From the Institute of Biomedical Sciences and the
¶ Institute of Molecular Biology, Academia Sinica, Taipei 115, and the § Department of Life Science, National Tsing-Hua
University, Hsin-Chu 300, Taiwan
Received for publication, July 6, 2002, and in revised form, December 19, 2002
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ABSTRACT |
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The Rana catesbeiana
(bullfrog) ribonucleases, which belong to the RNase A superfamily,
exert cytotoxicity toward tumor cells. RC-RNase, the most active among
frog ribonucleases, has a unique base preference for pyrimidine-guanine
rather than pyrimidine-adenine in RNase A. Residues of RC-RNase
involved in base specificity and catalytic activity were determined by
site-directed mutagenesis, kcat/Km analysis toward
dinucleotides, and cleavage site analysis of RNA substrate. The results
show that Pyr-1 (N-terminal pyroglutamate), Lys-9, and Asn-38
along with His-10, Lys-35, and His-103 are involved in catalytic
activity, whereas Pyr-1, Thr-39, Thr-70, Lys-95, and Glu-97 are
involved in base specificity. The cytotoxicity of RC-RNase is
correlated, but not proportional to, its catalytic activity. The
crystal structure of the RC-RNase·d(ACGA) complex was determined at
1.80 Å resolution. Residues Lys-9, His-10, Lys-35, and His-103
interacted directly with catalytic phosphate at the P1
site, and Lys-9 was stabilized by hydrogen bonds contributed by Pyr-1,
Tyr-28, and Asn-38. Thr-70 acts as a hydrogen bond donor for cytosine
through Thr-39 and determines B1 base specificity. Interestingly, Pyr-1 along with Lys-95 and Glu-97 form four hydrogen bonds with guanine at B2 site and determine B2
base specificity.
Ribonucleases are found widely within living organisms and are
thought to play an important role in the metabolism of RNA. Recently,
it has been shown that several members of the bovine ribonuclease
superfamily exhibit biological functions in addition to intrinsic
ribonucleolytic activities. Human eosinophil-derived neurotoxin
and eosinophil cationic protein exert neurotoxicity (1) as well as
antiparasitic activity (2), human angiogenin induces blood vessel
formation (3), and frog ribonuclease exhibits antitumor activity (4,
5). Ribonucleolytic activity is essential for the biological functions
of these proteins (6-12).
Bovine pancreatic ribonuclease, known as RNase A, in the ribonuclease
superfamily is well characterized and is a valuable model for the study
of structure-function relationships and protein refolding (13, 14). It
consists of 124 amino acid residues linked with four pairs of disulfide
bridges and possesses a substrate preference for pyrimidine-adenosine
in the RNA sequence but no cytotoxicity toward tumor cells. There are
three subsites within RNase A molecule: the P1 site, at
which phosphodiester bond cleavage occurs; the B1 site, for
binding pyrimidine, which donates oxygen via its ribose to the scissile
bond; and the B2 site, for binding the adenine ring on the
opposite site of the scissile bond. Three amino acid residues, His-12,
Lys-41, and His-119, at the P1 site are involved in
catalytic activity. Four amino acid residues, Thr-45, Asp-83, Phe-120,
and Ser-123, at the B1 site are involved in the binding of
the 5'-ribonucleoside, pyrimidine, whereas two residues, Asn-71 and
Glu-111, at the B2 site are involved in the binding of
the 3'-ribonucleoside, adenosine (14-18).
A new group of ribonucleases with antitumor activity has been found
mainly in frog, i.e. onconase from Rana pipiens
(9), RC-RNase, RC-RNase 2~RC-RNase 6, and RC-RNase L1 from Rana
catesbeiana (19), and a sialic acid-binding lectin from Rana
japonica (20). These frog ribonucleases are composed of 104-111
amino acid residues, which are similar to mammalian ribonucleases in
their amino acid sequence. The conserved amino acid residues for the
catalytic activity, i.e. His-12, Lys-41, and His-119 in
RNase A, are also found in frog ribonucleases. However, some distinct
properties are found mainly in frog ribonucleases rather than in
mammalian ribonucleases, i.e. cytotoxicity toward tumor
cells, substrate preference for pyrimidine-guanine, the presence of
pyroglutamate at the N terminus, a specific location of the fourth
disulfide bridge, and resistance to ribonuclease inhibitor from human
placenta (4-6).
To investigate the novel properties of frog ribonucleases, we mutated
the residue potentially involved in the catalytic activity and
substrate specificity of RC-RNase based on the alignment of amino acid
sequences in the ribonuclease superfamily (Fig.
