From the Center for Macromolecular Design, Institute
of Biosciences and Technology, and the ** Department of Biochemistry and
Biophysics, Texas A & M University, Houston, Texas 77030 and the
§ Structural and Computational Biology and Molecular
Biophysics Program, Howard Hughes Medical Institute and Department of
Biochemistry, Baylor College of Medicine,
Houston, Texas 77030
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ABSTRACT |
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A structure-based model describing the interaction of the two-domain PI-SceI endonuclease with its 31-base pair DNA substrate suggests that the endonuclease domain (domain II) contacts the cleavage site region of the substrate, while the protein splicing domain (domain I) interacts with a distal region that is sufficient for high affinity binding. To support this model, alanine-scanning mutagenesis was used to assemble a set of 49 PI-SceI mutant proteins that were purified and assayed for their DNA binding and cleavage properties. Fourteen mutant proteins were 4- to >500-fold less active than wild-type PI-SceI in cleavage assays, and one mutant (T225A) was 3-fold more active. Alanine substitution at two positions in domain I reduces overall binding >60-fold by perturbing the interaction of PI-SceI with the minimal binding region. Conversely, mutations in domain II have little effect on binding, reduce binding to the cleavage site region only, or affect binding to both regions. Interestingly, substitutions at Lys301, which is part of the endonucleolytic active site, eliminate binding to the cleavage site region but permit contact with the minimal binding region. This experimental evidence demonstrates that the protein splicing domain as well as the endonuclease domain is involved in binding of a DNA substrate with the requisite length.
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INTRODUCTION |
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The yeast PI-SceI endonuclease catalyzes the hydrolysis of two specific phosphodiester bonds within an asymmetrical recognition site (1). This enzyme is a homing endonuclease (for a review, see Ref. 2) that occurs as an intein situated within an H+-ATPase protein subunit. Like other homing endonucleases, PI-SceI recognizes an extremely long sequence (31 bp)1 and cuts DNA to yield 5'-phosphate and 3'-hydroxyl ends (3, 4). Mutagenesis and biochemical studies indicate that the PI-SceI recognition sequence can be divided into two regions (4, 5). Region I contains the cleavage site that is cut by the enzyme to generate a 4-base pair overhang, and region II includes an adjacent 17-bp sequence (the minimal binding sequence) that is sufficient for high affinity binding. Mutagenesis of the substrate reveals that PI-SceI tolerates substitutions at numerous positions, since substitutions at only nine positions in the substrate lead to severely reduced activity (4). Like the other homing endonucleases that have been studied, PI-SceI requires Mg2+ as a cofactor. The metal ion is likely to be required for the hydrolytic reaction, since it is required for catalysis but not for specific binding. Mn2+ can substitute for Mg2+, and it stimulates more efficient cleavage by the enzyme at cognate and noncognate sites (1, 5).
The three-dimensional structure of PI-SceI has been recently
determined by x-ray crystallography and reveals a bipartite domain structure (6). Domain I contains the protein splicing active site,
which is composed of the N- and C-terminal amino acids and two other
His residues that have been shown to be required for activity or have
been implicated in the reaction (7, 8). The residues that compose the
putative endonucleolytic active site, a lysine (Lys301) and
two aspartic acid residues (Asp218 and Asp326),
are present in domain II and form a catalytic triad that displays structural similarity to charged clusters found in restriction enzymes
(6, 9). By using the PI-SceI structural information and the
knowledge that the enzyme contacts two discrete regions of the
recognition sequence, a model for the docking of PI-SceI with its substrate was constructed where domains I and II of the protein contact regions II and I, respectively, of the substrate (Fig.
1). In this model, both domains are
proposed to contact the substrate, since the binding surface on the
endonuclease domain alone is insufficient to contact the entire 31-bp
recognition sequence. A bend of ~55° was introduced into the middle
of the substrate to accommodate the angular orientation of the two
domains with respect to each other, and experimental evidence confirms the existence of this distortion (4, 5). Furthermore, the scissile
bonds of the DNA were placed in close proximity to Asp218
and Asp326, which are thought to bind the Mg2+
co-factor. Two symmetry-related -sheets (sheets 7 and 9) in domain
II that flank the active site aspartic acid residues may serve as
platforms that contact the cleavage site region. Furthermore, we
speculate that a pair of
-hairpin loops between
15 and
16 and
between
21 and
22 that lie above the sheets contain amino acids
whose side chains mediate substrate binding. The interaction of domain
I with region II of the substrate may involve a cluster of positively
charged amino acids situated along the same face of PI-SceI
as the endonucleolytic active site. The structure of a second homing
endonuclease, I-CreI, was recently reported, and a model for
its binding to DNA bears similarity to that proposed for
PI-SceI (10). I-CreI is a homodimeric protein
that resembles domain II of PI-SceI, but it lacks the
protein splicing domain. Like PI-SceI, I-CreI
contains a set of
-sheet structures with interconnecting extended
loops that are proposed to form the protein interface that binds the
DNA substrate.
