The Canonical Protein Splicing Mechanism

In the protein-splicing reaction, the intein is excised and the N- and C-exteins are linked directly by a peptide bond. Early in vitro studies of protein splicing using inteins from extreme thermophiles expressed as fusion proteins in E. coli led to two general conclusions about the nature of protein splicing: (1) protein splicing is mediated entirely by amino acid residues within the intein and by the adjacent C-extein residue and requires no additional protein catalysts, and (2) protein splicing requires no coenzymes or sources of metabolic energy, suggesting that it occurs by bond rearrangements rather than by the cleavage and synthesis of peptide bonds. The nature of the amino acids that flank the scissile bonds in canonical inteins (Fig. 1) suggests a possible mechanism. The nucleophilic amino acids at the N-termini of the intein and the C-extein could function in nucleophilic attacks on amide and ester bonds, including attacks on amide bonds involving their own amino groups, and the

1 Throughout this chapter, we will refer to the nucleophilic residues at the N-terminus of the intein and the N-terminus of the C-extein as the upstream and downstream nucleophilic residues, respectively. The intein residues are numbered starting with the upstream nucleophilic residue as 1; the C-extein residues are numbered starting with the downstream nucleophilic residue as +1.

Fig. 1. Conserved elements in a canonical intein. The shaded areas are conserved intein motifs, identified by the revised nomenclature of Pietrokovski (1998). Motifs Nl, N3, CI and C2 correspond to motifs A, B, G, and F of the older nomenclature, respectively (Pietrokovski 1994; Perler 2002). Amino acid residues that play an essential role in the canonical protein-splicing mechanism are indicated. The conserved motifs and other intein sequences are to scale, based on the M. xenopi GyrA intein. The site of insertion of the homing en-donuclease domain or linker regions is indicated by the dark vertical line. In the S. cerevi-siae VMA intein, a DNA recognition region (DRR) is inserted just before motif N4

Fig. 1. Conserved elements in a canonical intein. The shaded areas are conserved intein motifs, identified by the revised nomenclature of Pietrokovski (1998). Motifs Nl, N3, CI and C2 correspond to motifs A, B, G, and F of the older nomenclature, respectively (Pietrokovski 1994; Perler 2002). Amino acid residues that play an essential role in the canonical protein-splicing mechanism are indicated. The conserved motifs and other intein sequences are to scale, based on the M. xenopi GyrA intein. The site of insertion of the homing en-donuclease domain or linker regions is indicated by the dark vertical line. In the S. cerevi-siae VMA intein, a DNA recognition region (DRR) is inserted just before motif N4

Asn residue at the intein C-terminus could undergo spontaneous cyclization coupled to peptide bond cleavage.

Detailed biochemical studies on protein splicing during the period 19931996 succeeded in defining the chemical reactions that underlie the canonical protein-splicing mechanism (reviewed in Perler et al. 1997; Paulus 1998,2000; Noren et al. 2000; Evans and Xu 2002). These reactions, which consist of the four steps illustrated in Fig. 2, are described in detail in the sections that follow.

2.1 First Step of Protein Splicing - N/O or N/S Acyl Shift

Protein splicing is initiated by attack of the nucleophilic side chain of the Ser or Cys residue at the intein N-terminus on its upstream peptide bond to generate an ester intermediate (Fig. 2, step 1). Spontaneous N/O and N/S acyl shifts have not been observed under physiological conditions, owing in part to the very unfavorable equilibrium position at neutral pH, but can be induced under strongly acidic conditions, and are thought to proceed through oxyoxazolidine or oxythiazolidine anion intermediates, respectively (Fig. 3; reviewed in Iwai and Ando 1967).

