Canonical Protein Splicing of a Class 1 Intein That Has a Class 3 Noncanonical Sequence Motif

Abstract

A Thermobifida fusca intein has two characteristics of class 3 inteins: a noncontiguous covariant Trp-Cys-Thr triplet and a Ser flanking its C terminus. However, it has Cys at position one, characteristic of class 1 inteins. Splicing does not require the internal Cys, which may instead coordinate the active site. Therefore, the intein is class 1.

Protein splicing is a posttranslational process by which an intervening polypeptide, or intein, is responsible for its own excision from the flanking polypeptides, or exteins, concomitant with extein ligation (9, 10).

The canonical mechanism of protein splicing has four steps (Fig. 1) (9, 10). First, the peptide bond linking the N-terminal extein (N extein) to the intein is converted to a thioester or ester by nucleophilic attack by the side chain of the first intein residue, Cys or Ser. Second, the first residue of the C-terminal extein (C extein) serves as a nucleophile to attack the nascent ester, resulting in transfer of the N extein from the side chain of the first intein residue to the side chain of the first C-extein residue. Third, the conserved C-terminal Asn of the intein cyclizes, and this is coupled to cleavage of the peptide bond linking the intein to the exteins. Finally, the ester linking the exteins converts to an amide and the intein C-terminal aminosuccinimide may be hydrolyzed.

FIG. 1.

Possible mechanisms to reach the branched-ester intermediate. The figure shows four mechanisms that reach the branched-ester intermediate: the class 1 (black arrows), class 2 (red arrow), and class 3 (green arrows) mechanisms and a mixed class 1/3 mechanism (purple arrows).

Two classes of inteins that lack an N-terminal nucleophile and can promote splicing without the canonical first step have been described previously (Fig. 1) (2, 14). Class 2 inteins can bypass the first step of splicing; splicing is initiated by attack of the downstream nucleophile on the N-terminal splice junction (13, 14). Class 3 inteins lack an N-terminal nucleophile and have a covariant Trp-Cys-Thr conserved triplet of noncontiguous residues. For the Mycobacterium phage Bethlehem DnaB and Deinococcus radiodurans Snf2 inteins, the Cys of the covariant triplet is responsible for initial attack on the N-terminal splice junction peptide bond, creating an internal branched thioester (2, 14). The N extein is then transferred from the side chain of the internal Cys to the side chain of the Ser flanking the intein C terminus. As most class 3 inteins are flanked by Ser or Thr, this mechanism has a compelling chemical logic: the more nucleophilic internal Cys attacks the amide, and the attack of the less nucleophilic downstream Ser is directed to the more electrophilic thioester, creating a more stable oxygen ester and driving the equilibrium of step two toward splicing.

The intein that interrupts a hypothetical gene (2914) of Thermobifida fusca (Tfu2914 intein) has sequence characteristics of both class 1 and class 3 inteins. The interrupted gene may encode a peptidoglycan recognition protein (7, 8) with sequence similarity to an N-acetylmuramyl-l-alanine amidase family 2 protein from Nocardiopsis dassonvillei (GenBank accession number CP002041) (1).

The Tfu2914 intein has an N-terminal Cys and therefore could promote the first step of splicing. However, it also has the covariant conserved triplet of class 3 inteins: Trp72 of intein block B, Cys320 of block F, and Thr339 of block G (Fig. 2). We wished to understand if either the internal or N-terminal Cys is required for splicing. That is, are the conserved class 3 elements of the Tfu2914 intein sufficient to allow it to promote splicing via a noncanonical mechanism?

FIG. 2.

Schematic of the intein fusion protein. The boxes indicate the extein and intein segments, to scale. The conserved intein blocks (A, B, F, and G) are indicated above the boxes, and the residue numbering scheme appears below them (12).