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To determine the identity of amino acid residues involved in contacting the PI-SceI recognition sequence, we used alanine-scanning mutagenesis to create a set of mutant proteins with single amino acid changes at numerous positions on the proposed DNA binding interface. These mutant proteins were purified and assayed for their substrate cleavage and DNA binding activities. The major finding of the work is that residues in both domains mediate DNA binding. Moreover, the binding behaviors of wild-type PI-SceI and several mutants provide compelling evidence for a high affinity interaction between domain I and the minimal binding region and for a substantially weaker association between domain II and the cleavage site region.
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EXPERIMENTAL PROCEDURES |
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Materials-- TALON metal affinity resin and TALONspin columns were obtained from CLONTECH. All oligonucleotides were synthesized by Genosys Biotechnologies, Inc.
Mutagenesis of PI-SceI Gene-- Wild-type and mutant PI-SceI proteins were expressed from plasmid pET PI-SceI C-His, which encodes a 479-amino acid PI-SceI derivative containing a polyhistidine C-terminal extension that facilitates rapid protein purification by metal affinity chromatography. To construct pET PI-SceI C-His, PCR mutagenesis (11) was used to insert six silent restriction sites (SpeI, ApaI, BssHII, BstEII, BsiWI, and MluI sites at positions 243, 406, 484, 717, 813, and 943, respectively, relative to the first codon) into plasmid pET23PI-Sce ESARC (9) to generate plasmid pET23PI-Sce-9. Plasmid pET23PI-Sce-9 was used as a template in a PCR reaction with two oligonucleotides (5'-TTCGGATCCGCGACCCATTTTGCATGGACGACAACCT-3' and 5'-CGGTACGCGTGAAACATTTCTG-3') to generate a 449-bp fragment. This product was digested with MluI and BamHI and ligated into MluI/BamHI-digested pET23PI-Sce-9 DNA to create pET PI-SceI C-His. The entire PI-SceI coding region of pET PI-SceI C-His was confirmed by DNA sequence analysis. Omitting the N-terminal methionine residue, pET PI-SceI encodes a 479-amino acid PI-SceI derivative with a C-terminal tail having the sequence KWVADPNSSSVDKLAAALEHHHHHH-COOH. Protein splicing-mediated cleavage of the C-terminal affinity tag was prevented by substituting Asn454 with alanine. To introduce mutations into the PI-SceI coding sequence, oligonucleotide primers were used in either cassette mutagenesis or two-step overlapping PCR amplification protocols (11). All introduced mutations and inserted sequences were confirmed by dideoxy sequencing.
