Study of the Micro-RNA Precursor Processing Mechanism by QM/MM Computational Simulations

PUZZOLO, JUAN L.; DRUSIN, SALVADOR I.; MORENO, DIEGO M.; RASIA, RODOLFO M.

Buenos Aires

Congreso; XLVI Reunión Anual de la Sociedad Argentina de Biofísica; 2017

Sociedad Argentina de Biofísica

p { margin-bottom: 0.25cm; direction: ltr; color: rgb(0, 0, 0); line-height: 120%; }p.western { font-family: "Times New Roman", serif; font-size: 10pt; }p.cjk { font-family: "Times New Roman", serif; font-size: 10pt; }p.ctl { font-family: "Times New Roman", serif; font-size: 10pt; }a:link { color: rgb(0, 0, 0); text-decoration: none; }Rnase III enzymes process doublestranded RNA in bacteria. It is composed by two domains: dsRBD, whichrecognizes and binds the target dsRNA, and RIIID, which bear theactive site. The processing by RIIID is performed through ahydrolysis reaction, in which there is a nucleophilic attack on thephosphate group of the RNA backbone by an activated H2Omolecule or a OH-ion previously coordinated to a Mg2+ion of the active site. The proposed mechanism for this reaction isbased on structural studies that are not able to capture thethermodynamics of the process, nor the protonation states of theresidues and solvent molecules involved in the mechanism withatomistic detail. In this regard, computational simulation techniquescan provide useful and detailed information about the reactionmechanism.To study the hydrolysis mechanismthrough hybrid quantum mechanics/molecular mechanics (QM/MM) methods,we performed 50 ns of classical molecular dynamics (MD) simulation onthe initial system. Next, we carried out 30 QM/MM steered moleculardynamics (SMD) simulations from different structures taken from theprevious MD, in which we forced the nucleophile to attack thephosphate group. The irreversible work obtained in each SMD was usedto calculate the free energy profile of the reaction, through theJarzynksi equality.Our results show that the nucleophileis an OH-ion and not a H2Omolecule, as the former´s energy barrier is considerably lower.Additionally, the network of hydrogen bridges around the active siteis important for its regeneration after the reaction. On the otherhand, by mutating key residues on the active site which bind the Mg2+ion, we observed that its role is not only activating thenucleophile, but arranging and stabilizing the active site byneutralizing the negative charge of the phosphate, nucleophile, andprotein residues.