Protein splicing elements: inteins and exteins — a definition of terms and recommended nomenclature

Perler, Francine B.; Davis, E. A.; Dean, Gary E.; Gimble, Frederick S.; Jack, William E.; Neff, Norma; Noren, Christopher J.; Thorner, Jeremy; Belfort, Marlene

doi:10.1093/nar/22.7.1125

Cited by 339 publications

(267 citation statements)

References 21 publications

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“…Protein splicing is a posttranslational processing event that involves the precise removal of an intervening sequence, an intein, from a protein precursor with concomitant ligation of the flanking protein sequences (N and C exteins) via a native peptide bond (1)(2)(3). In vitro splicing of inteins in heterologous proteins suggests that inteins with the first C-extein residue contain sufficient information needed for the autocatalytic splicing process without the requirement of exogenous energy or protein co-factor (4,5).…”

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confidence: 99%

Crystal Structure of a Mini-intein Reveals a Conserved Catalytic Module Involved in Side Chain Cyclization of Asparagine during Protein Splicing

Ding

Ghosh

et al. 2003

Journal of Biological Chemistry

135

186

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We have determined the crystal structure of a 154-residue intein derived from the dnaB gene of Synechocystis sp. strain PCC6803 and refined it to a 2.0-Å resolution. The x-ray structure suggests that this intein possesses two catalytic sites that appear to be separately responsible for splicing and cleavage of the Nand C-terminal scissile bonds. The conserved intein block F residues are the important components of a catalytic site for side chain cyclization of the last intein residue, Asn-154. The data suggest that the imidazole ring of His-143 is involved in the activation of the side chain N␦ atom of Asn-154, leading to a nucleophilic attack on the carbonyl carbon of Asn-154. Substitution of His-143 with Ala or Gln resulted in the inhibition of C-terminal cleavage. His-153, Asp-136, and a water molecule appear to constitute an oxyanion binding site by contacting the carbonyl oxygen of Asn-154 to stabilize the transition state. The structure and mutagenesis data also support that the close contact between the hydroxyl groups of Thr-138 and Ser-155, whose side chain participates in an S 3 O acyl shift, plays an important role in the nucleophile orientation. Our structural modeling suggests that this catalytic module is conserved in the C-terminal subdomains of inteins from diverse organisms.Protein splicing is a posttranslational processing event that involves the precise removal of an intervening sequence, an intein, from a protein precursor with concomitant ligation of the flanking protein sequences (N and C exteins) via a native peptide bond (1-3). In vitro splicing of inteins in heterologous proteins suggests that inteins with the first C-extein residue contain sufficient information needed for the autocatalytic splicing process without the requirement of exogenous energy or protein co-factor (4, 5).Among the more than 130 inteins identified so far, the majority contain two discrete functional domains, the homing endonuclease domain and the splicing domain (Fig. 1A) (InBase, the New England Biolabs Intein Data Base (6)). The protein splicing function of the intein does not depend on their homing endonuclease activity since splicing-proficient minimal inteins (mini-inteins) occur naturally (7-9) and have been generated by deleting the centrally located endonuclease domain. The splicing domain of a dual function intein appears to be split by the endonuclease domain into two segments, the N-and C-terminal subdomains. The N-terminal subdomain (in the range of 100 -150 residues) contains the conserved intein blocks A, B, N2, and N4, whereas the C-terminal subdomain of ϳ35-50 residues contains blocks F and G (6, 10). The N-terminal subdomain of inteins not only exhibits sequence homology with the hedgehog proteins of eucaryotes but also shares an acyl rearrangement mechanism of breaking the peptide bond preceding a nucleophile-containing residue (11). This raises the question of whether inteins share a common ancestor with other autoprocessing proteins and have evolved by acquiring a new catalytic capacity imbedde...

