From New England Biolabs, Inc., Beverly, Massachusetts 01915
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ABSTRACT |
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Protein splicing of the Saccharomyces
cerevisiae vacuolar membrane ATPase intein involves four highly
coordinated reactions that result in precise cleavage and formation of
peptide bonds. In this study, we investigated the roles of the last
N-extein residue (1 residue) and the intein penultimate residue in
modulating splicing reactions. Most of the 20 amino acid substitutions
at the
1 position had no effect on overall protein splicing but could
lead to significant accumulation of thioester intermediates when
splicing was blocked by mutation. A subset of
1 substitutions attenuated the initiation of protein splicing and enabled us to demonstrate in vitro splicing of a mesophilic intein
containing all wild-type catalytic residues. Substitutions involving
the intein penultimate residue allowed modulation of the branch
resolution and C-terminal cleavage reaction. Our data suggest that the
N-S acyl rearrangement, which initiates splicing, may also serve as the
rate-limiting step. Through appropriate amino acid substitutions, we
were able to modulate splicing reactions in vitro by change in pH or temperature or addition of thiol reagents. Both insertion and
deletion were tolerated in the central region of the intein although
splicing or structure of the intein may have been affected.
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INTRODUCTION |
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Protein splicing involves a precise excision of an internal
segment, the intein, from a protein precursor and a concomitant ligation of the flanking regions, the exteins, resulting in the production of two proteins (1). Since the initial discovery of protein
splicing, more than 40 inteins have been identified (2). Sequence
analysis reveals that inteins are bounded by Cys, Ser at N termini and
Asn at C termini and, with a few exceptions, have His as the
penultimate residue (2). The first
C-extein1 residues are
invariably Cys, Ser, or Thr (2). The chemical mechanism of protein
splicing of the inteins from the thermostable DNA polymerase of
Pyrococcus sp. GB-D (Psp pol-1 intein) and the 69-kDa vacuolar membrane ATPase subunit of Saccharomyces
cerevisiae (Sce VMA intein) suggests that a common
protein splicing pathway may have evolved in thermophilic archaea and
mesophilic eukaryotes (3-5). Protein splicing of the Sce
VMA intein consists of the following multi-step reactions: step 1, an
N-S rearrangement at the intein N terminus (Cys1) to form a
thioester bond between Cys1 and the last N-extein residue
(1 residue); step 2, a trans-esterification reaction by the first
C-extein residue Cys455 to form a branched intermediate;
step 3, succinimide formation at the intein C terminus
(Asn454) to resolve the branched intermediate; step 4, a
final S-N rearrangement to form a peptide bond linkage between the
ligated exteins (Fig. 1A) (5).
The recently solved crystal structure of the Sce VMA intein demonstrates that both intein terminal residues, Cys1 and Asn454, are in close proximity, forming a structure consistent with their proposed roles in the splicing pathway (6). Since the crystal structure represents the excised intein, the structure of the extein-intein precursor that involves at least one extein residue (i.e. Cys455) at the splicing active site is still unknown. Although the crystal structure supports our proposed splicing pathway, many mechanistic details of the splicing reactions have not been elucidated.
Previously, we examined the Sce VMA intein in an in vitro MYT splicing system (5). The rapid splicing of the Sce VMA intein in vivo precluded the isolation of the precursor and/or intermediates (5, 7, 8). Our studies utilized amino acid substitutions to arrest or attenuate the splicing process (5). A single substitution, Asn454 to Ala, was shown to block both splicing and C-terminal cleavage but not N-S acyl arrangement (5). Thiols such as dithiothreitol and cysteine were able to shift the N-S equilibrium by attacking the thioester bond and initiating N-terminal cleavage (5, 9). Previous studies have shown that the Sce VMA intein penultimate residue, His453, although conserved in almost all inteins, was not essential for protein splicing (5, 7, 8). A double substitution, H453L and C455S, was shown to attenuate protein splicing in vivo and allow in vitro splicing of the purified precursor (5). Although our data on the in vitro splicing reaction support our proposed splicing pathway (5), direct examination of in vitro splicing of the Sce VMA intein containing unaltered catalytic residues has been a challenge.
The Sce VMA intein also functions as a site-specific homing endonuclease which mediates gene mobility (10-12). The central conserved dodecapeptide motifs are directly involved in DNA recognition and cleavage (13, 14). The crystal structure of the Sce VMA intein suggests that the splicing and endonuclease functions may reside in two separate domains (6). We made large in-frame deletions which removed the domain containing the dodecapeptide motifs, demonstrating the remaining splicing domain was sufficient for efficient splicing (15).
