Modeling protein splicing: reaction pathway for C-terminal splice and intein scission

Protein splicing is a post-translational process where a biologically inactive protein is activated after the release of a so-called intein domain. In spite of the importance of this type of process, the specific molecular mechanism for the catalysis is still uncertain. In this work, we present a computational study of one of the key steps in protein splicing: the release of the intein due to the cyclization of an asparagine, the last amino acid of the intein. Density functional theory (DFT) calculations using the B3LYP functional in conjunction with the polarizable continuum model (PCM) were used to study the main stationary points along various possible reaction pathways. The results are compared with other DFT functionals and the MP2 ab initio method. In the first part of this work, the Asn-Thr dipeptide is analyzed with the aim of determining the specific requirements for the activation of the intrinsically slow Asn cyclization. The results show that the nucleophilic activation of the Asn side chain by removing one of its proton decreases the free energy barrier by approximately 20 kcal/mol. A full pathway of the reaction was also characterized in a larger model, including two imidazole molecules and two water molecules. The proposed reaction mechanism consists of two main steps: Asn side chain activation by a proton transfer to one of the imidazole groups, and cleavage of the peptide bond upon protonation of its nitrogen atom by the other imidazole. The overall free energy barrier in solution was determined to be 29.3 kcal/mol, in reasonable agreement with the apparent experimental barrier in the enzyme. The proposed mechanism suggests that the penultimate histidine stabilizes the tetrahedral intermediate and protonates the nitrogen of the scissile peptide bond, while a second histidine (located 10 amino acids upstream) activates the Asn side chain by deprotonating it.