  Riboswitches are cis-acting regulatory RNA elements prevalently located in
the leader sequences of bacterial mRNA. An adenine sensing riboswitch
cis-regulates adeninosine deaminase gene (add) in Vibrio vulnificus. The
structural mechanism regulating its conformational changes upon ligand binding
mostly remains to be elucidated. In this open framework it has been suggested
that the ligand stabilizes the interaction of the distal "kissing loop"
complex. Using accurate full-atom molecular dynamics with explicit solvent in
combination with enhanced sampling techniques and advanced analysis methods it
could be possible to provide a more detailed perspective on the formation of
these tertiary contacts. In this work, we used umbrella sampling simulations to
study the thermodynamics of the kissing loop complex in the presence and in the
absence of the cognate ligand. We enforced the breaking/formation of the
loop-loop interaction restraining the distance between the two loops. We also
assessed the convergence of the results by using two alternative initialization
protocols. A structural analysis was performed using a novel approach to
analyze base contacts. Our simulations qualitatively indicated that the ligand
could stabilize the kissing loop complex. We also compared with previously
published simulation studies. Kissing complex stabilization given by the ligand
was compatible with available experimental data. However, the dependence of its
value on the initialization protocol of the umbrella sampling simulations posed
some questions on the quantitative interpretation of the results and called for
better converged enhanced sampling simulations.