This paper extends our previous in vitro studies to focus on
residues that are in close proximity to the reaction center but not
directly involved in the splicing reactions, i.e. the last N-extein residue (the 1 residue) and the intein penultimate residue. This approach enabled us to modulate each of the first three splicing reactions by change in pH, temperature, and/or addition of thiol reagents and allowed direct examination of in vitro splicing
of the Sce VMA intein containing wild-type catalytic
residues. In addition, we investigated the effect of deletion and
insertion in the Sce VMA intein on splicing reactions. Our
results provide strategies for modulating protein splicing and insights
into the mechanism of protein splicing of a mesophilic intein.
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EXPERIMENTAL PROCEDURES |
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Numbering of Residues in the Intein Fusion Constructs-- Amino acid numbers refer to the position in the S. cerevisiae VMA intein, essentially the same as described previously (Fig. 1B) (5).
Constructions of pMYB130 Containing Different 1
Residues--
The construction of pMYB129 containing the wild-type
Gly
1 residue was described previously (9). pMYB129
contains an XhoI site and a KpnI site flanking
the N-terminal splice junction including the
1 position (9). These
unique sites allowed convenient substitution of the Gly
1
with the remaining 19 naturally occurring amino acids through linker
insertion. pMYB129 was digested with XhoI and
KpnI and then ligated with the complementary oligomers 5'-TC
GAG NNN TGC TTT GCC AAG GGT AC-3' and 5'-C CTT GGC AAA GCA NNN C-3'
that encoded each of the 19 amino acids (NNN) at the
1 position. The
resulting constructs were named pMYB130(X
1),
e.g. pMYB130(Ala
1) containing Ala as the
1
residue, pMYB130(Asn
1) containing Asn as the
1 residue,
etc. The substitutions of the
1 residues in pMYB130(X
1)
were further confirmed by DNA sequencing (New England Biolabs). Both
pMYB129 and pMYB130(X
1) contained the N454A mutation,
which blocked splicing and C-terminal cleavage and therefore were
fusion constructs for the study of the N-S acyl rearrangement and
N-terminal cleavage. Unless otherwise stated, all enzymes and plasmids
used were the products of New England Biolabs, Inc.
Construction of pMYK(X1) and pMYK Containing
His453 Substitutions--
The T4 polynucleotide kinase
gene (16) was synthesized by the polymerase chain reaction using the
primers 5'-GGT GGT ACC GGT AAA AAG ATT ATT TTG ACT ATT GGC-3' and
5'-GGT GGT CTG CAG TCA AAA ATC TCC CGA AGC GAC TTG CCA-3'. Polymerase
chain reaction mixtures (100 µl) contained Vent DNA polymerase buffer
(New England Biolabs), 3 mM MgSO4, 300 µM each of the 4 dNTPs, 10 µM each primer, 1 µl of T4 phage particle suspension, and 1 unit of Vent DNA
polymerase. Amplification was carried out for 25 cycles using a
Perkin-Elmer thermal cycler at 95 °C for 1 min, 50 °C for 1 min,
and 72 °C for 2 min. The product was digested with AgeI
and PstI and ligated with pMYB (15) digested with
AgeI and PstI, to yield pMYK. pMYK contained the
wild-type splice junction residues including Gly as the
1 residue. To
construct pMYK(X
1) containing different
1
substitutions, pMYB130(X
1) was digested with
XhoI and BamHI, and the resulting
XhoI-BamHI fragment was used to replace the
corresponding fragment from pMYK to yield pMYK(X
1).
pMYK(X
1) contained each of the 20 amino acid residues at
the
1 position and the wild-type splice junction residues and was
used to study the effect of the
1 substitutions on splicing.
Construction of pMYT4 and Its Mutant Derivatives--
The T4
phage DNA ligase gene (17) was synthesized by the polymerase chain
reaction using the primers 5'-GGT GGT ACC GGT ATT CTT AAA ATT CTG AAC
GAA ATA GCA-3' and 5'-GGT GGT CTG CAG TCA TAG ACC AGT TAC CTC ATG AAA
ATC ACC-3'. The reaction conditions were essentially as described
above. The product was digested with AgeI and
PstI and ligated with pMYB (15) that had been digested with
AgeI and PstI, yielding pMYT4, which contains the wild-type splice junction residues including Gly1. pMYT4
was digested with BamHI and AgeI and ligated with
the complementary oligomers, 5'-GA TCC CAG GTT GTT GTA CAG AAC GCA GGT
GGC CTG A-3' and 5'-CC GGT CAG GCC ACC TGC GTT CTG TAC AAC AAC CTG
G-3', to create pMYT4 (H453Q/C455A), in which His453 and
Cys455 were replaced with Gln and Ala, respectively.
Similarly, the same pMYT4 digest was also used with different pairs of
complementary oligomers to create pMYT4(H453Q/N454A/C455S). The pMYT4
mutant constructs were used for the study of C-terminal cleavage.