1) and the known structures of frog
ribonucleases, e.g. onconase (21) and RC-RNase (1KM8). Our
results show that the N-terminal pyroglutamate
(Pyr-1),1 Lys-9, and Asn-38
along with the conserved His-10, Lys-35, and His-103 residues are
involved in the catalytic activity of RC-RNase, whereas the residues
Thr-39 and Thr-70 are involved in B1 base recognition and
Pyr-1, Lys-95, and Glu-97 are involved in B2 base recognition. The cytotoxicity of RC-RNase is correlated, but not proportional to, its catalytic activity. To elucidate the molecular interaction between frog ribonuclease and possible RNA targets in tumor
cells, we grew the co-crystal of RC-RNase and substrate analog d(ACGA)
and illustrated the possible hydrogen bonds for the specific
recognition of RC-RNase on RNA substrate.
Construction of Expression Vector--
The cDNA of wild type
RC-RNase was cloned from bullfrog liver using a SmartTM
rapid amplification of cDNA ends kit from
Clontech (Palo Alto, CA) as described previously
(6). Various substitutional and deletional mutants were constructed
based on the alignment of amino acid sequence in the ribonuclease
family (Fig. 1) and the structure of frog ribonucleases,
e.g. onconase (21) and RC-RNase (1KM8). These mutants
included RC-Q1A, RC-Q1E, RC-Q1N, RC-Q1S, RCN2D, RC-K9A, RC-K9Q,
RC-T39A, RC-T39S, RC-N57A, RC-N59A, RC-T70A, RC-T70D, RC-T70Q, RC-T70S,
RC-K95A, RC-K95R, RC-E97A, RC-V102A, RC-V102D, RC-F104A,
dN2, and dQNW1-3, respectively. The RC-RNase
gene and its variants were inserted downstream of a modified pelB signal peptide sequence in a pET11d vector (6). The DNA sequences of mutated ribonuclease genes were analyzed by a PerkinElmer Life Sciences 377 automated DNA sequencer using the dideoxy chain termination method from a d-Rhodamine Terminator Cycle Sequencing Ready
Reaction kit (PerkinElmer Life Sciences).
Expression and Purification of Recombinant
Proteins--
Recombinant ribonuclease genes subcloned in a pET11d
containing the modified pelB signal peptide were transformed
into the Escherichia coli BL21(DE3) strain and cultured at
34 °C for 3 days. After centrifugation, the culture medium
containing secreted ribonuclease was concentrated by polyethylene
glycol 20,000 absorption and dialyzed against buffer A (20 mM Hepes, pH 7.9, 0.5 mM EDTA). The crude
samples were then purified by phosphocellulose and
carboxymethyl-cellulose chromatographies, and eluted with 0-0.4
M and 0-0.2 M KCl gradient in buffer A,
respectively (6).
CD and Mass Spectrum Analysis--
CD experiments were carried
out using an Aviv CD 202 spectrometer (AVIV, Lakewood, NJ) calibrated
with (+)-10-camphorsulfonic acid at 298 K. In general, a 2-mm path
length cuvette with 10-20 µM ribonuclease in 20 mM Na2HPO4, pH 7.2, was used for CD
experiments, and all protein solutions were made up to 1 ml. The
spectra were recorded from 180 nm to 260 nm. After background
subtraction and smoothing, all CD data were converted from CD signal
(millidegree) into mean residue ellipticity (degrees
cm2/dmol). The mass spectrometry analyses of C4-desalted
ribonucleases were performed on a Micromass Q-TOF UltimaTM
API spectrometer (Micromass, Wythenshawe, UK) equipped with an orthogonal electrospray source operated in the positive ion mode.
Ribonuclease Activity Assay--
Ribonuclease activity was
determined by the release of acid-soluble ribonucleotides from the RNA
substrate following ribonuclease digestion. The yeast total RNA (120 µg) was incubated with purified ribonuclease in 50 µl of 100 mM Tris-HCl buffer, pH 8.0, at 37 °C for 10 min, and the
reaction was terminated by the addition of ice-cold stop solution (7%
perchloric acid, 0.1% uranylacetate, 200 µl). The reaction mixtures
remained on ice for 30 min before centrifugation (12,000 × g, 4 °C for 20 min). The absorbance of the supernatant
was measured at 260 nm. One unit of ribonuclease activity is defined as
the amount of enzyme producing one A260 acid-soluble material at 37 °C for 10 min. Ribonuclease activities were also analyzed by zymogram assay on RNA-casting PAGE (22). Briefly,
after electrophoresis the gel was washed twice with 25% isopropyl
alcohol in 10 mM Tris-HCl, pH 7.5, to remove SDS for protein renaturation. The activity was visible after incubating the gel
at room temperature for 30 min in 10 mM Tris-HCl, pH
7.5, and staining by 0.2% toluidine blue O for 10 min.
