Mechanism of C-terminal intein cleavage in protein splicing from QM/MM molecular dynamics simulations

摘要: Protein splicing is a post-translational process in which biologically inactive protein activated by the release of segment denoted as an intein. The involves four steps. In third, scission intein takes place after cyclization last amino acid segment, asparagine. Little known about chemical reaction necessary for this cyclization. Experiments demonstrate that two histidines (the penultimate intein, and histidine located 10 acids upstream) are relevant We have investigated mechanism determinants GyrA focusing on requirements asparagine activation its First, influence protonation states these orientation side chain means molecular dynamics simulation. Molecular simulations using CHARMM27 force field were carried out three possible each histidines. results indicate only state conformation system suitable when fully protonated (positively charged), upstream His(e) neutral tautomeric form. free energy profile proton transfer to presented, computed hybrid quantum mechanics/molecular mechanics (QM/MM) umbrella sampling at SCCDFTB/CHARMM27 level theory. calculated barrier 19.0 kcal mol(-1). B3LYP/6-31+G(d) QM/MM single-point calculations give qualitatively similar profile, although with somewhat higher barriers, good agreement value derived from experiment 25 mol(-1) 60 °C. reactant, reactant intermediate highlight importance Arg181-Val182-Asp183 catalysing reaction. Overall, nucleophilic acting base plausible C-terminal cleavage splicing.