Modular organization of inteins and C‐terminal autocatalytic domains

Abstract

Analysis of the conserved sequence features of inteins (protein "introns") reveals that they are composed of three distinct modular domains. The N-terminal (N) and C-terminal (C) domains are predicted to perform different parts of the autocatalytic protein splicing reaction. An optional endonuclease domain (EN) is shown to correspond to different types of homing endonucleases in different inteins. The N domain contains motifs predicted to catalyze the first steps of protein splicing, leading to the cleavage of the intein N terminus from its protein host. Intein N domain motifs are also found in C-terminal autocatalytic domains (CADs) present in hedgehog and other protein families. Specific residues in the N domain of intein and CADs are proposed to form a charge relay system involved in cleaving their N-termini. The intein C domain is apparently unique to inteins and contains motifs that catalyze the final protein splicing steps: ligation of the intein flanks and cleavage of its C terminus to release the free intein and spliced host protein. All intein EN domains known thus far have dodecapeptide (DOD, LAGLI-DADG) type homing endonuclease motifs. This work identifies an EN domain with an HNH homing-endonuclease motif and two new small inteins with no EN domains. One of these small inteins might be inactive or a "pseudo intein." The results suggest a modular architecture for inteins, clarify their origin and relationship to other protein families, and extend recent experimental findings on the functional roles of intein N, C, and EN motifs.

The Deinococcus radiodurans Snf2 intein caught in the act: Detection of the Class 3 intein signature Block F branched intermediate

“…5 Asp is not commonly found at B:7 although it is present in a few other inteins. [1][2][3][4][5] Mutation of Asp61 to Ala inhibited reactions at the N-terminal splice junction reducing the amount of spliced products and increasing the amount of C-terminal cleavage products [ Fig. 4(B) and Table II].…”

“…1). [2][3][4][5] Intein residues are referred to using the Block letter and the Block position separated by a colon.…”

“…1). [1][2][3][4][5] Two variations in the intein-mediated protein splicing mechanism were previously described, which involve direct attack on an amide bond at the N-terminal splice junction to generate a BI. [5][6][7]12,13 In Class 2 inteins, exemplified by the Methanococcus jannaschii (Mja) KlbA intein, 12,13 the Block G BI (III) is formed by nucleophilic attack of Cysþ1 on the peptide bond at the N-terminal splice junction (Fig.…”

“…1) and the mechanistic roles that they play. [1][2][3][4][5][6][7] Putative Class 3 inteins can be identified by class-specific sequence features: (a) the absence of a Cys, Ser, or Thr N-terminal nucleophile at A:1, (b) a noncontiguous WCT triplet at B:12, F:4, and G:5, and (c) Thrþ1 or Serþ1 as the first C-extein residue at G:8 instead of the more common Cysþ1 (Fig. 1).…”

“…Conserved positions are populated by groups of residues that have similar properties except for the Block B His (B:10), which is almost invariant. [1][2][3][4][5] An exemplary sequence is shown for each intein class. The most common residue found at selected positions is underlined and the common variations for these positions are shown.…”