Protein Splicing of a Pyrococcus abyssi Intein with a C-terminal Glutamine

Mills, Kenneth V.; Manning, Jennifer S.; Garcı́a, Agustina; Wuerdeman, Lisa A.

doi:10.1074/jbc.m400887200

Cited by 56 publications

(69 citation statements)

References 34 publications

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“…However, our results showed that the N-intein linked to the N-extein could be as short as 11 aa and that a major portion (M-intein) of the intein could be unlinked from both exteins. In addition, other studies have shown that even the few conserved junction residues can be absent (35)(36)(37)(38). Taken together, these findings could suggest the possible existence of highly derived inteins that show no obvious sequence similarity to known self-splicing inteins.…”

Section: Discussionmentioning

confidence: 75%

Synthetic Two-piece and Three-piece Split Inteins for Protein trans-Splicing

Sun

Yang

Liu

2004

Journal of Biological Chemistry

111

109

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Inteins are protein-intervening sequences that can self-excise and concomitantly splice together the flanking polypeptides. Two-piece split inteins capable of protein trans-splicing have been found in nature and engineered in laboratories, but they all have a similar split site corresponding to the endonuclease domain of the intein. Can inteins be split at other sites and do transsplicing? After testing 13 split sites engineered into a Ssp DnaB mini-intein, we report the finding of three new split sites that each produced a two-piece split intein capable of protein trans-splicing. These three functional split sites are located in different loop regions between ␤-strands of the intein structure, and one of them is just 11 amino acids from the beginning of the intein. Because different inteins have similar structures and similar ␤-strands, these new split sites may be generalized to other inteins. We have also demonstrated for the first time that a three-piece split intein could function in protein trans-splicing. These findings have implications for intein structure-function, evolution, and uses in biotechnology.An intein is a protein-intervening sequence that catalyzes a protein-splicing reaction in which the intein sequence is precisely excised and its flanking sequences (N-and C-exteins) join with a peptide bond to produce the mature host protein (spliced protein) (1). The mechanism of protein splicing typically has four steps: two acyl rearrangements at the two splicing junctions, a trans-esterification between the two junctions, and a cyclization of the Asn residue at the C-terminal junction (2-4). Crystal structures of inteins revealed a splicing domain consisting of 11-12 ␤-strands and forming a compact horseshoe shape with the splicing junctions located in the central cleft (5-11). A majority of inteins also have a homing endonuclease domain inserted in the splicing domain sequence (12). These bifunctional inteins are ϳ350 -550 amino acids (aa) 1 long, although some extra large inteins are up to 1650 aa long and also contain tandem repeats (13,14). Nearly 200 intein and inteinlike sequences have been found in a wide variety of host proteins and in microorganisms belonging to bacteria, Archaea, and eukaryotes (12,15). Their sporadic phylogenetic distributions suggest lateral gene transfer through intein homing (16,17). Inteins generally share only low levels of sequence similarity, but they share striking similarities in structure, reaction mechanism, and evolution (4,18,19,21). It is thought that inteins first originated with just the splicing domain and then acquired the endonuclease domain, with the latter conferring genetic mobility to the intein. During intein evolution, however, some inteins lost their endonuclease domain to become mini-inteins consisting of just the ϳ130-aa protein-splicing domain plus a linker sequence of various lengths in place of the endonuclease domain (12,22).An interesting event of intein evolution is the loss of sequence continuity in some inteins, which apparently produced ...

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Section: Discussionmentioning

confidence: 75%

Synthetic Two-piece and Three-piece Split Inteins for Protein trans-Splicing

Sun

Yang

Liu

2004

Journal of Biological Chemistry

111

109

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“…Asp and Gln can undergo similar cyclization reactions. 6,7,[9][10][11] The intein penultimate His assists Asn cyclization. 6,7 A native peptide bond is formed between the exteins (VI) after a spontaneous [S/O]-N acyl shift.…”

