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Splicing of the mycobacteriophage Bethlehem DnaB intein: identification of a new mechanistic class of inteins that contain an obligate block F nucleophile.

Tori K, Dassa B, Johnson MA, Southworth MW, Brace LE, Ishino Y, Pietrokovski S, Perler FB - J. Biol. Chem. (2009)

Bottom Line: Several recently identified inteins cannot perform this acyl rearrangement because they do not begin with Cys, Thr, or Ser.These Class 3 inteins are characterized by a non-nucleophilic N-terminal residue that co-varies with a non-contiguous Trp, Cys, Thr triplet (WCT) and a Thr or Ser as the first C-extein residue.Based on biochemical data and confirmed by molecular modeling, we propose roles for these newly identified conserved residues, a novel protein splicing mechanism that includes a second branched intermediate, and an intein classification with three mechanistic categories.

View Article: PubMed Central - PubMed

Affiliation: New England Biolabs, Ipswich, Massachusetts 01938, USA.

ABSTRACT
Inteins are single turnover enzymes that splice out of protein precursors during maturation of the host protein (extein). The Cys or Ser at the N terminus of most inteins initiates a four-step protein splicing reaction by forming a (thio)ester bond at the N-terminal splice junction. Several recently identified inteins cannot perform this acyl rearrangement because they do not begin with Cys, Thr, or Ser. This study analyzes one of these, the mycobacteriophage Bethlehem DnaB intein, which we describe here as the prototype for a new class of inteins based on sequence comparisons, reactivity, and mechanism. These Class 3 inteins are characterized by a non-nucleophilic N-terminal residue that co-varies with a non-contiguous Trp, Cys, Thr triplet (WCT) and a Thr or Ser as the first C-extein residue. Several mechanistic differences were observed when compared with standard inteins or previously studied atypical KlbA Ala(1) inteins: (a) cleavage at the N-terminal splice junction in the absence of all standard N- and C-terminal splice junction nucleophiles, (b) activation of the N-terminal splice junction by a variant Block B motif that includes the WCT triplet Trp, (c) decay of the branched intermediate by thiols or Cys despite an ester linkage at the C-extein branch point, and (d) an absolute requirement for the WCT triplet Block F Cys. Based on biochemical data and confirmed by molecular modeling, we propose roles for these newly identified conserved residues, a novel protein splicing mechanism that includes a second branched intermediate, and an intein classification with three mechanistic categories.

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Stereo view of the arrangement of residues near the splice junctions in the theoretical model of the MP-Be DnaB intein. The heavy atoms of the backbone and side chains of the residues are shown in stick representations, with carbon atoms colored gray, nitrogen atoms in blue, and oxygen atoms in red. Distances between atoms of neighboring side chains are indicated by green dashed lines and labeled with the distance in Å. Cys320 and Trp67 are WCT triplet residues.
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Figure 7: Stereo view of the arrangement of residues near the splice junctions in the theoretical model of the MP-Be DnaB intein. The heavy atoms of the backbone and side chains of the residues are shown in stick representations, with carbon atoms colored gray, nitrogen atoms in blue, and oxygen atoms in red. Distances between atoms of neighboring side chains are indicated by green dashed lines and labeled with the distance in Å. Cys320 and Trp67 are WCT triplet residues.