Construction of pMYB(NG) (N454A) and p
MYB(Bam)--
The
construction of p
MYB(NG) was described previously (15). p
MYB(NG)
was digested with BamHI and AgeI and ligated with the complementary oligomers, 5'-GA TCC CAG GTT GTA GTA CAC GCT TGC GGT
GGC CTG A-3' and 5'-CC GGT CAG GCC ACC GCA AGC GTG TAC TAC AAC CTG
G-3', to create p
MYB(NG) (N454A), in which Asn454 was
replaced with Ala. The gene for BamHI (18) was amplified by
polymerase chain reaction following a previously described protocol
(9). The forward primer 5'-GTT GGT GCT AGC GGT GGT AAC AAC GAA GTT GAA
AAA GAA TTC ATC ACT GAT-3' encoded an NheI site, and the
reverse primer 5'-GGT GGT GAC GTC GGT GGT AAC AAC TTT GTT TTC AAC TTT
ATC TTT CCA TTT-3' encoded an AatII site. The polymerase
chain reaction products were digested with NheI and
AatII and ligated with the NheI-AatII
digested p
MYB(NG) to yield p
MYB(Bam).
Fusion Protein Expression and Purification by Amylose Affinity
Chromatography--
The proteins expressed from pMYB, pMYB, pMYK,
pMYT4, and their mutant derivatives, are referred to as MYB,
MYB,
MYK, MYT4 fusion proteins, respectively. Escherichia coli
strain ER2426 (15), harboring these plasmids or their mutant
derivatives, was used for fusion protein expression. The expression and
purification followed essentially the same procedures as described
previously (5). In the case of p
MYB(Bam), the plasmid was
used to transform E. coli strain pLD2263/m.bamH1, which
contained the BamHI methylase gene (19), and the expressed
fusion proteins were purified on amylose resin. The protein samples
were analyzed by SDS-PAGE, followed by Coomassie Blue staining and
Western blot analysis using antibodies raised against BamHI.
To test the ability of the splicing product MB fusion proteins to bind
chitin, an aliquot (1 ml) of amylose-purified protein was loaded onto a
chitin column containing 0.5 ml of resin. The resin was washed three
times with Hepes column buffer containing 30 mM Hepes, 0.5 M NaCl, pH 8.0, and the bound proteins were eluted with 2%
SDS and analyzed by SDS-PAGE.
DTT-induced Cleavage of Purified Fusion Proteins--
All
cleavage reactions of purified fusion proteins (0.5-1.0 mg/ml) were
conducted in Hepes column buffer (30 mM Hepes, 0.5 M NaCl) containing 40 mM dithiothreitol (DTT)
for up to 16 h. For native fusion proteins from pMYB129 and
pMYB130 (X1), cleavage reactions were conducted at 4 and
16 °C at pH 8.0. Subsequently, each reaction mixture (0.5 ml) was
loaded onto a chitin column containing 0.3 ml of resin. After washing
the resin three times with Hepes column buffer to remove the unbound
proteins and the residual DTT, the bound proteins were eluted with 2%
SDS. The SDS-eluted samples (40 µl) were mixed with 20 µl of SDS
Sample Buffer (New England Biolabs) and analyzed by SDS-PAGE. The
percentage of cleavage was determined by comparing the MYB precursors
from the DTT-treated samples with those from samples without DTT
treatment. For the MYB fusion proteins with Gly, Ala, Ile, Ser, or Gln
as the
1 residue, the half-times of the cleavage reaction were
determined by taking aliquots (40 µl) at appropriate times during the
incubation with DTT. The cleavage reaction was stopped by mixing the
samples with SDS Sample Buffer. To examine DTT-induced cleavage of
urea-denatured MYB fusion proteins, the proteins were dialyzed against
8 M urea at pH 5.5, 7.6, or 9.5 before incubation in 8 M urea at 23 °C in the absence of DTT for up to 16 h or in the presence of DTT for 4 h. The percentage of cleavage
under denaturing conditions was determined by comparing the MYB
precursors from the DTT-treated samples (at pH 7.6) with those from
samples without DTT treatment.
In Vitro Splicing of MYK Fusion Proteins--
Fusion proteins
from pMYK(Asn1), pMYK(H453L), pMYK(H453A/C455S),
pMYK(H453F/C455S), pMYK(H453L/C455S), pMYK(H453Q/C455S), or pMYK (H453Q/C455A) were purified on amylose resin in 30 mM Hepes, 0.5 M NaCl, pH 6.0. The purified
proteins (0.5-1 mg/ml) were immediately subjected to in
vitro splicing by incubating the samples in 30 mM
Hepes, 0.5 M NaCl, pH 6.0 at 16 and 23 °C. Samples from
pMYK(Asn
1) were also subjected to in vitro
splicing at pH 8.5. In these cases, the purified proteins were dialyzed
in 30 mM Hepes, 0.5 M NaCl, pH 8.5, prior to
incubation at 16 and 23 °C. At appropriate times, aliquots (40 µl)
were removed, mixed with 20 µl of SDS Sample Buffer, and analyzed by
SDS-PAGE.
