title: Kissing loop interaction in adenine riboswitch: insights from umbrella sampling simulations.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  Contacts between the two loops were progressively lost when larger inter-loop distances were enforced. Inter-loop Watson-Crick contacts survived at larger separation when compared with non-canonical pairing and stacking interactions. Intra-loop stacking contacts remained formed upon loop undocking. Our simulations qualitatively indicated that the ligand could stabilize the kissing loop complex. We also compared with previously published simulation studies.
  
  
  CONCLUSIONS
  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.
authors.forename: Francesco
authors.initials: F
authors.lastname: Di Palma
authors.forename: Sandro
authors.initials: S
authors.lastname: Bottaro
authors.forename: Giovanni
authors.initials: G
authors.lastname: Bussi
mesh_terms: D000225:Adenine / Q000737:chemistry*; D006801:Humans; D056004:Molecular Dynamics Simulation; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry*; D058928:Riboswitch / Q000235:genetics*; D013816:Thermodynamics
chemical_list: D058928:Riboswitch; D012313:RNA; D000225:Adenine