Mentions: Experimental data suggested novel reactivities in the MP-Be DnaB intein, which led to the new protein splicing mechanism proposed in under “Discussion.” A structural model of this intein was generated to determine whether the structure would support these conclusions. All intein splicing domains have a common fold termed the HINT (Hedgehog/intein) domain, which they share with Hedgehog autoprocessing domains (26). The HINT domain excludes the endonuclease domain and other domains inserted within the intein splicing domain. We performed comparative modeling of the MP-Be DnaB intein HINT domain based on the structure of a precursor of the Sce VMA intein ((20) Protein Data Bank (PDB) code 1JVA), which was hypothesized to most closely resemble an intein reactive conformation (12) despite the presence of mutations in 4 active site residues (20). Sequence identity between the MP-Be DnaB HINT domain and the HINT domains of other structurally characterized inteins ranges from 10 to 17%, which presents a significant challenge for homology modeling. A sequence alignment between the HINT domains of the MP-Be DnaB and Sce VMA intein was constructed using the program FFAS (Fold & Function Assignment System), which is a sequence profile alignment method that takes into account weak sequence similarity over the entire protein that is difficult to detect by other methods (21). The alignment contains 2 N-extein residues and 4 C-extein residues (Fig. 6). It has a sequence identity of 17% and is reasonable based on the conserved positions of the catalytic residues. The resulting model has a root mean square deviation with respect to the template Sce VMA intein of 1.7 Å for the backbone N, Cα, and C′ atoms of the 117 aligned residues. A PDB file of the modeled structure is included as supplemental File 1 along with Ramachandran data (supplemental Fig. 7A). A superposition of the model and template is shown in supplemental Fig. 7B, and a view of the MP-Be intein active site is shown in Fig. 7. The model shows that Ser62 (Block B, position 7) and His65 (Block B, position 10) are located near the N-terminal scissile bond between Gln−1 and Pro1. No unusual activating interactions of the N-terminal scissile bond are observed that could explain the lability of the N-terminal splice junction in the absence of Thr+1. The model reveals that the WCT triplet Cys320 residue (Block F, position 4) is located close to both the N-terminal and C-terminal splice junctions. The Cys320 side chain sulfur atom is located 4.9 Å from the Gln−1 carbonyl carbon and 2.6 Å from the Thr+1 side chain oxygen. This is slightly closer to the N-terminal scissile bond than the Thr+1 side chain oxygen atom, which is 5.2 Å from the Gln−1 carbonyl carbon. Thus, the model indicates that Cys320 is in position to (a) cleave the N-terminal splice junction equally well as Thr+1, (b) cleave the N-terminal splice junction in the absence of Thr+1, (c) transfer the N-extein from the side chain of Cys320 to the side chain of Thr+1, and (d) activate Thr+1 for attack on the N-terminal scissile bond. Any of these potential roles would explain the essential nature of Cys320.


Splicing of the mycobacteriophage Bethlehem DnaB intein: identification of a new mechanistic class of inteins that contain an obligate block F nucleophile.

Tori K, Dassa B, Johnson MA, Southworth MW, Brace LE, Ishino Y, Pietrokovski S, Perler FB - J. Biol. Chem. (2009)

Stereo view of the arrangement of residues near the splice junctions in the theoretical model of the MP-Be DnaB intein. The heavy atoms of the backbone and side chains of the residues are shown in stick representations, with carbon atoms colored gray, nitrogen atoms in blue, and oxygen atoms in red. Distances between atoms of neighboring side chains are indicated by green dashed lines and labeled with the distance in Å. Cys320 and Trp67 are WCT triplet residues.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2807308&req=5