Evidence for N/O or N/S Acyl Shift. The occurrence of N/O or N/S acyl shifts in protein splicing was demonstrated in vitro with a modified version of the Pyrococcus sp. GB-D (Psp) Pol intein in which the N-terminal Ser was replaced with Cys (Shao et al. 1996). In the presence of high concentrations of hydrox-ylamine or ethylenediamine, fusion proteins containing the modified intein undergo cleavage at the upstream scissile bond, consistent with the aminoly-sis of a thioester. The site of hydroxylaminolysis was identified by analysis of the C-terminus of the polypeptide cleavage product. Similar studies were performed using the S. cerevisiae (See) VMA intein, in which Cys occurs naturally at the upstream splice junction (Chong et al. 1996). Studies of the NIS acyl

N/O or N/S acyl shift and -fl N-terminal cleavage / |

/Asn cyclization and / C-terminal cleavage

Ester intermediate

Transesterification Step 2

/Asn cyclization and / C-terminal cleavage

N/O or N/S acyl shift and -fl N-terminal cleavage / |

Ester intermediate

Transesterification Step 2

y N-terminal cleavage

Branched Intermediate

y N-terminal cleavage

Asn cyclization and \ C-terminal cleavage ^

Asn cyclization and peptide cleavage

Branched Intermediate

Step 3

N-Extein .0

h?NM ML

O-N or S-N acyl shift and succinimide hydrolysis

Transient products

Step 4

O-N or S-N acyl shift and succinimide hydrolysis

Excised intein

Spliced exteins

Excised intein

Spliced exteins

Fig. 2. Canonical protein-splicing mechanism. The canonical protein-splicing pathway is shown on the left and possible side reactions are indicated by the dotted arrows on the right. The amino acid residues that participate directly in the chemical reactions are shown (X=0 or S). The remaining portions of the intein and the exteins are indicated schematically and are not to scale. RH indicates a nucleophile, which may be an organic thiol such as DTT or cysteine, an amine such as hydroxylamine or ethylenediamine, or water shifts of these inteins showed that the thioester intermediate can be cleaved by transesterification with a large excess of low molecular weight thiols such as cysteine or 1,4-dithiothreitol (DTT), which led to the development of a protein expression/purification system with self-cleavable affinity tags (Chong et al. 1997).

The Amide-Thioester Equilibrium in Protein Splicing. The equilibrium position of the NIS acyl shift involving the See VMA intein was measured after arresting further amide-ester interconversion by adding 8 M urea and then estimating the amount of ester intermediate by reaction with hydroxylamine (Chong et al. 1998b). Depending on the amino acid at the C-terminus of the N-extein, the Keq value for the NIS acyl shift ranges from less than 0.05 to as high aslO. A Keq value of about 1 was also observed with the Synechocystis sp. PCC6803 (Ssp) DnaB intein (Mathys et al. 1999). The large equilibrium constants for the NIS acyl shifts of the scissile bond are unexpected because SIN acyl shifts of typical peptide bonds at neutral pH are essentially irreversible (Iwai and Ando 1967; Shao and Paulus 1997).

Potential Use of Catalytic Strain To Promote N/S Acyl Shifts. Intein-promot-ed destabilization of the upstream scissile amide bond is a possible driving force for the first step of protein splicing and would also explain the large Keq value of the N/S acyl shift. The destabilization of this bond toward the oxythi-azolidine transition state would both enhance the reaction rate and make the equilibrium of the acyl shift more favorable in the direction of the thioester. Evidence for strain in the scissile bond was provided by analysis of the crystal structure of the Mycobacterium xenopi (Mxe) GyrA intein, which shows the peptide bond in question to be in the uncommon cis configuration (Klabunde et al. 1998), as well as one crystal structure of the See VMA intein, which reports main-chain distortion (Poland et al. 2000). The energy of cz's-peptide bonds is about 5 kcal moH higher than that of trans-peptide bonds (Ramach-andran and Mitra 1976), making the Keq value about 4000 times larger for N/S acyl shifts in which a cz's-peptide bond is rearranged to an ester.

Fig. 3. N/O or N/S acyl shift, with the postulated oxyoxazolidine or oxythiazolidine anion intermediate. X=0 or S