Expression and Purification of PI-SceI Proteins--
Plasmid pET
PI-SceI C-His encoding wild-type or mutant
PI-SceI proteins was transformed into Escherichia
coli strain BL21 (DE3). For most of the mutant proteins
characterized, a 200-ml culture was grown in LB medium (1%
Bacto-tryptone, 0.5% Bacto-yeast extract, 0.5% NaCl, 1 mM
NaOH) containing ampicillin (100 µg/ml) at 37 °C to an
A600 of 0.6-0.8. Expression of
PI-SceI protein was induced with 0.5-1.0 mM
isopropyl-1-thio--D-galactopyranoside, and growth was
continued overnight at 15 °C. The cells were harvested by
centrifugation, resuspended in 2 ml of sonication buffer (20 mM Tris-Cl (pH 8.0), 300 mM KCl, 10 mM MgCl2, 5% glycerol, 1 mM phenylmethylsulfonyl fluoride) containing 1 mM imidazole,
and lysed by sonication (3 × 1 min) at 4 °C. All further
manipulations were performed at 4 °C. Cell debris was pelleted by
centrifugation at 10,000 × g for 15 min. The clarified
lysate was applied to a TALON spin column (0.5 ml of TALON metal
affinity resin) pre-equilibrated with sonication buffer, and the metal
affinity columns were inverted for 5 min and centrifuged at 700 × g for 2 min. The resin was washed twice with 1 ml of
sonication buffer containing 1 mM imidazole, and
PI-SceI was eluted from the columns with sonication buffer containing 300 mM imidazole. Elution fractions containing
PI-SceI, as judged by SDS-polyacrylamide gel
electrophoresis, were pooled and dialyzed overnight in buffer D (10 mM potassium phosphate (pH 7.6), 5% glycerol, 0.1 mM EDTA, and 1.4 mM 2-mercaptoethanol) containing 40 mM KCl (buffer D40). The dialyzed protein was
applied to a 1-ml SP-Sepharose column equilibrated with buffer D40, the resin was washed with 2.5 ml of buffer D40, and protein was eluted with
10 × 1 ml of buffer D450. Elution fractions containing purified PI-SceI were pooled and stored in storage buffer (10 mM potassium phosphate (pH 7.6), 50 mM KCl, 2.5 mM 2-mercaptoethanol, and 50% glycerol) at
20 °C. For
some PI-SceI proteins, similar protocols were used to purify
the enzyme from 1-liter cultures. The PI-SceI proteins were
purified to greater than 95% as judged by SDS-polyacrylamide gels. The
affinity-tagged PI-SceI (Mr = 53,800)
concentration was determined using the extinction coefficient of
5.03 × 104/M/cm as determined by
published methods (12). The wild-type protein had the same specific
activity as native
PI-SceI.2
Native Gel Mobility Shift Assay of DNA Binding-- To detect protein-DNA complexes in DNA mobility shift analyses, a 219-bp duplex DNA fragment containing a single PI-SceI recognition site was synthesized by PCR and labeled with [32P]ATP as described previously (4). Nonspecific binding was measured using a 189-bp duplex DNA fragment that was identical in all respects except that it lacked the PI-SceI recognition site. Each reaction mixture (20 µl) contained 25 mM Tris-HCl (pH 8.5), 100 mM KCl, 10% glycerol, 50 µg/ml bovine serum albumin, 2.5 mM 2-mercaptoethanol, 5 fmol of 219-bp substrate (5'-32P-labeled at both ends), and PI-SceI as specified and was incubated at 25 °C for 10 min. The samples were subjected to electrophoresis through a 7% native polyacrylamide gel in 0.5 × TBE at 210 V for 5 min and then at 120 V for 2-4 h at 4 °C. The amounts of bound and unbound substrate were determined using a PhosphorImager and FragmeNT Analysis software (Molecular Dynamics, Inc.). Autoradiographic exposure of the dried gel to film was used to visualize the unbound DNA and the PI-SceI-DNA complexes.
The PI-SceI reaction pathway can be described by Scheme I (Fig. 2), where the free protein (Pf) and DNA (Df) interact to form the lower protein-DNA complex (PDLC) that involves PI-SceI contacts to region II of the substrate (4, 5, 9). PDLC is in equilibrium with the upper complex, PDUC, where PI-SceI contacts both regions I and II of the substrate. A second pathway for PDUC formation is possible involving a complex where PI-SceI contacts region I only (PDx), but this complex has not been observed. The PDUC complex binds Mg2+ and forms the putative pentavalent phosphate transition state that undergoes double-stranded scission. PI-SceI is proposed to remain tightly bound to the region II cleavage product following the reaction.
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(Eq. 1) |
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(Eq. 2) |
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(Eq. 3) |
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(Eq. 4) |
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(Eq. 5) |
PI-SceI Cleavage Analysis-- In an initial characterization of the PI-SceI proteins, purified enzyme (50-150 nM) was incubated with XmnI-linearized pBS-PISce36 (7 nM) (4) in 15 µl of cleavage buffer (100 mM KCl, 25 mM Tris-HCl (pH 8.5), 2.5 mM 2-mercaptoethanol, 2.5 mM MgCl2) for 30 min and 1 h at 37 °C. On the basis of these assays, mutant proteins that were determined to be partially or fully defective in cleavage activity were assayed with purified PI-SceI proteins (100 nM) under the same conditions for various lengths of time. Reactions were terminated by the addition of 5 µl of stop buffer (5 mM Tris-HCl (pH 7.5), 10 mM EDTA, 0.05% (w/v) SDS, 2.5% (w/v) Ficoll). Samples were subjected to electrophoresis in 1 × TBE on a 0.9% agarose gel, which was stained with ethidium bromide and photographed. The amounts of undigested plasmid DNA and the two cleavage products were determined using a scanning densitometer (Molecular Dynamics). Cleavage rates were calculated from curve fitting of the linear portions of the reaction using KaleidaGraph (Synergy Software).
