Molecular Insights into the Substrate-Assisted Mechanism of Viral DNA 3′-End Processing in Intasome of Prototype Foamy Virus Integrase from Molecular Dynamic and QM/MM Studies

Abstract

Integrases participate in two important steps for virus replication such as 3′-end processing of viral DNA (vDNA) and nuclear entry of host DNA (hDNA). In this work, insight into the structural changes in intasome of prototype foamy virus integrase (PFV-IN) complexed with vDNA from classical molecular dynamic (MD) simulations are done. Analysis of the results reveal the existence of alternative conformations of the enzyme active site indicating that the 3′-end processing reaction can occur according to three different pathways and taking place with the possible participation of aspartate 185 of a neighboring phosphate group or involving an internal phosphate group of the substrate. In this work, one of them, the so-called substrate-assisted mechanism was explored by QM/MM methods. The free energy barriers of 34.4 kcal mol–1 for the first and 35.3 kcal mol–1 for the second step of reaction computed with free energy perturbation (FEP) methods at the M06-2X/AMBER level show that 3′-end processing has to proceed via a different mechanism than studied herein. Nevertheless, the obtained results are in good agreement with the experimental observations that the substitution of the key atom for this mechanism, oxygen by sulfur, did not influence the catalysis. Additionally, the obtained mechanism reveals significant similarities to the previously studied substrate-assisted mechanism in twister ribozyme. The possible role of Mg2+ in the active site is discussed.