Branching out of the intein active site in protein splicing

Inteins perform a macromolecular vanishing act that continues to impress as its secrets are revealed. Dispersed through all domains of life, these autocatalytic domains exit the folds of ostensibly unrelated proteins soon after translation. Inteins bust at the seams, severing peptide bonds that immediately precede and follow their sequence. Critically, they also sew up the damage; the two polypeptide segments that first flanked the intein, known as exteins, are concurrently joined. Extein ligation renders the transformation traceless while adding a significant exception to the one gene/one protein rule. The act proceeds without assistance from cofactors, accessory proteins, or energy source—just a small catcher’s mitt structure of ∼130 amino acids (Fig. 1 A ). Since the discovery of inteins (1, 2), efforts to understand their protein splicing activity have inspired not only mechanistic insight but biotechnology applications galore. Fig. 1. Intein structure/function and the branched intermediate. ( A ) Intein architecture. Characteristic β structure of a protein splicing intein domain (red), shown with short extein fragments (blue). Image rendered using Pymol with Protein Data Bank code 4OZ6. ( B ) Protein splicing pathway: ( 1 ) acyl shift to form the linear intermediate; ( 2 ) transesterification to generate the branched intermediate; ( 3 ) succinimide formation to separate the intein from linked exteins; and ( 4 ) spontaneous acyl shift to regenerate peptide backbone in the spliced exteins. N, asparagine. ( C ) Resolution of the branched intermediate. Succinimide formation (step 3 in B ) via attack by the intein’s catalytic asparagine residue (red) at the downstream extein junction (blue), presumably accelerated by general base (B:) and general acid (HB) catalysis. ( D ) Amide-imidate equilibrium. Tautomerization and deprotonation converts an asparagine carboxamide to a less stable imidate. Following a proposal put forward by Perler, Paulus, and coworkers more than 20 y ago, the … [↵][1]1To whom correspondence may be addressed. E-mail: mbelfort{at}albany.edu or callahan{at}binghamton.edu. [1]: #xref-corresp-1-1