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RESULTS |
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Mutagenesis of PI-SceI--
To identify the amino acid residues
that participate in substrate binding, we introduced amino acid
substitutions into the domain II platform and loop regions and into the
positively charged region of domain I that is predicted from the model
to contact the DNA. In domain II, substitutions were made in 14,
15, and
16 in one of the two symmetry-related platforms and in
19,
20,
21, and
22 in the other (Table
I). Substitutions were also made at the
active site at Lys301 and Pro304, two highly
conserved residues situated in block D, a conserved motif found in
homing endonucleases and maturases (7, 8). In domain I, substitutions
were introduced at amino acids Arg90, Arg91,
Arg94, and Lys97, which comprise the cluster of
positive charges thought to bind the DNA. In general, amino acid
residues with side chains containing putative hydrogen bond donors or
acceptors were targeted, since hydrogen bonds are frequently important
components of protein-DNA interactions. Alanine substitutions were
introduced, since this residue lacks hydrogen bond partners and it
would be expected to exert minimal steric or electrostatic effects on
structure. To investigate the effect of charge changes at
Lys301, substitution was made at this position with
arginine, which maintains the positive charge, and with glutamic acid,
which introduces a negative charge.
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Characterization of DNA Cleavage Activity of Mutant Proteins-- The 48 mutant proteins that were successfully purified were tested for their ability to cleave a PI-SceI recognition site on linearized plasmid pBS-PISce36. In initial experiments designed to quickly identify mutant derivatives that were partially or completely defective in cleavage activity, an approximately 50-150 nM concentration of PI-SceI protein was incubated with a 7 nM concentration of linearized substrate under standard reaction conditions in buffer containing MgCl2. Table I shows that 34 of 48 mutant proteins tested had at least 25% of the activity of wild-type PI-SceI. These results reveal that mutations can be made at numerous positions in the protein proximal to the active site with little or no effect on activity. Of the remaining mutants examined, 12 (R90A, R94A, D229A, R231A, D232A, K301R, Y328A, T338A, K340A, H343A, K369A, and H377A) were partially active (activity levels less than 25% of wild-type activity) and two displayed no activity (K301A and K301E). Surprisingly, one mutant protein (T225A) was at least 3 times more active than wild-type PI-SceI.
More detailed rate experiments were performed for the partially or fully defective proteins and for the enhanced activity protein in reaction buffers containing either MgCl2 or MnCl2. These experiments were carried out under single turnover conditions (excess enzyme relative to substrate). Steady state conditions could not be achieved, since PI-SceI remains tightly bound to one of the two cleavage products (5, 9), yielding a low turnover number. The amount of linearized substrate that was cleaved to form the two products was measured as described under "Experimental Procedures," and the cleavage activities are shown in Table II. In the buffer containing Mg2+, 25% of the substrate was cleaved by wild-type PI-SceI in approximately 5 min. By contrast, for two of the mutant proteins (K301A and K301E), no cleavage activity was apparent after 4 h of incubation, and for two others (D229A and K340A), only trace amounts of cleavage products were detected. The reaction rates for these mutant proteins were too slow to measure accurately, and we estimate that their activities are at least 500 times lower than that of wild-type PI-SceI. Of the remaining 10 defective mutants, four were >20-fold less active than wild-type PI-SceI (R90A, R94A, Y328A, and H377A), and five were 4-20-fold less active (R231A, D232A, K301R, T338A, H343A, and K369A). The PI-SceI protein with enhanced activity, T225A, cleaved the DNA substrate over 3 times faster than the wild-type protein in the presence of Mg2+.