Section: Introductionmentioning

confidence: 99%

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

Brace¹,

Southworth²,

Tori³

et al. 2010

Protein Science

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Inteins are the protein equivalent of introns. They are remarkable and robust single turnover enzymes that splice out of precursor proteins during post-translational maturation of the host protein (extein). The Deinococcus radiodurans Snf2 intein is the second member of the recently discovered Class 3 subfamily of inteins to be characterized. Class 3 inteins have a unique sequence signature: (a) they start with residues other than the standard Class 1 Cys, Ser or Thr, (b) have a noncontiguous, centrally located Trp/Cys/Thr triplet, and (c) all but one have Ser or Thr at the start of the C-extein instead of the more common Cys. We previously proposed that Class 3 inteins splice by a variation in the standard intein-mediated protein splicing mechanism that includes a novel initiating step leading to the formation of a previously unrecognized branched intermediate. In this mechanism defined with the Class 3 prototypic Mycobacteriophage Bethlehem DnaB intein, the triplet Cys attacks the peptide bond at the N-terminal splice junction to form the class specific branched intermediate after which the N-extein is transferred to the side chain of the Ser, Thr, or Cys at the C-terminal splice junction to form the standard intein branched intermediate. Analysis of the Deinococcus radiodurans Snf2 intein confirms this splicing mechanism. Moreover, the Class 3 specific Block F branched intermediate was isolated, providing the first direct proof of its existence.

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“…This led the authors to hypothesize that the newly formed amino group at the intein N-terminus in the branched intermediate cleaves the C-terminal splice junction (17). This is inconsistent with the Pab PolII intein data yielding C-terminal cleavage in the absence of N-terminal cleavage (18). Another possibility is that the Cterminal scissile bond is activated for cleavage by an oxygen nucleophile (Asp) and is thus very susceptible to hydrolysis.…”

Section: Inteins Ending In Gln or Aspmentioning

confidence: 96%

“…Asp and Gln can undergo similar rearrangements to Asn that would result in peptide bond cleavage. C-terminal cleavage in the Gln containing Pyrococcus abyssi (Pab) PolII intein was facilitated by the intein penultimate His (18). A C-terminal aminoglutarimide has not been observed, possibly because of instability or because another mechanism is being utilized (17).…”

Section: Inteins Ending In Gln or Aspmentioning

confidence: 99%

“…A C-terminal aminoglutarimide has not been observed, possibly because of instability or because another mechanism is being utilized (17). Studies of the Chilo iridescent virus (CIV) RNR and the Pab PolII inteins yield differing results when the intein C-terminal Gln was 'reverted' to Asn; the yield of spliced product was reduced 7-fold in the CIV RNR intein (17), while the rate of splicing increased 4-fold with the Pab PolII intein (18). Inteindependent variation was also seen when the penultimate residue of inteins naturally lacking a penultimate His was 'reverted' to His.…”

Section: Inteins Ending In Gln or Aspmentioning

confidence: 99%

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Protein Splicing Mechanisms and Applications

Perler

2005

IUBMB Life (International Union of Biochemistry and Molecular B

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SummaryInteins are protein splicing elements that employ standard enzyme strategies to excise themselves from precursor proteins and ligate the surrounding sequences (exteins). The protein splicing pathway consists of four nucleophilic displacements directed by the intein plus the first C-extein residue. The intein active site(s) are formed by folding of the intein within the precursor, which brings together the splice junctions and internal intein residues that assist catalysis. Inteins with non-canonical catalytic residues splice by modified pathways. Understanding intein proteolytic cleavage and ligation activities has led to the development of many novel applications in the fields of protein engineering, enzymology, microarray production, target detection and activation of transgenes in plants. Recent advances include intein-mediated attachment of proteins to solid supports for microarray or western blot analysis, linking nucleic acids to proteins and controllable splicing, which converts inteins into molecular switches. IUBMB Life, 57: 469 -476, 2005

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Protein Splicing of a Pyrococcus abyssi Intein with a C-terminal Glutamine

Cited by 56 publications

References 34 publications

Synthetic Two-piece and Three-piece Split Inteins for Protein trans-Splicing

Synthetic Two-piece and Three-piece Split Inteins for Protein trans-Splicing

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

Protein Splicing Mechanisms and Applications

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