A more definitive examination of the conformation of the upstream scis-sile bond made use of nuclear magnetic resonance (NMR), which can be carried out on a much shorter time scale than X-ray crystallography and therefore permits the use of functional inteins (Romanelli et al. 2004). Using protein semisynthesis (Muir 2003), a segmental isotopically labeled Mxe GyrA intein was constructed, which allows for unequivocal assignment of the resonances of the scissile amide bond. The wild-type intein is able to promote thi-ol-induced N-terminal cleavage and hence promote the NIS acyl shift, but a His75Ala intein can promote only trace amounts of N-terminal cleavage. The possibility of a hydrogen bond between His75 and the scissile amide is suggested by an unusually large upfield chemical shift for the amide proton of the wild-type intein, together with a downfield shift in the His75Ala mutant. In addition, a possible change in the polarization of the scissile bond, perhaps due to a loss of amide resonance due to bond rotation, is suggested by the complementary observation that the one-bond dipolar coupling constant (1JNC') for the scissile bond is abnormally low in the wild-type intein, but not in the His75Ala mutant. However, the spectral overlap of the 1H{15N} HSQC spectra suggests that the His75Ala mutation does not significantly alter the overall structure of the intein. These data could be interpreted to suggest that His75 stabilizes a locally distorted scissile amide bond conformation.

2.2 Second Step of Protein Splicing - Transesterification

Intramolecular transesterifications are freely reversible and relatively rapid reactions that require good nucleophiles. In protein splicing, transesterification occurs either between a thioester and a thiol or alcohol, or between an oxygen ester and an alcohol, but never between an oxygen ester and a thiol. In the first two cases, the equilibrium constant at neutral pH is about 1; in the thioester-alcohol case it is about 50 (Jencks et al. 1960). The transesterification step involves attack by the nucleophilic side chain of the N-terminal amino acid of the C-extein on the ester linking the N-extein and intein, yielding a branched ester intermediate (Fig. 2, step 2).

Evidence for Branched Intermediate Formation. A branched intermediate was first detected in the in vitro splicing reaction involving the Psp-GBD Pol intein, which has Ser residues at both splice junctions (Xu et al. 1993). The intermediate was detected by its unusually slow migration upon sodium do-decyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) and by the detection of two N-termini, corresponding to those of the N-extein and the intein. Mild alkaline hydrolysis of the branched intermediate trapped with 6 M guanidinium chloride shows that the N-extein is esterified to the downstream

Ser hydroxyl group (see Fig. 2; Xu et al. 1994). Transesterification can be reversed by shifting the equilibrium of the N/O acyl shift to the amide by raising the pH (Xu et al. 1993), and the branched ester can be restored by lowering the pH, indicating full reversibility (Xu et al. 1994). Branched intermediates were also characterized in protein-splicing systems involving a thioester rather than an oxygen ester (Chong and Xu 1997; Chen et al. 2000), and the equivalent structure has tentatively been identified in the naturally split Syne-chocystis sp. (Ssp) DnaE intein (Nichols et al. 2003). On the other hand, the occurrence of branched intermediates has not been reported in every extensively studied protein-splicing system.

Mechanistic Aspects of Transesterification. The first intein crystal structures lack C-exteins (Duan et al. 1997; Klabunde et al. 1998), although modeling of their N-terminal Cys residues suggests that the nucleophilic side chains of the N-terminal residues of the intein and the C-extein are closely juxtaposed to allow facile transesterification. This was confirmed by the crystal structure of the See VMA intein with adjacent extein residues, in which the two nucleophilic side chains are 3.6 A apart (Mizutani et al. 2002). It is likely that this intein is functional because the crystals are isomorphic with those of a mutant See VMA intein that can undergo splicing in the crystal lattice (Mizutani et al. 2002, 2004). This contrasts with the structures of the inactive Zn2+-complex of the See VMA intein (Poland et al. 2000) and of the Ssp DnaB intein (Ding et al. 2003), in which the two nucleophilic side chains are separated by a distance of 9 and 8.5 A, respectively, implying either that a conformational change has to occur before transesterification can proceed or that these crystal structures correspond to inactive forms of the intein.

A general base catalyst may serve to increase the nucleophilicity of the side chain that initiates transesterification, especially when a hydroxyl group is involved, but the intein crystal structures available at this time provide no insights into this matter. Modeling of the linear ester intermediate, based on a crystal structure of the See VMA intein, suggests that the neutral amino group of the N-terminal Cys produced in the NIS acyl shift can activate the N-ter-minal thiol of the C-extein for nucleophilic attack (Mizutani et al. 2002,2004). In the Mycobacterium tuberculosis (Mtu) RecA intein, the apparent pKa of the intein N-terminal Cys, which initiates the NIS acyl shift, is approximately 8.2, whereas that of the C-extein N-terminal Cys, which initiates the transesterification reaction, is about 5.8 (Shingledecker et al. 2000). This suggests that transesterification may be facilitated by the induction of an unusually low pKa for the attacking thiol group by the unique electrostatic and dielectric environment of the protein-splicing active center rather than by general base catalysis.