Analytical Methods-- SDS-PAGE was performed in 12% Tris glycine gels (Novex, San Diego, CA), followed by staining with Coomassie Blue. For Western blot analyses, the SDS-polyacrylamide gels were blotted onto nitrocellulose membranes and analyzed by probing with polyclonal antibodies against the Sce VMA intein (New England Biolabs) or BamHI (gifts of Jurate Bitinaite and Rebecca B. Kucera) as described previously (3). The stained gels were digitized with a Microtec Scanmaker 600 ZS, and the scanned images were analyzed to determine relative protein concentration with NIH Image 1.47 software. Variations in protein sample loading (normally <10%) were normalized prior to comparisons. Protein concentrations were estimated by the method of Bradford (20).
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RESULTS |
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Fusion Constructs of the Sce VMA Intein for Modulation of Protein
Splicing--
Protein splicing of the Sce VMA intein was
examined by fusing the intein between two independent extein domains
(Fig. 1B). E. coli
maltose-binding protein (21) (MBP) was used as the N-extein to
facilitate purification of splicing products that contained the MBP
moiety. C-exteins of varying molecular masses were used for easy
resolution of splicing products on SDS-PAGE. The MYB, MYK, or MYT4
fusion systems contained Bacillus circulans chitin-binding domain (22) (CBD or B), T4 polynucleotide kinase (K), or T4 DNA ligase
(T4) as the C-extein, respectively (Table
I). In the case of the wild-type
Sce VMA intein with Gly1 and Cys as the first
C-extein residue (residue 455), complete in vivo splicing
occurred in all three fusion systems (data not shown). The MYB
fusion system, including pMYB129, pMYB130(X
1),
p
MYB1(NG) (N454A), and p
MYB(Bam), was constructed to
study the effect of the
1 substitutions on thiol-induced N-terminal cleavage and N-S acyl rearrangement and the effect of deletion and
insertion mutations (Table I). The MYK fusion system, including pMYK(X
1), pMYK(H453X/C455S), was constructed to examine
the effect of substituting the
1 residue and the intein penultimate
residue on protein splicing (Table I). The MYT4 fusion system,
including pMYT4(H453Q/C455A) and pMYT4(H453Q/N454A/C455A), was for the
study of succinimide formation coupled to C-terminal cleavage (Table I).
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Effect of the 1 Substitutions on Induction of N-terminal
Cleavage--
An N-S acyl rearrangement at the N terminus of the
Sce VMA intein forms a thioester bond between
Cys1 and the
1 residue (Fig. 1A) (5).
Exogenous thiols, e.g. DTT and cysteine, can attack the
thioester bond to induce cleavage at the intein N terminus (Fig.
1A) (5, 9). This thiol-induced cleavage has been previously
examined in pMYB129 (9). In this study, we determined the effect of pH
and temperature on the half-time and efficiency of the cleavage
reaction (Table II). The rate of the
DTT-induced cleavage of the fusion proteins from pMYB129 increased 4-fold at pH 8.0 versus pH 6.0 at 4-16 °C and almost
50-fold at 23 °C. At pH 8.0, the rate of cleavage increased 30-fold
at 23 versus 4 °C (Table II).
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Effect of the 1 Substitutions on Protein Splicing--
Since N-S
acyl rearrangement initiates protein splicing (Fig. 1A) (5),
it is probable that the
1 substitutions that shift the N-S
equilibrium also affect protein splicing. This was examined in the MYK
fusion system using pMYK(X
1), in which the intein
contained all wild-type catalytic residues for splicing. After
expression of pMYK(X
1) in E. coli, the
splicing products were purified on amylose resin. The predicted
splicing products were identified by their apparent molecular masses on
SDS-PAGE. As shown in Fig. 3A,
the majority of
1 substitutions in MYK resulted in the production of
a predominant 75-kDa protein whose size corresponded to the ligated
exteins, MK, indicating that efficient splicing occurred in
vivo. The spliced intein (Y) was identified in the crude extract
and the flow-through (data not shown). However, some
1 substitutions
produced significant amounts of the linear precursors, MYK
(e.g. Val (lane 3), Leu (lane 4), Ile
(lane 5), Cys (lane 8), Asn (lane 11),
and Pro (lane 19)), or C-terminal cleavage products, MY and
K (e.g. Leu (lane 4), Ile (lane 5)).
In the case of the Cys
1 substitution, significant
splicing also occurred (lane 8). A minor component,
corresponding to the size of the intein (Y), was detected in samples of
many
1 substitutions (e.g. the Cys
1 and
Asn
1) possibly due to splicing of the MYK precursors
during purification.
