posted on 2021-06-17, 09:14authored byChristian Götz, Gerald Hinze, Andrea Gellert, Hannah Maus, Franziska von Hammerstein, Stefan J. Hammerschmidt, Luca M. Lauth, Ute A. Hellmich, Tanja Schirmeister, Thomas Basché
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.