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DNA Binding Properties of the Mutant Proteins-- To test whether defects in substrate binding by the mutant proteins account for the reduction in cleavage rates, gel shift analyses were performed as described under "Experimental Procedures" using a 219-bp linear fragment containing a single PI-SceI site. Wild-type PI-SceI forms two complexes with this substrate in the absence of metal ion co-factor; a lower complex (PDLC) in which the protein binds solely to a 17-bp minimal binding region distal to the cleavage site (region II) and an upper complex (PDUC) in which PI-SceI binds to both the minimal binding region and to the cleavage site region (region I). Fig. 3 shows that wild-type PI-SceI forms both complexes in the binding experiment. In this report, we used the data to measure two equilibrium dissociation constants, K1 and K2, that describe PI-SceI binding to its substrate (see "Experimental Procedures"). Overall binding can be expressed as the product of these parameters and is approximately 0.7 nM (Table III). As suggested previously (4, 5, 9), it appears that the major contributing factor to this tight affinity stems from the interaction of PI-SceI with region II of the substrate, since K1 is only about 10-fold higher than K1 × K2. The high value of K2, which reflects the partitioning between the lower and the upper complexes, suggests that the binding energy released by the interaction of domain II with the DNA is used to stabilize the energetically unfavorable distorted DNA conformation that is present in the upper complex. Furthermore, as predicted from the model, the ratio of the upper and lower complexes, which reflects K2, is independent of protein concentration.3 The equilibrium dissociation constant for binding of PI-SceI to a nonspecific DNA fragment of similar size is over 300-fold lower than to the specific probe (~200 nM compared with 0.67 nM).4
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DISCUSSION |
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In this report, we employed alanine-scanning mutagenesis to generate mutations in regions of PI-SceI endonuclease that are believed to contact the DNA substrate. This type of strategy has been successfully used to probe protein-DNA recognition for several other DNA-binding proteins, including the Arc repressor (13) and the E. coli Tyr B protein (14). Alanine-scanning mutagenesis has the advantage of only substituting a single methyl group for the wild-type side chain, which effectively removes any important functional group that is normally present. Random mutagenesis followed by genetic selection is more likely to cause a loss-of-function phenotype by introducing a deleterious moiety that alters the protein conformation. A drawback of alanine-scanning mutagenesis is that unless a complete mutagenesis profile is performed for a given protein, there is the possibility that functionally important residues may not be tested. It is also possible that main chain functional groups contribute to binding free energy, which would not be probed by our strategy.
In the absence of a crystal structure that includes the DNA substrate, it remains unclear whether the functionally important residues identified here by mutation act directly by removing a critical contact or indirectly by modifying the protein conformation. However, these mutations probably do not cause any gross structural perturbations, since all of the mutant proteins could be purified in soluble form using the same procedures as for wild-type PI-SceI, suggesting they are correctly folded. Furthermore, and most importantly, in the presence of Mn2+, all of the mutant proteins are active to some degree, with some being nearly as active as wild-type PI-SceI.
According to Scheme I (Fig. 2), mutations that modify cleavage activity can exert their effects by altering the catalytic machinery of the protein (i.e.. they can affect k1) and/or by affecting the substrate binding determinants (they can affect K1-K4). We show here that there is a good correlation between the decrease in the level of cleavage activity and the decrease in substrate binding, suggesting that binding interactions have been disrupted. The mutants that display the lowest levels of cleavage activity, i.e. R90A, R94A, D229A, K301A, K301E, Y328A, K340A, and K377A, yield either little or no apparent PDUC complex or produce complexes that migrate faster than that of the wild-type enzyme. The absence of the PDUC complex suggests that important contacts near the cleavage site have been disrupted and that no interaction occurs between PI-SceI and region I, while the appearance of faster migrating complexes indicates possible conformational differences in the complex that may affect the cleavage activity. The T225A mutant, which is approximately 3-fold more active than wild-type PI-SceI, has a K1 × K2 value similar to wild type, but we cannot rule out a small binding enhancement.
The main finding of this report, that both PI-SceI domains
contact the recognition sequence, is supported by consideration of the
thermodynamic binding parameters of the various mutant enzymes together
with the positions of the substituted residues in the crystal
structure. Fig. 4 shows an overview of
the entire protein that indicates the positions of the amino acids
where mutations lead to a loss or gain of activity. Two domain I
residues, Arg90 and Arg94, are strong
candidates for amino acids that contact region II, since proteins with
mutations at these positions have K1 × K2 values that are significantly higher than that of
wild-type PI-SceI. Residue Arg90 is exposed to
solvent and lies on the same face of the protein as the active site in
domain II, which might be expected if both regions contact the DNA.