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Effect of Substituting the Intein Penultimate Residue on Protein Splicing-- Succinimide formation by the last intein residue, Asn454, is coupled to peptide bond cleavage at the intein C terminus, resulting in branch resolution (Fig. 1A) (5). It is conceivable that the intein penultimate residue, His453, may assist the function of Asn454. The effects of single and double substitutions of His453 were therefore examined in the MYK fusion system (Table IV). Analysis of the purified proteins from the single substitution, H453L, indicated partial in vivo splicing and in vivo N-terminal cleavage (Table IV). Incubation of the purified precursors at pH 8.0 resulted in no in vitro splicing; however, significant cleavage at both splice junctions occurred in the presence of DTT (Table IV). Efficient splicing was observed when the H453L substitution was combined with the C455S substitution (Fig. 4). Expression of pMYK(H453L/C455S) resulted in the production of a major 125-kDa protein corresponding to the linear precursor MYK (Fig. 4, lane 1). A slowly migrating component corresponding to the branched intermediate (MYK*) was also observed along with splicing products, MK and Y (Fig. 4, lane 1). Incubation of the purified proteins at pH 8.5 resulted in significant in vitro splicing of the linear precursor MYK, as indicated by an increase of MK and Y (Fig. 4, lanes 7-10). The rate of splicing was much lower at pH 6.0 as only a slight increase of MK and Y was observed (Fig. 4, lanes 1-6). The amount of branched intermediate (MYK*) remained unchanged after incubation at pH 6.0 but decreased rapidly at pH 8.5 (Fig. 4), suggesting that the branch resolution reaction was favored at pH 8.5 but inhibited at pH 6.0. Although more in vivo splicing was observed resulting in less linear precursors, the double substitutions, H453Q/C455S, produced similar results as described above (data not shown). In comparison, the double substitutions, H453A/C455S and H453F/C455S, blocked splicing in vivo and allowed partial splicing and some accumulation of the branched intermediate in vitro (Table IV, SDS-PAGE data not shown). These results suggest that substitution of His453 attenuated succinimide formation by the adjacent Asn454, which, in conjunction with the C455S substitution, resulted in accumulation of the branched intermediate. Consistent with this explanation, the H453Q substitution blocked both splicing and cleavage in vivo when a second substitution, C455A, rendered the intein incapable of undergoing trans-esterification, i.e. branch formation (Fig. 1A) (Table IV). However, efficient cleavage at both splice junctions could occur in vitro in the presence of DTT (Table IV). This DTT-induced cleavage at both splice junctions was further examined in the MYT4 fusion system as described below.
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Induction of C-terminal Cleavage in Vitro-- Expression of the fusion construct pMYT4 (H453Q/C455A) resulted in the production of a single 150-kDa protein corresponding to the linear precursor MYT4 (Fig. 5A, lane 1). The MYT4 precursors were very stable in vitro as no significant cleavage at both splice junctions was observed after incubation at 4 °C for 72 h or 23 °C for 16 h (Fig. 5, A and B, lane 2). However, treatment of MYT4 with DTT at 23 °C immediately induced cleavage at the intein N terminus to yield YT4 and M (Fig. 5A, lanes 3-5). In addition, cleavage at the intein C terminus was observed, as indicated by the appearance of T4 and Y (Fig. 5A, lanes 4-10). The amount of YT4 declined after an initial increase (Fig. 5A, lanes 3-10) suggesting that T4 and Y were produced from the C-terminal cleavage of YT4 and that the C-terminal cleavage occurred after the initiation of the N-terminal cleavage. Consistent with this explanation, no significant amount of MY, the product of an exclusive C-terminal cleavage, was observed (Fig. 5A).
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Effect of Deletion and Insertion on Protein Splicing--
We have
demonstrated efficient splicing in pMYB1(NG) in which the
endonuclease domain of the intein was removed by deletion (15). To
determine if the intein deletion mutant could be modified to undergo
cleavage reactions similar to the full-length intein in the MYB fusion
system, the N454A substitution was introduced in p
MYB1(NG) to yield
p
MYB1(NG) (N454A). Expression of p
MYB1(NG) (N454A) resulted in
the production of a predominant linear precursor (Fig.
6A, lane 1). Induction of
N-terminal cleavage by DTT was conducted at different temperatures. As
shown in Fig. 6A, incubation of the purified precursors with
DTT at 4 and 16 °C resulted in significant N-terminal cleavage,
yielding M and
YB(NG) (N454A) (lanes 2-7), whereas
incubation at 23 °C blocked DTT-induced cleavage (lanes
8-10). As a control, the linear precursors were incubated without
DTT at 4-23 °C for 16 h, resulting in no significant
N-terminal cleavage (data not shown). The double substitution,
H453Q/C455A, which led to the DTT-induced splice junction cleavage in
MYT4 (Fig. 5), was also introduced in the p
MYB1(NG) construct.
