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

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