Base Specificity of Ribonucleases--
The specific cleavage
sites of ribonucleases were determined by incubation of ribonucleases
with 5'-32P-labeled synthetic 18-mer RNA with the known
sequence 5'-AAGGUUAUCCGCACUGAA-3', followed by denaturing gel
electrophoresis and autoradiography (22). The
kcat/Km values of
ribonucleases toward dinucleotides, e.g. CpG, UpG, UpU, CpU,
and UpA, were measured by HPLC separation and quantification (23, 24).
Briefly, the dinucleotides were digested with ribonucleases at 37 °C
for 10 min in buffer containing 100 mM MES, pH 6.0, 50 mM NaCl, and 0.1 mg/ml RNase-free bovine serum albumin.
Digested nucleotides were separated by reverse-phase HPLC using 1-5%
acetonitrile (depending on products) in 0.1% trifluoroacetic acid on a
Vydac C18 column with a Waters automated gradient controller. The
kcat/Km values were
calculated after quantification of the substrate and product. Values
were obtained from at least two independent experiments.
Assay of Cytotoxicity by ATP Lite-M Measurement--
HeLa cells
(5 × 103) were grown in 100 µl of Dulbecco's
modified Eagle's medium containing 10% fetal calf serum in 96-well plates and treated with 2 µM ribonuclease for 48 h.
50 µl of lysis buffer was added to each well and incubated for 2 min
before 50 µl of substrate solution was added. The luminescence was
measured in a dark adapt plate by Top Count Microplate Scintillation
Counter (Packard A Canberra Company) according to the manufacturer's
instructions (ATP Lite-M assay system, Packard BioScience Company, The
Netherlands) (6). The cytotoxicity was expressed in percentage of loss
in cell viability caused by RC-RNase treatment compared with control cells. The value was determined from the mean of six wells.
Crystallization of the RC-RNase·d(ACGA)
Complex--
Adequate RC-RNase·d(ACGA) co-crystals were obtained
using a three-step seeding method. Briefly, 10 mg/ml RC-RNase was
dissolved in 4 mM Tris-HCl, pH 8.0, and 4 mM
NaCl. Crystals were grown at room temperature using the hanging drop
vapor diffusion method. 1.5 µl of reservoir solution (0.1 M potassium sodium tartrate tetrahydrate, 24% (w/v)
polyethylene glycol 8000, and 0.05 M sodium citrate, pH
5.6) in a 1:1 ratio was incubated with 1.5 µl of protein solution.
Minute RC-RNase crystals were obtained and transferred to the mixture
of RC-RNase and d(ACGA) (0.81 mM:1.62 mM) and
incubated with the reservoir solution in a 1:1 ratio as described
above. The newly formed small RC-RNase·d(ACGA) crystals were further transferred to a new RC-RNase·d(ACGA) mixture with the same
composition as described above and incubated for 3 weeks.
RC-RNase·d(ACGA) co-crystals up to 0.05 × 0.3 × 0.3 mm
were obtained for data collection.
Data Collection, Structure Determination, and
Refinement--
Before data collection, RC-RNase·d(ACGA) complex
crystals were immersed in a cryoprotectant consisting of 4, 8, 12, 16, and 20% (w/v) glycerol in a reservoir solution for 5 min at each step. The diffraction data were collected at 100 K using wavelength 1.05 Å at beamline X4A on an Area Detector Systems Corporation Q4 CCD area
detector at the Brookhaven National Laboratory. Data reduction was
performed using the Denzo/Scalepack software package (25). The
resulting data are provided in Table
I.
The structure of the RC-RNase·d(ACGA) complex was solved by the
molecular replacement method using the software package AmoRe (26), and
the structure of RC-RNase (Protein Data Bank code 1KM8) was used as the
probe. All data with I/ Preparation and Analysis of Recombinant Ribonucleases--
RNase
A, onconase (AF332139), cloned in this laboratory, the wild type
RC-RNase gene, and the mutated RC-RNase genes were expressed in
E. coli BL21(DE3). The secreted proteins in the media were
purified to homogeneity by phosphocellulose and carboxymethyl column
chromatographies (Fig. 2A).
Onconase and most of the recombinant RC-RNases, except for those with
N-terminal mutants, possessed Pyr at their N termini as did
that of native frog RC-RNase detected by mass spectrum analysis and
Edman degradation after Pfu aminopeptidase treatment (6).
RC-Q1E had two kinds of product, ended with pyroglutamate or glutamate
at the N terminus, which were separated by FPLC Mono S column
chromatography using 0.07-0.11 N NaCl salt gradient
elution (Amersham Biosciences).
The CD spectra of most RC-RNase mutants were analyzed to rule out the
possibility of incorrect folding. There was no significant difference
among native RC-RNase, wild type secretory RC-RNase, RC-Q1A, RC-Q9A,
RC-N38Q, RC-T39A, RC-T70A, RC-T70D, RC-K95A, RC-E97A, RC-V102A,
and RC-V102D, some CD spectra of which are shown in Fig.