Figure 7: Stereo view of the arrangement of residues near the splice junctions in the theoretical model of the MP-Be DnaB intein. The heavy atoms of the backbone and side chains of the residues are shown in stick representations, with carbon atoms colored gray, nitrogen atoms in blue, and oxygen atoms in red. Distances between atoms of neighboring side chains are indicated by green dashed lines and labeled with the distance in Å. Cys320 and Trp67 are WCT triplet residues.
Mentions: Experimental data suggested novel reactivities in the MP-Be DnaB intein, which led to the new protein splicing mechanism proposed in under “Discussion.” A structural model of this intein was generated to determine whether the structure would support these conclusions. All intein splicing domains have a common fold termed the HINT (Hedgehog/intein) domain, which they share with Hedgehog autoprocessing domains (26). The HINT domain excludes the endonuclease domain and other domains inserted within the intein splicing domain. We performed comparative modeling of the MP-Be DnaB intein HINT domain based on the structure of a precursor of the Sce VMA intein ((20) Protein Data Bank (PDB) code 1JVA), which was hypothesized to most closely resemble an intein reactive conformation (12) despite the presence of mutations in 4 active site residues (20). Sequence identity between the MP-Be DnaB HINT domain and the HINT domains of other structurally characterized inteins ranges from 10 to 17%, which presents a significant challenge for homology modeling. A sequence alignment between the HINT domains of the MP-Be DnaB and Sce VMA intein was constructed using the program FFAS (Fold & Function Assignment System), which is a sequence profile alignment method that takes into account weak sequence similarity over the entire protein that is difficult to detect by other methods (21). The alignment contains 2 N-extein residues and 4 C-extein residues (Fig. 6). It has a sequence identity of 17% and is reasonable based on the conserved positions of the catalytic residues. The resulting model has a root mean square deviation with respect to the template Sce VMA intein of 1.7 Å for the backbone N, Cα, and C′ atoms of the 117 aligned residues. A PDB file of the modeled structure is included as supplemental File 1 along with Ramachandran data (supplemental Fig. 7A). A superposition of the model and template is shown in supplemental Fig. 7B, and a view of the MP-Be intein active site is shown in Fig. 7. The model shows that Ser62 (Block B, position 7) and His65 (Block B, position 10) are located near the N-terminal scissile bond between Gln−1 and Pro1. No unusual activating interactions of the N-terminal scissile bond are observed that could explain the lability of the N-terminal splice junction in the absence of Thr+1. The model reveals that the WCT triplet Cys320 residue (Block F, position 4) is located close to both the N-terminal and C-terminal splice junctions. The Cys320 side chain sulfur atom is located 4.9 Å from the Gln−1 carbonyl carbon and 2.6 Å from the Thr+1 side chain oxygen. This is slightly closer to the N-terminal scissile bond than the Thr+1 side chain oxygen atom, which is 5.2 Å from the Gln−1 carbonyl carbon. Thus, the model indicates that Cys320 is in position to (a) cleave the N-terminal splice junction equally well as Thr+1, (b) cleave the N-terminal splice junction in the absence of Thr+1, (c) transfer the N-extein from the side chain of Cys320 to the side chain of Thr+1, and (d) activate Thr+1 for attack on the N-terminal scissile bond. Any of these potential roles would explain the essential nature of Cys320.

Bottom Line: Several recently identified inteins cannot perform this acyl rearrangement because they do not begin with Cys, Thr, or Ser.These Class 3 inteins are characterized by a non-nucleophilic N-terminal residue that co-varies with a non-contiguous Trp, Cys, Thr triplet (WCT) and a Thr or Ser as the first C-extein residue.Based on biochemical data and confirmed by molecular modeling, we propose roles for these newly identified conserved residues, a novel protein splicing mechanism that includes a second branched intermediate, and an intein classification with three mechanistic categories.

View Article: PubMed Central - PubMed

Affiliation: New England Biolabs, Ipswich, Massachusetts 01938, USA.

ABSTRACT
Inteins are single turnover enzymes that splice out of protein precursors during maturation of the host protein (extein). The Cys or Ser at the N terminus of most inteins initiates a four-step protein splicing reaction by forming a (thio)ester bond at the N-terminal splice junction. Several recently identified inteins cannot perform this acyl rearrangement because they do not begin with Cys, Thr, or Ser. This study analyzes one of these, the mycobacteriophage Bethlehem DnaB intein, which we describe here as the prototype for a new class of inteins based on sequence comparisons, reactivity, and mechanism. These Class 3 inteins are characterized by a non-nucleophilic N-terminal residue that co-varies with a non-contiguous Trp, Cys, Thr triplet (WCT) and a Thr or Ser as the first C-extein residue. Several mechanistic differences were observed when compared with standard inteins or previously studied atypical KlbA Ala(1) inteins: (a) cleavage at the N-terminal splice junction in the absence of all standard N- and C-terminal splice junction nucleophiles, (b) activation of the N-terminal splice junction by a variant Block B motif that includes the WCT triplet Trp, (c) decay of the branched intermediate by thiols or Cys despite an ester linkage at the C-extein branch point, and (d) an absolute requirement for the WCT triplet Block F Cys. Based on biochemical data and confirmed by molecular modeling, we propose roles for these newly identified conserved residues, a novel protein splicing mechanism that includes a second branched intermediate, and an intein classification with three mechanistic categories.

Show MeSH
Related in: MedlinePlus