Branched intermediate formation stimulates peptide bond cleavage in protein splicing

Abstract

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.

Structure of the branched intermediate in protein splicing

“…This circumvented insurmountable synthetic problems (SI Appendix) associated with the preparation of the branched peptide containing both a backbone ester (at Val182) and a side-chain ester (connecting the exteins). Fortunately, we have previously shown that the rate of branched intermediate resolution is unaffected by the substitution of Dapa for the +1 Thr (15). Thus, we could assess the effect of the backbone amide-to-ester substitution at Val182 using this system.…”

“…Mxe GyrA is the most commonly used intein in protein engineering applications (9). Solution NMR studies on this semisynthetic construct suggest that branched intermediate formation affects the local structure around the +1 scissile amide bond connecting the intein to the C-extein, whereas kinetic studies indicate that inteinsuccinimide formation is greatly stimulated in the context of this intermediate (15). These studies also suggest an approach to trap the branched structure for high-resolution structure studies, based on mutation of a key histidine residue in the intein, His187 within block F (Fig.…”

“…These studies also suggest an approach to trap the branched structure for high-resolution structure studies, based on mutation of a key histidine residue in the intein, His187 within block F (Fig. 1A); the structure of the Mxe GyrA intein splicing precursor (herein referred to as the linear Mxe GyrA intein structure) reveals this residue is well positioned to act as a general base during succinimide formation (7), and consistent with this role, mutation of this residue selectively blocks this step (15). With this in mind, we used protein semisynthesis to prepare branched construct 1 comprising the Mxe GyrA intein, four native N-and C-extein residues linked through the requisite side-chain ester bond and, critically, the isosteric but noncatalytic unnatural amino acid β-thienyl-alanine (ThA) in place of His187 within the intein (Fig.…”

“…The study highlights the power of the combined structural and semisynthesis methods for dissecting protein catalysis. xenopi DNA Gyrase A (Mxe GyrA) intein-splicing reaction (15). Mxe GyrA is the most commonly used intein in protein engineering applications (9).…”

Branching out of the intein active site in protein splicing

“…The new results of Liu et al should be viewed in context of an earlier study, where this semisynthetic branched intermediate (ssBI) was first analyzed with solution NMR (10). Comparison of NMR spectra of the ssBI and the starting precursor indicated repositioning of some catalytic residues in the intein.…”