3. This similarity indicates that the
secondary structures of these RC-RNase mutants are similar to that of
native RC-RNase.
Relative Ribonuclease Activity of RC-RNase and Its
Variants--
The ribonuclease activities of RC-RNase and its mutants
were analyzed using an acid-insoluble method as shown in Table
II. The specific activity of RC-RNase
toward yeast total RNA was slightly less than that of RNase A (19),
whereas the activity of native and recombinant wild type RC-RNase was
similar. When Pyr-1, Lys-9, or Asn-38 was mutated, most of the
enzymatic activity was destroyed (RC-Q1A, 1.5%; RC-K9A, 2.2%;
RC-N38A, 1.0% left) (Table II). However, RC-Q1E retained 28% of its
activity. These results indicate that Pyr-1, Lys-9, and Asn-38 along
with His-10, Lys-35, and His-103 are involved in catalytic
activity.
Residues adjacent to the predicted B1 site based on the
structure of native RC-RNase (1KM8), Val-37, Thr-39, Thr-70, or Phe-104, were mutated and assayed for catalytic activity (Table II). We
found that 5.8% of activity remained in RC-T39A, 41.0% in RC-T39S,
65.8% in RC-T70A, 79.6% in RC-T70S, 21.5% in RC-F104A, and 79.2% in
RC-V37A. Similarly, residues adjacent to the hypothesized B2 site were also mutated and assayed for catalytic
activity. The RC-K95A (24.2% remained), RC-E97A (11.6%), RC-V102A
(19.1%), and RC-V102D (3.4%) had a marked reduction in activity,
whereas RC-N57A (48.7%) and RC-N59A (76.2%) did not exhibit marked
reduction. These findings suggest that Thr-39, Lys-95, Glu-97, Val-102,
and Phe-104 may participate in the catalytic activity of RC-RNase through base recognition.
The relative activity of most RC-RNase mutants was also determined by
zymogram as shown in Fig. 2B. Most of the recombinant RC-RNases had the same mobility as native RC-RNase, except for RC-K9A
and RC-K95A, which likely have a more compact structure than native
RC-RNase. The relative activity of these recombinant ribonucleases
shown by zymogram is in good agreement with the activity obtained by
the acid-insoluble method (Table II). One exception was RC-K95A, which
was less active than native RC-RNase when assayed by the acid-insoluble
method, but it was more active than native RC-RNase when assayed by
zymogram. This discrepancy in relative activity may result from the
difference in molecular weight of the nucleotides released from the gel
and that remained in aqueous solution. Because of the broader base
specificity of the RC-K95A (see below) and complete digestion of RNA
substrate in the gel, the shorter oligonucleotide produced by RC-K95A
tends to diffuse from gel. In contrast, the RNA was only partially
digested by the ribonuclease to obtain the absorbance of soluble
oligonucleotides within the linear range (A260
0.1-0.3).
Residues Involved in B1 Base Recognition--
The
residues adjacent to the predicted B1 site, Val-37, Thr-39,
Thr-70, and Phe-104, were mutated and analyzed for base specificity. We
found that both CpG and UpG of an 18-mer RNA were cleaved by native and
wild type recombinant RC-RNase, whereas CpG was preferentially cleaved
by the T39A or T70A mutant (Fig. 4).
However, both CpG and UpG were cleaved if Thr-39 or Thr-70 was replaced
by a cognate residue in RC-T39S or RC-T70S. Interestingly, UpG of
18-mer RNA was more susceptible than CpG in T70D. The catalytic
activity and substrate binding affinities of Thr-39 and Thr-70 mutants toward these dinucleotides CpG or UpG were determined by
kcat/Km value analyses
(Table III). RC-T70A had a decreased
Km for CpG (17%) and increased
Km for UpG (213%), whereas RC-T70D had an increased Km for CpG (178%) and decreased
Km for UpG (54%). These findings indicate that
the hydroxyl group of Thr-70 allows the binding of both cytosine and
uracil at B1 site, whereas the carboxyl group of Asp-70
favors the binding of uracil. The base specificity of RC-RNase was not
changed significantly in RC-F104A.
Residues Involved in B2 Base Recognition--
The
residues close to the predicted B2 site, Asn-57, Asn-59,
Lys-95, Glu-97, and Val-102, were mutated and analyzed for base specificity. The results shown in Table III and Fig. 4A
indicate that the base specificities were not significantly changed in N57A and N59A mutants. CpU, along with the original scissile substrate CpG and UpG, was cleaved by RC-E97A. The
kcat/Km value of E97A for
CpG and UpG decreased to 7 and 6, respectively, whereas their Km values increased 2- and 5-fold, respectively.
