PMC

Potential intein reactions. Protein splicing results in ligation of the N-extein (E_N) and C-extein (E_C), as directed by the intein (I). When inteins are mutated or inserted in heterologous contexts, off-pathway reactions can occur resulting in N-terminal, C-terminal, or double cleavage products that are unable to splice. Off-pathway N-terminal cleavage can occur in both the linear and the branched (thio)ester intermediates. Off-pathway C-terminal cleavage occurs when cyclization of the intein C-terminal residue precedes branch intermediate formation.

The intein-mediated class 1 protein splicing mechanism. Class 1 inteins with a C-terminal Asn and a Cys, Ser, or Thr at the first position in both the intein and the C-extein splice using the standard four-step protein splicing mechanism depicted in this figure. Inteins with C-terminal Glu, Gln, or Asp use this same mechanism except for Glu, Gln, or Asp cyclization in step 3, although other mechanisms are possible. Succinimide hydrolysis can also produce iso-Asn. X represents an oxygen or a sulfur atom. For clarity, tetrahedral intermediates and residues facilitating each step are omitted. Although the definition of an intein is the excised sequence (), for brevity we will include the C-extein nucleophile when discussing mechanisms.

Variations in splicing mechanisms. Inteins missing the standard N-terminal nucleophile use various strategies to get to the same block G branched intermediate formed after step 2 in class 1 inteins. Class 2 inteins form the block G branched intermediate after direct attack on the amide bond at the N-terminal splice site by Cys⁺¹. Class 3 inteins first form a block F branched intermediate with Cys^F:4 as the branch point and then transfer the N-extein to the +1 residue to form the block G branched intermediate. Once the block G branched intermediate is formed, class 2 and class 3 inteins follow the same steps (3 and 4) to complete splicing as in class 1 inteins. Abbreviations used are: E_N, N-extein; E_C, C-extein; I, intein; BI, branched intermediate; X, an oxygen or a sulfur atom.

Precursor domains and conserved motifs. A, a precursor with an intein containing a homing endonuclease domain (gold) is depicted with intein splicing domain motifs (red) listed above and conserved residues that participate in catalysis listed below. Residues in intein motifs are numbered based on their position within each motif (green) as defined in InBase (). Residues specific to class 2 or 3 inteins are in lowercase, and only a subset of residues found at A:1 is depicted. Motifs A, B, F, and G have also been called N1, N2, C2, and C1, respectively (). Motifs C, D, E, and H are specific to certain homing endonucleases and are not shown. To simplify discussion of inteins in various precursors, residues in each part are numbered independently. Intein residues are numbered from the N to C terminus beginning with 1. Residues in the N- and C-exteins (blue) are numbered from the splice site outwards and include a minus sign for N-extein and a plus sign for C-extein residues. B, folding of the precursor forms the intein active site and initiates protein splicing. Homing endonuclease domains in larger inteins fold separately from the intein and extein domains. Association of extein fragments can influence precursor folding and active site architecture. X represents an oxygen or a sulfur atom.