To study this intein, we amplified the gene for the entire fusion protein from genomic DNA of T. fusca strain YX (ATCC, Manassas, VA). The gene was amplified by PCR using primers TFUSPCRU (5′-TCCGCTTGGAGATCTCATGCCCAAACCC) and TFUSPCRL (5′-GCAGGGCGGCAAGCTTGCGCAGCCAGAC). The PCR product was digested with BglII and HindIII and inserted between the same sites in pET-29b(+) to generate plasmid pSNICH. The DNA sequence was confirmed by Macrogen, Inc., and is consistent with that from the NCBI database (accession number YP_290970) except for two mutations in the C extein that result in Met+63Thr and His+179Arg (6, 11). Mutants generated for this study were made using appropriate oligonucleotide pairs via site-directed mutagenesis using PfuTurbo DNA polymerase.

Plasmid pSNICH encodes a fusion protein of an N-terminal S peptide (designated S here), followed by the fusion gene (designated NIC, for N extein [N], intein [I], and C extein [C]) and a C-terminal His tag (designated H). The full-length fusion protein is designated SNICH (predicted molecular mass, 77,086 Da). Splicing would result in the fused exteins (SNCH, 39,081 Da) and excised intein (I, 38,005 Da). Cleavage of the ester from step one or two would result in N-terminal cleavage, yielding SN (10,081 Da) and ICH (67,005 Da). Asn cyclization uncoupled from splicing would result in C-terminal cleavage, yielding SNI (48,086 Da) and CH (29,000 Da).

We overexpressed SNICH in Escherichia coli BL21(DE3) by inducing mid-log-phase cultures with IPTG (isopropyl-β-d-thiogalactopyranoside) to 1 mM and incubating the cells with shaking for 16 h at 20°C. We lysed the pelleted cells by using BugBuster reagent (EMD, Gibbstown, NJ), adding phenylmethylsulfonyl fluoride (PMSF) to 0.4 mM and 10 μl/sample of protease inhibitors (P8849; Sigma Aldrich). The clarified supernatant was added to 0.5 ml of settled HisLink resin (Promega), washed with 10 ml of buffer A (100 mM bis-Tris propane [pH 7.5], 500 mM NaCl, 10 mM imidazole) supplemented with 0.1% Tween 20, and then washed with 10 ml of buffer A. His tag-containing proteins were eluted with 200 mM imidazole in buffer A. Alternatively, proteins were purified from clarified supernatant by S-protein agarose by the manufacturer's instructions (EMD). Protein concentrations were determined by the Bradford method (3).

To study the products of splicing of overexpressed SNICH, proteins were analyzed by SDS-PAGE using 4 to 20% gradient Tris-glycine gels (Lonza, Rockland, ME) (5) or by Western blotting. The molecular weights of expressed and purified proteins were verified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) at the Tufts University Core Facility.

The wild-type Tfu2914 intein splices efficiently. SDS-PAGE reveals a band of the appropriate molecular mass for the spliced product (SNCH) (Fig. 3). Analysis by MALDI-TOF MS showed a peak at an m/z of 39,189, within 0.3% of the expected value. It is notable that the intein splices with Gly in place of the conserved penultimate His.

FIG. 3.

SDS-PAGE analysis of protein splicing. Coomassie blue-stained SDS-PAGE analysis using 1.5 μg of total purified protein. Protein was purified via the C-terminal His tag (WT-His) or N-terminal S peptide (WT-S).

Products of intein side reactions and the precursor fusion protein are found in inclusion bodies, so we analyzed the influence of site-directed mutations via Western blotting of whole cells disrupted by boiling in SDS loading buffer (New England Biolabs). We detected the products of splicing, N- and C-terminal cleavage, and branched ester formation by Western blotting with an antibody directed against the C extein (Fig. 4). We also observed the corresponding bands on Western blots using an antibody against the N extein except for protein SN, which we did not routinely detect, perhaps due to its low molecular mass (data not shown).

FIG. 4.

Western blot analysis of protein splicing and side reactions. Western blot analysis was performed using an anti-His tag antibody; all proteins with the C extein should be labeled. The identity of each protein sample is indicated above each lane; the vector lane was derived from cells harboring pET-29b(+). For analysis by Western blotting, gels were blotted onto polyvinylidene difluoride (PVDF). The membranes were blocked using 1% bovine serum albumin (BSA) in buffer W (phosphate-buffered saline [PBS] and 0.1% Tween 20) and incubated with a 1:5,000 dilution of alkaline phosphatase (AP)-conjugated S protein (not shown) or a 1:10,000 dilution of mouse anti-His monoclonal antibody (GenScript, Piscataway, NJ). The blots were washed in buffer W, and the anti-His blot was incubated with a 1:8,000 dilution of anti-mouse IgG-AP conjugate (EMD) and washed again. The blots were developed with Western Blue-stabilized substrate (Promega). WT, wild type.