These results indicate that the binding affinities of E97A for CpG and
UpG decrease. When the nearby residue Lys-95 was mutated to
Ala, CpC, CpA, CpU, UpA, and UpU, along with the original scissile
substrate CpG and UpG, were cleaved. The
kcat/Km value for CpG and
UpG was dramatically decreased to 17 and 36, respectively, whereas the
kcat/Km value for UpA
increased 1.6-fold compared with that of the wild type RC-RNase. With
regard to the substrate binding affinity, the Km of the K95A for UpA decreased to 45%, whereas that for UpG increased to 564%. Similar results were also found in K95G, K95T, and K95R mutants (data not shown). These results indicate that Lys-95 mutation induced the RC-RNase to change its B2 base specificity from
guanine to adenine. Although the base specificity of V102A did not
change, the kcat/Km
values for five dinucleotides decreased dramatically (5-9% left)
(Fig. 4 and Table III).
N-terminal Pyroglutamate Involved in B2 Base
Specificity--
The CpA, CpC, CpU, UpA, UpU as well as the original
CpG and UpG of 18-mer RNA were cleaved by Pyr-1 substitutional mutants (RC-Q1A, RC-Q1N, and RC-Q1S) or deletion mutants (dN2 and
dQNW1-3), however, only CpG, UpG, and UpU were cleaved by
substitution mutants (RC-Q1E, RC-N2D, and RC-K9A) (Fig. 4B).
Therefore, it is concluded that Pyr-1 in the right position plays a
dual role in both catalytic activity (Table II and Table III) and
B2 base specificity (Fig. 4B), and the cognate
residue Glu-1 may exert a function similar to that of Pyr-1.
Residues Involved in Cytotoxicity--
In general, the
cytotoxicity of an RC-RNase variant is correlated with its catalytic
activity except Lys-9, Asn-38, and Val-102 mutations (Table II). Both
RC-N38Q and RC-V102A retained 14.6 and 19.1% activity while still
exerting 78.2 and 84.4% cytotoxicity, respectively. In contrast,
RC-N38A and RC-V102D lost both catalytic activity (1.0%, 3.4%
remained) and cytotoxicity (1.5%, 2.0% remained). Both RC-K9A and
RC-K9Q lost most of their catalytic activity (2.2%, 1.9% remained),
but they retained differential cytotoxicity (66.7% and 9.2% left, respectively).
Quality and Statistics of the Structure of the RC-RNase·d(ACGA)
Complex--
To determine the detail of interactions between RC-RNase
and RNA substrate for catalytic reaction and base recognition, the co-crystal of the RC-RNase·d(ACGA) complex was grown, and its structure was solved by molecular replacement and refined to 1.80 Å resolution. The final model contained 1928 non-hydrogen, non-solvent atoms and 323 water molecules. The crystallographic R-factor
and free R-factor of the final model were 18.9 and 22.9%,
respectively. The statistics of refinement are listed in Table I. The
crystals belong to the space group P21 and have two 1:1
complexes per asymmetric unit. The two independent molecules are
related by a noncrystallographic 2-fold axis (Fig.
5). The structures of all of the residues
were defined unambiguously except the weak electron density region at
the 5'-end adenosines of d(ACGA). The 3'-end adenosine has two
conformational disorders. The average temperature factors for the main
chain and side chain atoms are 20.0 and 22.2 Å2,
respectively. However, there was a difference noted in the average temperature factors between molecule A (16.8 Å2) and
molecule B (25.3 Å2) (Fig.
6). The difference is caused largely by
the small difference in packing force between these two monomers.
Molecules A and B are very similar according to their superimposition.
The side chain orientation of the 14 residues between these two
molecules was slightly shifted, and the total root mean square
deviation for all C The Co-crystal Structure of the RC-RNase·d(ACGA)
Complex--
The overall dimension (backbone to backbone) of the two
monomers is about 68 Å × 43 Å × 38 Å (Fig. 5). Interestingly, the base stacking and continuity of the phosphate backbone of the two
d(ACGA) molecules are clearly seen in the central area of the diagram. This suggests that a continuous RNA is bound to an RC-RNase. The secondary structures of the RC-RNase·d(ACGA) complex crystal were analyzed using the PROMOTIF program (33). There are three
Conformational Changes of RC-RNase upon Binding with
d(ACGA)--
The crystal structure of RC-RNase in the complex is
almost identical to that of the free form (1KM8), except that two
All of the side chain atoms of the complex RC-RNase have the same
conformation as the free form except for Lys-35 and Val-37. The side
chain of Lys-35 moves to form a strong hydrogen bond with O2P of the
backbone phosphate (2.60 Å) and a hydrogen bond with O3' of the ribose
(3.02 Å) (Fig. 7D). The side
chain of Val-37 rotates about 60o thus allowing the two
methyl groups to have a better hydrophobic contact with the cytosine at
the B1 site. Some of the side chain movements on the
surface of RC-RNase are caused primarily by hydrogen bonds from
neighboring molecules in the crystal lattice.
