Branched intermediate formation stimulates peptide bond cleavage in protein splicing

Protein splicing is a posttranslational modification in which an intein domain excises itself out of a host protein. Here, we investigate how the steps in the splicing process are coordinated so as to maximize the production of the final splice products and minimize the generation of undesired cleavage products. Our approach has been to prepare a branched intermediate (and analogs thereof) of the Mxe GyrA intein using protein semi-synthesis. Kinetic analysis of these molecules indicates that the high fidelity of this protein splicing reaction results from the penultimate step in the process (intein-succinimide formation) being rate-limiting. NMR experiments indicate that formation of the branched intermediate affects the local structure around the amide bond cleaved during succinimide formation. We propose that this structural change reflects a re-organization of the catalytic apparatus to accelerate succinimide formation at the C-terminal splice junction.