Unveiling the Catalytic Role of B-Block Histidine in the N–S Acyl Shift Step of Protein Splicing

Abstract

Protein splicing is a post-translational modification that involves the excision of a segment denoted as "intein" and the joining of its two flanking segments. The process is autocatalytic, making inteins appealing for many applications in biotechnology, bioengineering, or medicine. The canonical mechanism of protein splicing is composed of four sequential steps, and is initialized by an N-S or N-O acyl shift to form a linear ester. It is well-established that a histidine, the most conserved amino acid in all inteins, catalyzes this initial step, even though its role remains to be understood. In this study, we combine molecular dynamics simulations and quantum mechanics/molecular mechanics (QM/MM) hybrid calculations to investigate the alternative reaction pathways proposed for the N-S acyl shift in Mycobacterium tuberculosis RecA intein. The results rule out the histidine acting as a base and activating the side chain of Cys1; instead, an aspartate performs this action. In the reaction mechanism proposed herein, denoted as the "Asp422 activated" mechanism, two sequential roles are attributed to the histidine: (i) ground-state destabilization by straining the scissile peptide bond and (ii) protonation of the leaving amide group. In summary, the study provides relevant data to understand the catalytic role of this histidine, and proposes a reaction pathway for the N-S acyl shift reaction in protein splicing that fits with the available experimental data.