Structure of the RC-RNase·d(ACGA) Complex--
The crystal
structure of RC-RNase·d(ACGA) complex and the critical residues
involved in catalytic activity and base recognition are illustrated in
Fig. 7A. At the B1 site, two hydrogen bonds were
found between Thr-39 and cytosine, one between the side chain hydroxyl
group of Thr-39 and N3 of cytosine (2.72 Å), and the other between the
backbone amide and O2 of cytosine (2.75 Å). Furthermore,
hydroxyl oxygen of Thr-39 was linked with the hydroxyl oxygen of Thr-70
by a hydrogen bond (2.85 Å). The side chains of Val-37 and Phe-104
provide hydrophobic contacts with the substrate cytosine at the
B1 site. The Ile-107 of RC-RNase, homologous to Ser-118 of
angiogenin and Ser-123 of RNase A, is far from the substrate (Fig.
7B).
At the B2 site, the carboxyl group of Glu-97, equivalent to
Glu-111 of RNase A, forms two hydrogen bonds with the N1 and amine (N2)
of guanine (2.66 and 3.03 Å, respectively). Moreover, the side chain
amine (N
With regard to residues near the P1 site, His-10 forms a
hydrogen bond with P1 phosphate (2.74 Å), Lys-35 forms two
hydrogen bonds with O2P of the phosphate backbone (2.60 Å) and O3' of
the ribose (3.02 Å), His-103 forms two hydrogen bonds with
P0 and P1 phosphate (3.19 and 3.13 Å,
respectively) (Fig. 7D). The Pyr-1 forms three hydrogen
bonds near the active site. First, the side chain O RC-RNase possesses a conserved amino acid sequence and tertiary
structure similar to those of ribonuclease in the RNase A superfamily
including three catalytic residues, two triple-stranded antiparallel
Regarding base specificity, all members of the RNase A superfamily
prefer pyrimidine at the B1 site (38). Some show equal preference for cytosine and uracil, e.g. bovine RNase A,
human pancreatic ribonuclease, bullfrog RC-RNase and RC-RNase L1 (19, 36, 39). Others prefer uracil, e.g. human RNase 4, frog
onconase, and RC-RNase 2 (19, 40, 41), and the third group prefer cytosine, e.g. human RNase 2, human RNase 5 and frog
RC-RNase 4 (19, 23, 42). For RNase A, it is known that Thr-45, Asp-83, Phe-120, and Ser-123 are involved in the B1 base
recognition. Thr-45 and Phe-120 are conserved in the RNase A
superfamily and play a role in pyrimidine binding through hydrogen
bonds and van der Waals interactions, respectively (43). Although
Asp-83 and Ser-123 are not conserved in the RNase A superfamily, they
determine the enzyme configuration and B1 base specificity
of RNase A through hydrogen bonds to Thr-45 and uracil, respectively
(43).
In RC-RNase, Thr-39, Phe-104, Thr-70, and Ile-107 are the equivalent
residues of RNase A Thr-45, Phe-120, Asp-83, and Ser-123, respectively.
In this study, we found that the wild type RC-RNase has an equal
preference for uracil and cytosine at the B1 site, RC-T70A
prefers cytosine, and RC-T70D prefers uracil (Table
IV and Fig. 4A). T70A has a
higher affinity (low Km, 17%) and RC-T70D a
lower affinity (high Km, 178%) for CpG compared
with that of wild type RC-RNase. From x-ray
crystallography, we found that the hydroxyl oxygen of
Thr-39 may serve as a hydrogen bond donor for N3 of cytosine and
simultaneously serve as hydrogen bond acceptor from the hydroxyl oxygen
of Thr-70; therefore, the Thr-39 may serve as a hydrogen bond acceptor
from uracil and donor for Thr-70. In contrast, some ribonucleases
possessing Asp at the equivalent Thr-70 residue, e.g.