Little can be concluded from the positioning of residue
Arg94 since it is part of a disordered loop in the crystal
structure, but it is in the same vicinity as Arg90. The
residues in domain II that alter activity cluster in groups that
neighbor the active site. For example, the Tyr328,
Lys340, and Thr338 side chains are in close
proximity in the crystal structure. The Tyr328 phenolic
group and the -amino group of Lys340 are situated within
4 Å of one another and extend upward into the solvent-exposed region
above the platform formed by
-sheet 9 that is thought to contain the
DNA (Fig. 5A). The Y328A
protein exhibits a ~25-fold reduction in cleavage activity that
probably results in part from its reduced DNA binding affinity.
However, binding defects alone cannot account for the large reduction
in cleavage activity of the Y328A mutant, and there may be effects on
catalysis as well. Even more striking is the nearly total absence of
activity of the Lys340 mutant, which can be easily
accounted for by its binding defect. According to our model (Fig. 1),
it might be predicted that PI-SceI domain I and domain II
mutations affect K1 and
K2, respectively. Within domain I, this
prediction is borne out by the R90A and R94A proteins. However, Y328A
is an example of a domain II substitution that alters
K1, which suggests that rather than being
independent, the domains communicate with each other. Alanine
substitution at the third residue in this group, Thr338,
increases the K2 value to unity, resulting in
equal partitioning between the complexes. The Thr338 side
chain is not solvent-exposed and would not be expected to contact the
substrate. In the other half of the binding platform, which originates
from
-sheet 7, the Thr225 side chain also extends above
the platform surface (Fig. 5B). Removal of most of the
threonine side chain by alanine substitution does not have a major
effect on binding. Residues His343 and His377
are located above one another in two loops that are part of an extended
structure that rises above one side of the active site. Both
2
nitrogens are pointed toward the opening above the active site where
the DNA is thought to be located (Fig. 5B). The behavior of
the H377A mutant protein nicely fits our model, since it yields no
PDUC complex (high K2 value), and is
>50-fold reduced in activity compared with wild-type
PI-SceI. Somewhat surprisingly, the
K1 value is nearly 10-fold higher compared with
wild-type PI-SceI, which again suggests synergy between the
two domains. The Lys369 residue is situated in the same
loop as His377, but its orientation is uncertain due to
disorder in the structure. However, stereochemical refinement of the
structure indicates hydrogen bonding between the Lys369 and
Lys340
-amino groups, and the K369A substitution may
affect the structure of the binding platform. Diametrically opposite to
His343 and His377 on the other side of the
active site are residues Asp229, Arg231, and
Asp232, which form a tight cluster where the side chains
are oriented toward the putative substrate binding cavity. A hydrogen
bond exists between the Arg231
-guanidino group and the
Asp229 carboxyl group. The binding constants for the D229A
mutant could not be accurately determined, but it is clear that the
large decrease in cleavage activity cannot be accounted for solely by
reductions in overall binding (Fig. 3). What is certain is that the
mutation alters the mobility of the PDUC complex, which may
indicate conformational differences in the DNA. Alternatively, as with
any of the mutants described here, there may be conformational changes
in the catalytic center that affect activity.
