Protein Splicing of Inteins and Hedgehog Autoproteolysis: Structure, Function, and Evolution

The Mechanism of Protein Splicing Splicing is extremely rapid and to date, precursors have not been identified in native systems. The mechanism of protein splicing (Figure 2) has been extensively reviewed begins when the side chain hydroxyl or thiol of the con-Protein splicing is a posttranslational editing process served intein N-terminal Ser1 or Cys1 attacks the car-that removes an internal protein fragment (intein) from bonyl (C ϭ O) of the preceding amino acid, resulting in a precursor and ligates the external protein fragments an ester or thioester bond at the N-terminal splice site (exteins) to form the mature extein protein. Inteins, once (step 1, acyl rearrangement). This carbonyl is then at-considered an oddity, are now known to be widely dis-tacked by the hydroxyl/thiol group of the Ser/Thr/Cys tributed. The excised intein is also a stable protein that at the beginning of the C extein (the ϩ1 residue) resulting can have homing endonuclease activity (reviewed in Bel-in N-terminal cleavage and formation of a branched in-fort and Roberts, 1997). Homing endonucleases are site-termediate (step 2, transesterification). The branched specific enzymes that make double-strand breaks in intermediate is resolved by cleavage of the peptide bond intronless or inteinless alleles, initiating a gene conver-at the C-terminal splice site due to cyclization of the sion process that results in insertion of the mobile intron intein C-terminal Asn (step 3, Asn cyclization). Finally, or intein gene. They are grouped on the basis of signa-a spontaneous ON or S-N acyl rearrangement estab-ture motifs such as the LAGLIDADG (dodecapeptide) or lishes a peptide bond between the exteins (step 4, acyl HNH motifs. The intein plus the first downstream extein rearrangement). residue mediates both splicing and DNA cleavage. When Although protein splicing was once thought to follow inteins were first described, we thought it remarkable a unique pathway, the initial acyl rearrangement has that one protein could direct two proteolytic cleavages, now been linked to activation of autocatalytic reactions protein ligation and DNA cleavage. We now know that in diverse biological processes including autoprote-protein cleavage and ligation are mediated by a protein olysis, protein targeting, and addition of prosthetic splicing element that is ‫051ف‬ amino acids (aa) and that groups (reviewed in Beachy et al., 1997, and Perler et the core endonuclease is a separate structural domain. al., 1997b). Each step requires assistance from a proton The mystery of how a single protein can accomplish donor and acceptor to …