2.3 Third Step of Protein Splicing - Asparagine Cyclization

Certain Asn residues in peptides and proteins can undergo slow cyclization to aminosuccinimide (reviewed in Clarke 1987). This reaction usually results in deamidation of the side chain by attack of the peptide bond nitrogen on the carbonyl carbon of the asparagine P-amide. Another mode of asparagine cyclization, the attack of the P-amide nitrogen on the carbonyl carbon of the peptide bond, leads to peptide bond cleavage and can occur at high temperatures (Geiger and Clarke 1987) or in unusually long-lived proteins such as the lens crystallins (Voorter et al. 1988). It is this second type of asparagine cyclization that occurs in the course of protein splicing and leads to the concomitant cleavage of the downstream scissile bond (Fig. 2, step 3).

Evidence for Asparagine Cyclization. The occurrence of asparagine cyclization in protein splicing was demonstrated by the identification of an aminosuccinimide residue at the C-terminus of the excised Psp-GBD Pol in-tein by high-performance liquid chromatography (HPLC), mass spectrometry and colorimetric analysis (Xu et al. 1994; Shao et al. 1995). The excised See VMA intein also has a C-terminal aminosuccinimide residue, suggesting that the same cleavage mechanism occurs in the inteins of hyperthermophil-es and mesophiles, regardless of whether protein splicing involves oxygen or thioesters (Chong et al. 1996). The essential role of asparagine cyclization in protein splicing was shown by the replacement of the C-terminal Asn by Asp, which completely blocks splicing and leads to the accumulation of the branched intermediate (Xu and Perler 1996).

Mechanistic Aspects of Asparagine Cyclization. The mechanism of asparagine cyclization and the attendant cleavage of the peptide bond linking the intein to the C-extein is not yet clear. Some biochemical evidence suggests that the adjacent conserved His residue plays a role in Asn cyclization. For instance, its replacement with other amino acids can inhibit protein splicing (Cooper et al. 1993) and lead to the accumulation of the branched ester intermediate (Xu and Perler 1996). Nevertheless, 27 inteins are known with a penultimate amino acid residue other than His (Perler 2002), at least three of which undergo efficient splicing in Escherichia coli. In these cases, the replacement of the penultimate residue with His either slightly reduces splicing efficiency, enhances splicing, or has no significant effect (Mills and Paulus 2001; Nichols and Evans 2004). On the other hand, the Chlamydomonas eugametos (Ceu) ClpA intein, which has a Gly residue in place of the penultimate His, is unable to splice in E. coli unless the Gly residue is replaced by His (Wang and Liu 1997).

Examination of the current intein crystal structures sheds little light on the role of the penultimate His residue in asparagine cyclization. The bond dis tances reported in the crystal structure of the Mxe GyrA intein are consistent with protonation of the scissile bond by His197, which would facilitate its cleavage concomitant with the cyclization of Asn198 (Klabunde et al. 1998). On the other hand, the crystal structure of the Ssp DnaB intein and adjacent extern fragments is consistent with an H-bond between the Asn carbonyl oxygen of the scissile bond and the imidazole ring of the adjacent His residue (Ding et al. 2003). Asparagine cyclization and cleavage of the downstream scissile bond can still occur when the other steps in protein splicing are blocked by mutations, such as replacement of either the upstream or downstream nucle-ophilic residue (Hirata and Anraku 1992; Cooper et al. 1993; Chong et al. 1996, 1998a; Xu and Perler 1996; Mathys et al. 1999; Southworth et al. 1999). However, in some cases, the N/O or NIS shift must occur prior to Asn cyclization (Xu and Perler 1996).

2.4 Finishing Reaction

O/N or S/N Acyl Shift. After cyclization of the C-terminal Asn residue and the attendant cleavage of the downstream scissile bond, protein splicing is complete in the sense that the intein is excised and the exteins are linked. However, an ester bond links the extein segments and the excised intein contains an unnatural C-terminal residue, aminosuccinimide. Especially critical for the completion of the splicing process is the O/N or S/N shift of the peptide ester intermediate to yield a spliced product composed entirely of peptide bonds. This is essentially the reverse of the first step of protein splicing, the N/O or N/ S acyl shift, which now operates in the thermodynamically favored direction.