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mentioning

confidence: 99%

Crystal Structure of a Mini-intein Reveals a Conserved Catalytic Module Involved in Side Chain Cyclization of Asparagine during Protein Splicing

Ding

Ghosh

et al. 2003

Journal of Biological Chemistry

135

186

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“…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)(3)(4).…”

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confidence: 99%

“…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).…”

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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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“…An intein is a protein sequence embedded inframe within a precursor protein sequence that excises itself out, ligating the flanking sequences together through a peptide bond (25)(26)(27). This process is known as intein-mediated protein splicing, which is an autocatalytic event (25-27).…”

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confidence: 99%

In situ growth of a PEG-like polymer from the C terminus of an intein fusion protein improves pharmacokinetics and tumor accumulation

Gao

Liu

Christensen³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

145

131

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This paper reports a general in situ method to grow a polymer conjugate solely from the C terminus of a recombinant protein. GFP was fused at its C terminus with an intein; cleavage of the intein provided a unique thioester moiety at the C terminus of GFP that was used to install an atom transfer radical polymerization (ATRP) initiator. Subsequent in situ ATRP of oligo(ethylene glycol) methyl ether methacrylate (OEGMA) yielded a site-specific (C-terminal) and stoichiometric conjugate with high yield and good retention of protein activity. A GFP-C-poly(OEGMA) conjugate (hydrodynamic radius (R h ): 21 nm) showed a 15-fold increase in its blood exposure compared to the protein (R h : 3.0 nm) after intravenous administration to mice. This conjugate also showed a 50-fold increase in tumor accumulation, 24 h after intravenous administration to tumor-bearing mice, compared to the unmodified protein. This approach for in situ C-terminal polymer modification of a recombinant protein is applicable to a large subset of recombinant protein and peptide drugs and provides a general methodology for improvement of their pharmacological profiles.drug delivery | polymer bioconjugate | protein engineering P olymer modification of therapeutically relevant proteins is important because it can improve their stability, increase their solubility, enhance their systemic circulation, and also potentially reduce their immunogenicity and antigenicity (1-4). Traditionally, synthetic polymers have been conjugated to proteins by reaction of the polymer with the reactive side chains of protein residues such as lysine or cysteine (5-10). Attachment of PEG to proteins, termed PEGylation, is the most widely used polymer conjugation methodology to improve the pharmacological profiles of proteins (11)(12)(13)(14). Recently, in situ polymerization directly from the surface of a protein, in which a polymerization initiator is conjugated to the protein, followed by in situ growth of the polymer has emerged as a promising alternative to postpolymerization conjugation to synthesize protein-polymer conjugates with high yield (15)(16)(17)(18)(19)(20). However the chemically reactive residues in most proteins are typically promiscuously distributed on their surface, which can result in the growth of polymers from many sites from the protein, resulting in heterogeneous protein-polymer conjugates with poorly controlled stoichiometry and decreased biological activity compared to the protein, thereby limiting the utility of these methods. Hence, the major challenge in protein-polymer conjugation is to create: (i) site-specific, (ii) stoichiometric protein-polymer conjugates, with (iii) high yield, (iv) good retention of protein activity, and (v) significantly improved pharmacological properties over the native protein.Recently, we demonstrated a general strategy for the in situ growth of a stoichiometric, PEG-like polymer from the N terminus of a protein by a N-terminal transamination reaction followed by a chemoselective aldehyde-hydroxylamine reaction to ins...

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Protein splicing elements: inteins and exteins — a definition of terms and recommended nomenclature

Cited by 339 publications

References 21 publications

Crystal Structure of a Mini-intein Reveals a Conserved Catalytic Module Involved in Side Chain Cyclization of Asparagine during Protein Splicing

Crystal Structure of a Mini-intein Reveals a Conserved Catalytic Module Involved in Side Chain Cyclization of Asparagine during Protein Splicing

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

In situ growth of a PEG-like polymer from the C terminus of an intein fusion protein improves pharmacokinetics and tumor accumulation

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