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The PI-SceI mutants containing substitutions at
Lys301 fall into a separate category, since this amino
acid, unlike the other residues characterized here, is highly conserved
among homing endonucleases (7, 8) and, together with Asp218
and Asp326, forms a "catalytic triad" that comprises
the PI-SceI active site (6). Similar clusters of two acidic
residues and a lysine residue are found at the active sites of several
restriction endonucleases (15). Lys301 is situated at the
C-terminal end of 18 in the PI-SceI crystal structure,
and the side chain extends into the putative substrate binding cavity
that is also occupied by the two aspartic acid side chains (Figs. 4 and
5). Substitution of Lys301 with alanine or glutamic acid
dramatically increases K2 and consequently eliminates all activity. Similar substitutions at Lys92 of
EcoRV, which may be an analogous residue to
Lys301, reduce substrate binding and cleavage activities
(16). The basic character of the Lys301 side chain is
critical for the PI-SceI binding interaction, since a K301R
mutant is partially active in binding and cleavage assays. By contrast,
arginine substitution at Lys92 of EcoRV
abolished DNA cleavage activity with either Mg2+ or
Mn2+ (17). We also found that cleavage activity of the
PI-SceI K301A and K301E mutants could be partially rescued
by Mn2+ (Table II). A similar effect was observed for the
EcoRV K92E mutant protein but not for the K92A protein,
which led to speculation that the binding of a second Mn2+
ion to the Glu residue restored the positive charge normally contributed by the Lys92 side chain. This is unlikely to be
the case for PI-SceI, since we observe rescue of activity to
the K301A mutant as well. In fact, the activity of all of the mutant
proteins is partially rescued by substitution of Mn2+ for
Mg2+. It is also worth noting that a set of substrate
mutants that are catalytically inactive in Mg2+ also have
activity restored by the presence of Mn2+ (4). Similar
instances of activity "rescue" by Mn2+ have been
observed with EcoRV mutants that have low levels of activity
in Mg2+ but have nearly wild-type activity levels in
Mn2+ (16, 18). However, unlike the restriction enzymes,
PI-SceI normally displays greater activity in the presence
of Mn2+ than with Mg2+. One EcoRV
mutant has been identified for which this is also the case (19). Taken
together, our data are consistent with the Lys301 side
chain establishing an important binding contact within region I,
perhaps to a phosphate oxygen near the scissile phosphodiester bond.
The substrate binding properties of the protein mutants characterized
here complement those of a set of loss-of-function DNA substrate
mutants that contain substitutions in regions I and II. Point mutations
at positions A+16, G+18, and A+19
in region II dramatically reduce all binding to wild-type
PI-SceI (4). According to our model, these base pairs are
located in the same general vicinity as the R90A and R94A mutant
proteins, which display similar binding defects. By contrast,
substitutions in the PI-SceI substrate near the cleavage
site at positions A9, T
1, G+1,
G+3, and G+4 only eliminate PDUC
complex formation or produce a complex that migrates faster than that
of wild-type PI-SceI (4). These binding properties are similar to those of some domain II mutants described here. Thus, there
is a good correlation between the DNA binding properties of both the
substrate and protein mutants that strongly supports the conclusions of
the PI-SceI docking model. However, a convincing demonstration that these proposed interactions occur must await the
determination of the PI-SceI structure complexed to its
recognition site.
The results presented here are the first to show that the PI-SceI protein splicing domain is involved in site-specific substrate binding. We hypothesized that the PI-SceI intein gene originally arose by the fusion of two pre-existing genes, one that encoded an endonuclease and the other that encoded a splicing protein (6). Surprisingly, the recently determined structure of the autoprocessing domain of the Drosophila Hedgehog protein is very similar to domain I of PI-SceI, but it lacks the PI-SceI DNA recognition region. Instead, it contains an unrelated region that binds cholesterol (20). This suggests that the protein splicing domain existed previously as a core protein that acquired new functions in different instances by associating with new sequences (20). In the case of PI-SceI, it raises the possibility that the DNA binding region was acquired after the intein was assembled. Presumably, the acquired ability of the PI-SceI intein to make base-specific interactions to two distant regions of its recognition site provided increased selectivity to the enzyme.
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FOOTNOTES |
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* This work was supported by National Institutes of Health (NIH) Grant GM50815 (to F. S. G.), by funds from the Institute of Biosciences and Technology (to F. S. G.), and funds from the Offices of Research and Information Technology of Baylor College of Medicine (to F. A. Q.).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.
¶ Supported by a National Institutes of Health NIGMS Grant GM08280 to the Houston Area Molecular Biophysics Program.
An Investigator of the Howard Hughes Medical Institute.
To whom correspondence should be addressed: Center for
Macromolecular Design, Inst. of Biosciences and Technology,
2121 W. Holcombe Blvd., Houston, TX 77030. Tel.: 713-677-7605; FAX:
713-677-7641; E-mail: fgimble{at}ibt.tamu.edu.
1 The abbreviations used are: bp, base pair(s); PCR, polymerase chain reaction; TBE, Tris borate-EDTA.
2 F. S. Gimble, unpublished results.
3 Z. He and F. S. Gimble, unpublished results.
4 M. Crist and F. S. Gimble, unpublished results.
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