Uncatalyzed O/N and S/N shifts have been studied in model peptides. The rates of O/N and S/N acyl shifts of peptide esters are extremely rapid and increase strikingly with pH (Shao and Paulus 1997). Thioesters involving the side chain of Cys residues rearrange about 1000 times more rapidly than oxygen esters. The extremely rapid rates and the virtual irreversibility of O/N and S/N acyl shifts at neutral pH make these acyl shifts ideal finishing reactions that rapidly drive protein splicing to completion under physiological conditions without the need for a catalyst. On the other hand, recent crystal-lographic studies on a slowly splicing variant of the See VMA intein, which undergoes protein splicing in the crystal lattice, show the spliced extein to be positioned relative to the excised intein so that its N-terminal amino group can stabilize the oxythiazolidine anion intermediate in the S/N acyl shift (Mi-zutani et al. 2004). Although facilitation of the acyl shift may not be a critical factor in accelerating the protein-splicing process, its occurrence in the relatively hydrophobic environment of the protein-splicing active center may serve to reduce side reactions such as the hydrolysis of the relatively unstable thioester bond.

Succinimide Hydrolysis. The rate of hydrolysis of C-terminal aminosuc-cinimides was measured using synthetic tetrapeptides corresponding to the C-terminus of the Psp-GBD Pol intein (Shao et al. 1995). The rate of succinimide hydrolysis at 37 °C is strikingly pH-dependent, with a t1/2 of 350 h at pH 5.5, which declines to 17 h at pH 7.4. At 90 °C, the t1/2 at pH 7.4 is 4 min. These rates are considerably slower than the rates of hydrolysis of N-substi-tuted cyclic imides produced at internal positions of polypeptide chains by nucleophilic attack of the peptide bond N on the carbonyl C of the Asn P-amide (summarized in Shao et al. 1995). The relatively slow rate of hydrolysis of C-terminal aminosuccinimide implies that, at least in mesophilic organisms, a substantial fraction of the excised inteins carry C-terminal aminosuccinimide residues.

2.5 Association of Split Inteins

Protein splicing is ordinarily an intramolecular process and therefore does not involve a reactant association step. However, some inteins can be artificially split, expressed as separate proteins, and reconstituted to yield a functional protein-splicing complex (Mills et al. 1998; Southworth et al. 1998; Yamazaki et al. 1998). The reconstitution of artificially split inteins requires a denaturation/renaturation procedure, probably owing to misfolding that occurs when the intein fragments are expressed separately. It is therefore difficult to measure the parameters of the reassociation reaction, but the affinity of the intein fragments must be quite high, as indicated by nearly quantitative reassociation of the split Mtu RecA intein in the 10 |iM range with only a twofold molar excess of the C-terminal fragment (Lew et al. 1999).

However, the discovery of naturally split inteins in cyanobacteria (Wu et al. 1998), in which they presumably serve to reconstitute the separately expressed fragments of the DnaE protein, makes intein fragment association an essential first step in the splicing of DnaE. The association of the N- and C-termi-nal segments of the Ssp DnaE intein occurs with high efficiency without the need for prior denaturation (Evans et al. 2000). The association of the N-ter-minal segment (N-extein-EN) with the C-terminal intein segment (Ec) was studied using a competition assay, which suggested that the dissociation of N-extein-EN from Ec is a very slow and rate-limiting process, consistent with a very low EN-EC dissociation constant. In addition, even with a large molar excess of N-extein-EN over Ec, no turnover of the intein as an active catalyst is observed (Nichols and Evans 2004). This suggests that the product EN has a much higher affinity than N-extein-EN for Ec and that its tight binding to Ec blocks further reaction. The binding of N-extein-EN to Ec may be diminished by the strain energy required to destabilize the scissile bond into a conformation resembling the oxythiazolidine transition state. Since the scissile bond is absent in EN, strain energy will not need to be diverted from binding energy, and the binding energy should therefore be more favorable for EN than for N-extein-EN.

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