There are four mechanisms that could lead to the branched-ester intermediate, which would then proceed to splice (Fig. 1). Path 1 (black arrows) is the class 1 mechanism, the canonical mechanism without the internal thioester. Path 2 (red arrow) is the class 2 mechanism, direct attack of the downstream nucleophile on the N-terminal amide bond. Path 3 (green arrows) is the class 3 mechanism, attack of the internal nucleophile on the N-terminal amide bond, followed by transesterification to the branched ester. Path 4 (purple arrows) is a mixed class 1/3 mechanism, with formation of the linear thioester followed by transesterification to the internal branched thioester and then transesterification to the branched ester.

We made a series of mutant Tfu2914 inteins to test these models (Fig. 4). Mutant inteins with Ser+1Ala or Cys320Ala can promote N-terminal cleavage. This suggests that the linear thioester can form. A mutant intein with Cys1Ala cannot splice or facilitate N-terminal cleavage, even with a second mutation, Ser+1Cys, to improve the nucleophilicity of the downstream nucleophile, which suggests that the linear thioester is essential. These data suggest that the class 2 and 3 mechanisms are unlikely.

It is possible that a class 2 or 3 mechanism could occur with a mutation to an N-terminal residue other than Ala. Therefore, we tested mutants with Cys1 mutations to Pro, Met, Gln, and Asn (present in other class 2 or 3 inteins), Val and Leu (common replacements for Cys), and Ser. None of these mutant inteins promoted splicing or N-terminal cleavage except for Cys1Ser, which promoted a small extent of splicing (Fig. 4).

It is unlikely that an internal thioester is an obligate intermediate following either the class 3 or class 1/3 path. A mutant Tfu2914 intein with Cys320Ala can promote N-terminal cleavage and a small extent of splicing (Fig. 4), so the internal thioester is not essential. There was a double band near where the precursor migrated in the Cys320Ala and Asn341Ala mutants; we attribute the slower-migrating band to a branched ester. Both bands reacted with both antibodies. This suggests that the branched ester can form without a prior internal thioester. Also, because the Cys1Ser intein can splice, albeit poorly, the class 1/3 mechanism is unlikely because it would be thermodynamically unfavorable for an N-terminal linear ester to form and then convert to the internal thioester. Alternatively, it is possible that the inteins that splice poorly with Cys320Ala or Cys1Ser are able to bypass an internal thioester that would ordinarily make splicing more efficient.

The data suggest that Cys320 plays a role in promoting Asn cyclization. Mutations of Cys1 to Ala or Ser+1 to Ala result in Tfu2914 inteins that can promote C-terminal cleavage (Fig. 4). Therefore, Asn cyclization can occur without the prior steps prevented by these mutations. However, if these mutations are coupled with a Cys320Ala mutation, the intein cannot promote C-terminal cleavage. Even if a branched ester can form, as we believe we observed with the intein with a single Cys320Ala mutation, uncoupled C-terminal cleavage is still prevented. Perhaps Cys320 is required to properly coordinate the active site to promote Asn cyclization, as with the Asp at that position in the Mycobacterium tuberculosis RecA intein (15, 16). Alternatively, the Cys320 could promote proper branched-ester formation, with Asn cyclization being coupled to splicing in the context of a branched ester, as with the Mycobacterium xenopi GyrA intein (4).

Although the Tfu2914 intein has characteristics of a class 3 intein, including the conserved covariant Trp-Cys-Thr triplet and a flanking Ser residue (in place of the more common Cys), it splices by a class 1 mechanism. If the internal Cys is mutated to Ala, N-terminal cleavage can be promoted and a small extent of branched ester and spliced product are formed, suggesting that a linear thioester forms and that an internal thioester is not absolutely essential. However, the internal Cys contributes to catalysis, likely by playing a role in coordinating the steps of splicing.