fluorescence Correlation and Single-Molecule FRET Studies

Abstract

The dengue virus protease (DENV-PR) represents an attractive target for counteracting DENV infections. It is generally assumed that DENV-PR can exist in an open and a closed conformation and that active site directed ligands stabilize the closed state. While crystal structures of both the open and the closed conformation were successfully resolved, information about the prevalence of these conformations in solution remains elusive. Herein, we address the question of whether there is an equilibrium between different conformations in solution which can be influenced by addition of a competitive inhibitor. To this end, DENV-PR was statistically labeled by two dye molecules constituting a FRET (fluorescence resonance energy transfer) couple. Fluorescence correlation spectroscopy and photon-burst detection were employed to examine FRET pair labeled DENV-PRs freely diffusing in solution. The measurements were performed with two double mutants and with two dye couples. The data provide strong evidence that an equilibrium of at least two conformations of DENV-PR exists in solution. The competitive inhibitor stabilizes the closed state. Because the open and closed conformations appear to coexist in solution, our results support the picture of a conformational selection rather than that of an induced fit mechanism with respect to the inhibitor-induced formation of the closed state.

A competition smFRET assay to study ligand‐induced conformational changes of the dengue virus protease

“…NMR or single molecule Förster resonance energy transfer (smFRET) have proven to be suitable tools for studying population distributions of conformational substrates in solution. 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 …”

“… 28 , 29 Using this method, it was already shown that the DENV protease in solution is in an equilibrium between two conformations and that the presence of a competitive inhibitor stabilizes the closed conformation. 16 …”

“…NMR or single molecule Förster resonance energy transfer (smFRET) have proven to be suitable tools for studying population distributions of conformational substrates in solution. [9][10][11][12][13][14][15][16] The dengue virus (DENV) NS2B-NS3 protease is essential for viral replication of the virus. 17 Besides host proteases, it is responsible for the cleavage of the viral precursor polyprotein, which is translated from the single-stranded RNA genome.…”

Fragment-based design of α-cyanoacrylates and α-cyanoacrylamides targeting Dengue and Zika NS2B/NS3 proteases

“…51 Considering these structural and functional aspects of the NS2B/NS3 pro , our research team has published several reviews 10,31,52 and original studies focused on the development of DENV and ZIKV NS2B/NS3 pro inhibitors. 53–56…”

Viruses: A Physical Chemistry Perspective

“…25 In the antiviral work category, Goẗz et al employ singlemolecule FRET in solution to reveal the dynamics of the dengue virus protease, which is an appealing antiviral target because it takes multiple conformations at equilibrium that can be shifted with the aid of inhibitors. 26 Because of area expansion of virus vectors caused by climate change, the dengue virus and related flaviviruses are an increasing global health concern. In the same category enters the Lassa virus, a pathogen from West Africa responsible for lethal hemorrhagic fever.…”