Incubation of the purified precursors with DTT resulted in no
significant cleavage at either splice junction (data not shown),
suggesting that the H453Q/C455A substitution may disrupt the structure
of the intein deletion mutant for efficient cleavage.
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DISCUSSION |
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In this study, we present a comprehensive analysis of the first
three reactions in the protein splicing pathway of the Sce VMA intein: N-S acyl rearrangement, trans-esterification, and succinimide formation coupled to C-terminal cleavage (Fig.
1A). By substituting the 1 residue and the intein
penultimate residue, we were able to attenuate the splicing process and
allow investigation of each splicing reactions in in vitro
systems. Appropriate substitution of the
1 residue enabled us to
examine in vitro splicing of the Sce VMA intein
containing all wild-type catalytic residues. Induction of branch
resolution and C-terminal cleavage in vitro was made possible by substitution of the intein penultimate residue in conjunction with substitution of the first C-extein residue. In addition, the study of the effect of insertion and deletion provides the first evidence suggesting that intein can tolerate the insertion of
a foreign protein domain and that the structure of the intein for
protein splicing may be destabilized by the deletion or insertion mutation. Our data yield further insights into the mechanism of protein
splicing and strategies for modulating protein splicing reactions.
Roles of the Last N-extein Residue and Modulation of Protein
Splicing with 1 Substitutions--
Efficient splicing occurs when
inteins are transferred into heterologous proteins, suggesting that the
inteins plus the first C-extein residue contain sufficient information
for catalyzing splicing reactions (3, 5, 7, 23, 24). On the other hand,
it is conceivable that splicing in a foreign context can be affected by
the proximal extein sequences since the catalytic residues of protein
splicing are located at the intein termini (4, 5). It has been recently
proposed that splicing of the Sce VMA intein involves
interactions between the intein residues upstream of the C-terminal
splice junction and the proximal extein residues upstream of the
N-terminal splice junction (25). The N-S acyl rearrangement initiates
protein splicing, forming a thioester bond between Cys1 and
the
1 residue (5). Glycine is the
1 residue in the 69-kDa vacuolar
ATPase subunit of S. cerevisiae in which the Sce
VMA intein is embedded prior to protein splicing (26). The question of
how substitution of the
1 residue may affect protein splicing has not
been examined by previous studies. In this study, we addressed this
question by substituting the
1 residue with each of the 20 naturally
occurring amino acids.
Effect of Substituting the Intein Penultimate Histidine-- Substitution of the penultimate histidine of the Psp pol-1 intein (His536) has been shown to block both splicing and C-terminal cleavage and lead to accumulation of branched intermediate (4). Substitution of the penultimate histidine of the Sce VMA intein (His453), on the other hand, produced different results. Neither splicing nor cleavage was completely inhibited by the H453L substitution (Table IV). Double substitutions, such as H453L/C455S, are required in order for efficient splicing to occur without in vivo cleavage. The observations of the accumulation of a branched intermediate and in vitro splicing of the precursors in the double substitutions, H453L/C455S (Fig. 4) and H453Q/C455S (data not shown), suggest that the His453 substitution attenuates but does not block succinimide formation or C-terminal cleavage, whereas the C455S substitution may help to allow normal splicing to proceed without side reactions. In vitro protein splicing was less efficient when His453 was substituted with Ala or Phe (i.e. in H453A/C455S and H453F/C455S) (Table IV). Splicing was completely blocked, and efficient DTT-induced cleavage was observed when Cys455 was substituted with Ala to block the trans-esterification in the double substitution, H453Q/C455A (discussed below). It is apparent that substituting the intein penultimate histidine residues in conjunction with the first C-extein residue provides an alternative for modulating protein splicing and converting splicing to cleavage. These results illustrate the intricate coordination of the active site residues of the Sce VMA intein in catalyzing protein splicing and the possible role of His453 in assisting succinimide formation and branch resolution.
Induction of C-terminal Cleavage and Simulation of Protein Splicing Reactions in Vitro-- The double substitution, H453Q/C455S, resulted in accumulation of a branched intermediate and in vitro splicing (Table IV) suggesting that, albeit at an attenuated rate, the H453Q substitution could still allow C-terminal succinimide formation and cleavage. However, C-terminal cleavage in the H453Q/C455A double mutant was completely blocked resulting in isolation of linear unspliced precursors (Fig. 5). As the C455A substitution rendered the mutant incapable of undergoing trans-esterification, the results suggested that by inducing N-terminal cleavage to mimic trans-esterification, we were be able to induce C-terminal cleavage. This was indeed the case when the linear precursors carrying the H453Q/C455A double substitution were treated with DTT or cysteine to generate both N-terminal and C-terminal cleavage products (Fig. 5). Unlike thiol-induced N-terminal cleavage (9), C-terminal cleavage was independent of thiols as it could occur after the removal of thiols and was inhibited at pH 6.0 but not at pH 8.0 (Fig. 5B). In addition, no C-terminal cleavage was observed before thiol treatment (Fig. 5A, lanes 1 and 2), suggesting that C-terminal cleavage occurred after the induction of N-terminal cleavage. We speculate that thiol-induced N-terminal cleavage in the H453Q/C455A double mutant triggers a conformational change in the intein structure thereby allowing succinimide formation and C-terminal cleavage to proceed at basic pH. Whether or not this reflects the actual protein splicing of the wild-type Sce VMA intein remains unanswered. Nevertheless, the induction of cleavage at both splice junctions by exogenous cysteine simulated each step of the splicing reactions as follows: the exogenous cysteine functioned as Cys455 to attack the thioester bond formed by the N-S acyl rearrangement; C-terminal cleavage occurred after the N-terminal cleavage; and the exogenous cysteine formed a covalent bond with the N-extein through an S-N acyl rearrangement (Fig. 1A) (5).
