Design of an intein that can be inhibited with a small molecule ligand.
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.
Brenzel
Steffen
S
Philipps-Universität Marburg, Fachbereich Chemie/Biochemie, Hans-Meerwein-Strasse, 35032 Marburg, Germany.
Mootz
Henning D
HD
D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D047668:Inteins / Q000187:drug effects* / Q000235:genetics; D008024:Ligands; D058619:Maltose-Binding Proteins; D019154:Protein Splicing / Q000187:drug effects* / Q000235:genetics; D017434:Protein Structure, Tertiary; D020123:Sirolimus / Q000494:pharmacology*; D022061:Tacrolimus Binding Protein 1A / Q000235:genetics / Q000378:metabolism
D002352:Carrier Proteins; D008024:Ligands; D058619:Maltose-Binding Proteins; D022061:Tacrolimus Binding Protein 1A; D020123:Sirolimus