RC-T70D, onconase, RC-RNase 2, and human RNase 4, prefer uracil at the
B1 site because the carboxyl group of Asp-70 only serves as
a distal hydrogen bond acceptor for uracil but not as a hydrogen bond
donor for cytosine. It is concluded that Thr-70 is the key residue that
determines B1 base specificity through its dual function of
hydrogen bond donor or acceptor.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Alignment of the amino acid sequences of
RNase A and the human and frog ribonuclease superfamily. Sequence
alignment was carried out using the multiple sequence alignment
function of the BCM Search Launcher (49), and gaps were included for
optimal alignment and maximum homology of the sequences. The
number at the end of each sequence indicates the
total residues of the protein. The number with a
dot above the sequence of RC-RNase and bRNase A indicates
the putative residue involved in catalytic activity and base
recognition. RC-RNase (AF039104), RC-RNase 2-4 (AF242553-5), RC-RNase
6 (AF242556), and RC-RNase L1 (AF288642) are the ribonucleases from
R. catesbeiana. Onconase (AF332139) is the ribonuclease from
R. pipiens. bRNase A (X07283) is the bovine pancreatic
ribonuclease. hRNase 1 (D26129) is the human pancreatic ribonuclease.
hRNase 2 (X16546) is the human eosinophil-derived neurotoxin. hRNase 3 (X16545) is the human eosinophil cationic protein. hRNase 4 (D37931) is
the human RNase 4. hRNase 5 (M11567) is the human angiogenin. Conserved
catalytic residues are boxed, and cysteines for disulfide
bridges are shown in gray.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Data collection and refinement statistics of RC-RNase·d(ACGA)
complex
>0 were used for the subsequent
refinement using crystallography NMR software (27), and manual
adjustments were performed using molecular graphics package O (28).
Most of the substrate analog d(ACGA) was located at the electron
density map and added manually. Two 3'-end adenosines of d(ACGA) have
two conformation disorders, and the adenosine models were adjusted
manually and fixed in the refinement. Water molecules were added in
areas of electron density in difference Fourier maps that were <3.2 Å from a hydrogen bond partner. Assessment of the quality of the
coordinates was performed using the programs PROCHECK (29) and MOLEMAN
(30). Molecular graphics were generated with MOLSCRIPT (31), Insight II
and QUANTA (Accelrys, Inc. San Diego) programs. The coordinates have been deposited in the Protein Data Bank (code 1M07) (32).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (31K):
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Fig. 2.
Purity and catalytic activity of RNase A,
RC-RNase, and RC-RNase variants. A, SDS-PAGE analysis.
Ribonucleases (2 µg) were separated by 13.3 SDS-PAGE and stained with
Coomassie Blue. B, zymogram analysis. Ribonucleases (1 ng)
were separated by RNA casting 13.3% nonreducing SDS-PAGE and stained
by toluidine blue O (19).
View larger version (21K):
[in a new window]
Fig. 3.
CD spectrum analysis of RC-RNase and its
variants. CD spectra are presented as mean residue ellipticity,
expressed in degrees cm2/dmol. nRC and
sRC indicate native RC-RNase isolated from bullfrog oocytes
and secretory RC-RNase purified from culture medium,
respectively.
Specific activities, cell survival rate, and relative cytotoxicity of
RC-RNase and its variants
View larger version (51K):
[in a new window]
Fig. 4.
Base specificity of RNase A, onconase,
RC-RNase, and RC-RNase variants. A 5'-end-labeled
oligoribonucleotide was partially digested with ribonucleases (3-386
pg) at 37 °C for 10 min, separated by 8 M
urea-15% PAGE and visualized by autoradiography. RNA,
5'-32P-labeled RNA substrate; OH , RNA
substrate treated with 0.05 M sodium bicarbonate-carbonate,
pH 9.2, at 90 °C for 12 min. The RNA sequence and cleavage sites
(arrows) are shown in the left and right
margins, respectively. A, RC-RNase mutants involved in
B1 and B2 base specificity. B,
RC-RNase mutants involved in catalytic activity including N terminus
mutants and the K9A mutant.
Substrate specificities of RC-RNase and its variants
atoms was 0.19 Å. The mean temperature factor
of the 323 water molecules was 38.9 Å2. The average
temperature factor for the d(ACGA) molecule was high (40.0 Å2) because of the flexibility of the two 5'-end
adenosines and the disorder of the two 3'-end adenosines of d(ACGA).
The average temperature factor of the central d(CG) was only 24.2 Å2, which is located clearly in the electron density
map.
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Fig. 5.
Ribbon diagram of the
three-dimensional structure of two RC-RNase·d(ACGA) complex
molecules. Two d(ACGA) molecules are shown by liquorice
representation viewing down along the noncrystallographic 2-fold axis.
The -helix and
-sheet strand are colored green and
blue, respectively. The base stacking and continuity of the
phosphate backbone are clearly seen in the central area of
the diagram.
View larger version (28K):
[in a new window]
Fig. 6.
Graphic representation of the B factors for
molecule A and molecule B as a function of the residue number. The
B-factors in molecule B (dashed line) are higher
than that of molecule A (solid line).