Further Examination of the Mechanism of Protein Splicing of the Sce
VMA Intein--
Studies of protein splicing of a thermophilic archeal
intein, i.e. Psp pol-1 intein, have been facilitated by the
fact that in vivo splicing of the wild-type intein in a
foreign protein context could be attenuated by low growth temperatures
(12-15 °C) (3, 27). Consequently, sufficient precursors could be
isolated to demonstrate in vitro splicing (3). The
Sce VMA intein, on the other hand, underwent rapid splicing
in vivo even at low growth temperatures (5, 7). We utilized
amino acid substitutions of catalytic residues of the Sce
VMA intein to block or attenuate the splicing process to isolate
sufficient precursors and intermediates (5). Since the mechanism of
protein splicing was deduced from analyses of mutants containing
altered catalytic residues (5, 7), little is known about the close
coupling and intricate balancing of the wild-type splicing reactions of
the Sce VMA intein. In this study, we modulated protein
splicing through appropriate substitutions of the 1 residue and the
intein penultimate residue. As a result, we were able to examine
protein splicing of the Sce VMA intein without altering its
essential catalytic residues. In particular, we focused on the effect
of pH on splicing reactions. Thiol-induced cleavage at the N terminus
proceeded efficiently at pH values ranging from 5.5 to 9.0, but the
rate of cleavage was significantly higher at pH 8.0 than pH 6.0 (Table
II) (9). Similarly, our results suggest that branch resolution (Fig. 4) and C-terminal cleavage (Fig. 5B) were inhibited at pH 6.0 but proceeded efficiently at pH 8.0-8.5. It is possible that the
nucleophilic displacements at the intein upstream and downstream splice
junctions are assisted by similar groups of residues that act as acid
or base catalysts. Completely different pH effects were observed when
in vitro splicing of the fusion precursors from
pMYK(Asn
1) was examined. The rate of splicing was higher
at pH 6.0 than pH 8.0 (Fig. 3B). As no catalytic residues of
the intein in pMYK(Asn
1) were altered, it is possible
that a similar pH profile may also apply to protein splicing of the
wild-type intein in vivo. Since it has been shown that the
N-S (or N-O) rearrangement favors ester bond formation at low pH and
amide bond formation at high pH (28, 29), the results suggest that the
rate of overall splicing is primarily determined by the N-S acyl
rearrangement. Consistent with this explanation, we have shown that
thiol-induced N-terminal cleavage (Table II), branch resolution (Fig.
4), and C-terminal cleavage (Fig. 5B) proceed more
efficiently at high pH than low pH. Therefore, both
trans-esterification and branch resolution are unlikely to be the
rate-limiting step in the protein splicing pathway. In addition, we
were able to modulate the rate of protein splicing by substituting the
1 residue that directly affects the N-S acyl rearrangement (Table
II). It has been shown that the rate of splicing for the thermophilic
Psp pol-1 intein is also favored at pH 6 and inhibited at pH
9 or above (3). Our data are consistent with the proposal that both the
thermophilic Psp pol-1 intein and the mesophilic
Sce VMA intein follow the same protein splicing pathway
(5).
