Design of an Intein that Can Be Inhibited with a Small Molecule Ligand

Abstract

Protein splicing is a process in which an intervening sequence, the intein, catalyzes its own excision out of a larger polypeptide precursor by joining the flanking sequences, the exteins, with a native peptide bond. Inteins are almost completely promiscuous toward the nature of their extein sequences and can be inserted into virtually any host protein. The intein-mediated formation of a peptide bond between two polypeptides offers great potential to modulate protein structure and, hence, protein function on the post-translational level. In this work, we report the design of an intein that can be inhibited by the addition of a specific small molecule ligand. Our design strategy involved the generation of a trans-splicing intein, in which the intein domain is split into two-halves that are located on two separate polypeptides, each joined with the respective N- or C-terminal extein. To turn these fragments into an active intein with an incorporated "off" switch, each was fused at its newly created terminus with the F36M mutant of FKBP12, referred to as the FM domain. The F36M substitution was reported to effect a homodimerization of the usually monomeric FKBP12 protein; however, addition of the small molecule ligand, rapamycin, or synthetic derivatives thereof leads to a dissociation of the dimer. This phenomenon was exploited by first reconstituting the active intein on the basis of FM domain dimerization. Second, addition of the small molecule ligand prevented formation of the active intein complex and inhibited protein trans-splicing. This intein exhibited unexpected kinetic properties and provides a new and potentially very general means to control protein function on the post-translational level.

Interaction Studies and Alanine Scanning Analysis of a Semi-synthetic Split Intein Reveal Thiazoline Ring Formation from an Intermediate of the Protein Splicing Reaction

“…Other more recently emerging exciting avenues for protein engineering approaches rely on protein trans-splicing. This reaction is performed by split inteins, which are found in a few native examples (10,11) or can be created artificially from a regular intein (12)(13)(14)(15)(16)(17)(18)(19). Protein trans-splicing enables the post-translational linkage of proteins or polypeptides originating from two separate molecules.…”

Recent advances in in vivo applications of intein-mediated protein splicing

“…This has found in vivo applications such as the controllable generation of firefly luciferase in cultured cells and in Drosophila melanogaster [58], and PTS of a tobacco etch virus protease in yeast [59]. A mutated form of FKBP12 can be used to induce spontaneous reassociation and PTS of the split intein; in this case, the addition of rapamycin prevents reassocation and inhibits splicing [60]. Recently, Silver and coworkers demonstrated that the FKBP12 and FRB domains could be replaced with complementary coiled coil domains to induce luciferase activity in mammalian cells via specific coiled coil interactions rather than addition of a small molecule, presumably by inducing PTS of the luciferase segments [61].…”

Split Inteins: Nature’s Protein Ligases

“…While such modules have been developed with intact inteins, [38] split inteins provide an optimal platform for CPS, given that control over fragment complementation inherently implies control over protein splicing. Indeed split intein CPS systems have been engineered to be responsive to small molecules, [39, 40] light, [41-43] and proteolysis. [41] In an interesting recent study from Mootz and coworkers, an artificially split SspDnaB intein was outfitted with an uncleavable isopeptide bond bearing a peptidyl side-chain at position 1 of the N-intein and a photo-cleavable 4,5-dimethoxy-2- nitrobenzyl (DMNB) group capping the N-terminus in place of an N-extein.…”