Effect of Deletion and Insertion in the Sce VMA
Intein--
Previously, we deleted the central region of the
Sce VMA intein and showed that the remaining intein
structure (splicing domain) is sufficient to catalyze protein splicing
(15). In this study, we examined how the deletion might affect the
structure and catalysis of the splicing domain. Our results indicated
several differences between the deletion mutant (Y(NG)) and the
full-length intein. First, splicing efficiency of the intein deletion
mutant was affected by heterologous extein domains. For instance, when
the intein deletion mutant was fused between MBP and CBD in
MYB(NG),
more than 80% of the fusion precursors spliced in vivo
(15). However, changing the C-extein into the E. coli
thioredoxin (T) in
MYT (NG) resulted in only 50% of the precursors
splicing in vivo (data not shown). In comparison, the
full-length intein underwent complete splicing in vivo in
all fusion systems that we constructed (i.e. MYB, MYT, MYK,
and MYT4) (5). Second, the induction temperature for protein expression
affected protein splicing of the deletion mutant but not the
full-length intein. In vivo splicing of the intein deletion
mutant was efficient when induction of protein expression of
p
MYB(NG) in E. coli was conducted at 15-20 °C but was
completely blocked when the induction was at 30 °C or above (data
not shown). The full-length intein, on the other hand, catalyzed efficient splicing in vivo at induction temperatures ranging
from 15 to 37 °C (5, 9). The sensitivity of the intein deletion mutant to induction temperature could be due to the effects of the
deletion on protein folding in vivo. Consistent with this explanation, expression of p
MYB(NG) at induction temperatures of
30 °C or above resulted in only unspliced linear precursors incapable of undergoing in vitro splicing or cleavage (data
not shown). Third, the amino acid substitutions that resulted in
thiol-induced splice junction cleavage of the full-length intein had
different effects on the intein deletion mutant. The rate of
DTT-induced cleavage of the full-length intein with the N454A
substitution increased significantly upon increase of the incubation
temperature (between 4 and 23 °C) (Table II). In contrast,
DTT-induced cleavage of the deletion mutant was efficient only at
4-16 °C and was completely inhibited at 23 °C (Fig.
6A). It is possible that the deletion mutation destabilized
the structure of the deletion mutant, and consequently an increase in
temperature perturbed the alignment of catalytic residues. The active
site structure of the full-length intein, on the other hand, was more
stable, and higher temperatures simply increased the rate of
trans-esterification reaction by DTT.
Perspectives-- The work presented here has advanced our understanding of the mechanism of protein splicing. As analogous reactions have been found in the autoprocessing of some biologically important proteins, e.g. hedgehog proteins (31-33), glycosylasparaginases (34), etc., our results should enhance the understanding of the biological functions of these processes. Although the crystal structure of the excised Sce VMA intein has been solved, the active site structure at the extein-intein junctions prior to splicing has yet to be determined. It is apparent that complete characterization of the residues involved in four nucleophilic displacements of the splicing pathway requires further mutagenesis and crystallographic studies.
Protein splicing illustrates a highly specific and efficient way to cleave and religate peptide bonds. It is possible that all inteins may share a similar splicing pathway. As more inteins are being identified in various organisms (2), our study of the Sce VMA intein may reveal common strategies for modulating the rate of protein splicing, converting splicing into efficient and controllable peptide cleavage, and re-engineering inteins through deletion and insertion. The results presented in this study suggest many potential applications. The endonuclease activity of an intein is responsible for the intein-mediated gene mobility that allows site-specific insertion of an intein into an intein-less allele (12). By replacing the endonuclease domain of an intein with an endonuclease of different specificity, we can potentially redirect intein lateral transmission to a designated site. Intein insertion into a heterologous protein could be used to regulate its biological function through protein splicing. Our studies of the modulation of protein splicing and the effect of the ![]() |
ACKNOWLEDGEMENTS |
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We thank George A. Garcia, William E. Jack, Ira Schildkraut, Donald G. Comb, Elisabeth A. Raleigh, Jack Benner, Eric Cantor, and Fana B. Mersha, Richard D. Whitaker, Maurice W. Southworth, and Francine B. Perler for valuable discussions and reading of the manuscript; Jurate Bitinaite and Rebecca B. Kucera for the gifts of BamHI antisera and E. coli strain pLD2263/m.bamH1.
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Note Added in Proof |
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The crystal structure of GyrA
intein has recently been solved that showed the active site structure
of the intein plus Ala as the 1 residue (35).
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FOOTNOTES |
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* This work was supported by New England Biolabs.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence may be addressed: New England Biolabs, 32 Tozer Rd., Beverly, MA 01915. Tel.: 978-927-5054 (ext. 324 or 372);
Fax: 978-921-1350.
1
The abbreviations used are: C-extein, intein
C-terminal flanking region; N-extein, intein N-terminal flanking
region; M or MBP, maltose-binding protein; Sce VMA intein,
intein from the vacuolar ATPase subunit of S. cerevisiae; Y,
full-length Sce VMA intein; Y, the Sce VMA
intein with a deletion mutation;
Y(Bam), the
Sce VMA intein whose endonuclease domain is replaced by
restriction endonuclease BamHI; CBD or B, chitin-binding
domain; K, phage T4 polynucleotide kinase; T4, phage T4 DNA ligase; MK,
the ligated exteins; PAGE, polyacrylamide gel electrophoresis; DTT,
1,4-dithiothreitol; GFP, green fluorescent protein; X
1,
one of 20 amino acid residues; VMA, vacuolar membrane ATPase.
2 S. Chong, unpublished results.
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REFERENCES |
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