-helices at
1 (3-10),
2 (19-22), and
4 (45-49), and two
310 helices at
3 (26-28) and
5 (50-52). The four
-strands
4 (37-42),
6 (66-73),
7 (83-90), and
1
(11-12) form sheet A. The
-sheet B contains strand
5 (57-61),
8 (92-97), and
9 (100-107). Two additional short strands,
2
(29-30) and
3 (33-34), form a small sheet. There is one inverse
-turn at 23-25. There are nine
-turns at residues 16-19,
29-32, 30-33, 52-55, 55-58, 78-81, 96-99, 97-100, and 106-109.
There are two bulges: one at Lys-95 to bridge with Val-102 and His-103,
and one at Cys-93 to bridge with Ala-105 and Gly-106.
-turns (17-19 and 31-33) in the free form are changed to two
-turns (16-19 and 30-33) in the complex form. Superimposing the
two complex molecules A and B with free form of RC-RNase, the root mean
square deviation, 0.43 and 0.46 Å, respectively, indicates that they closely resemble one another. The conformations of the main chain atoms
differ only in two regions (residues 13-18 and 62-68) because of the
close contacts between the neighboring molecules in the crystal
lattices (data not shown).
View larger version (41K):
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Fig. 7.
Stereo diagrams of RC-RNase and
oligonucleotide complex. A, ribbon diagram of RC-RNase
with d(CG), the -helix,
-sheet, and d(CG) are shown in
green, blue, and red, respectively.
The relevant residues in the B1, B2, and
P1 sites are numbered. B,
C, and D show the hydrogen bonds between
respective residues and substrates in B1, B2,
and P1 sites, respectively. The hydrogen bonds are shown as
blue dashed lines. The substrate in red was shown
within the electronic density omit map (contoured at 1.5
). The
image was generated by SwissPDBviewer (50), Turbo-Frodo, and Photoshop
(Adobe Systems, Inc.). <E indicates pyroglutamate.
) of a nearby residue, Lys-95, formed a hydrogen bond with
the side chain carboxyl oxygen of Glu-97 (2.70 Å) and another weak
hydrogen bond with carbonyl oxygen (O6) of guanine (3.26 Å). In RNase
A, residue Asn-71 contributes to the B2 base specificity
and substrate binding (34), but the possible Asn-71-equivalent residue
in RC-RNase, Asn-57 or Asn-59, is apart from B2 substrate (Fig. 7C), and the loop between Cys-65 and Cys-72 of RNase A
is not found in RC-RNase.
oxygen of Pyr-1
binds to the Lys-9 side chain amine (N
) (2.95 Å), of which hydrogen
bonds to the catalytic phosphate (2.80 Å). Furthermore, the Lys-9
backbone carbonyl oxygen was bound to the Asn-38 side chain amide and
Tyr-28 hydroxyl group by hydrogen bonds (2.85 and 2.71 Å,
respectively) (Fig. 7D). Second, the Pyr-1 backbone amide
binds to the carbonyl oxygen of Val-102 (2.63 Å), whereas the backbone
amide of Val-102 hydrogen bonds to backbone carbonyl oxygen of Lys-95
(2.85 Å), which interacts directly with guanine as described above.
Third, the carbonyl oxygen of Pyr-1 binds directly to the amine (N2) of
guanine (3.35 Å) (Fig. 7C).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-sheets, and three
-helices (4, 5, 35). However, the frog
ribonucleases in the superfamily possess a distinct base preference and
antitumor activity (4, 5). Based on a comparison of the primary
structure, the RC-RNase has special residues, e.g. Pyr-1,
Lys-9, Thr-70, and Lys-95, which differ from those of mammals. In this
report, these residues were mutated and analyzed for catalytic
activity, base specificity, and cytotoxicity. The secondary structures
of these mutated RC-RNases were similar to native RC-RNase based on the
CD spectrum, and thus these residues may be responsible for these novel
properties rather than conformational changes. The structure of
ribonuclease-oligonucleotide complex has been solved, e.g.
RNase A complexed with d(ATAAG), whereas retrobindings were observed in
the crystal structure of RNase A complexed with dinucleotide,
2',5'-UpG, 2',5'-CpG, or 3',5'-d(CpG) or in the solution structure of
RC-RNase complexed with 2',5'-CpG or 3',5'-d(CpG) (17, 35-37). In
retrobinding form, the orientation of substrate differs from that in
the catalytic binding form. In the case of CpG, sulfate or phosphate
occupies the P1 site, and guanine locates in the
B1 site, whereas cytosine is not clearly observed. In this
report, we grew the RC-RNase·d(ACGA) complex in the catalytic binding
form and analyzed the molecular interaction between RC-RNase and the
substrate analog.
Comparison of selected residues in the subsites of RNase A and
RC-RNases
Although Val-37 in the B1 site of RC-RNase exhibits a dramatic chemical shift in the RC-RNase·d(CG) complex as detected by NMR study (35), RC-V37A and RC-V37K did not reveal significant changes in catalytic activity (79.2 and 91%, respectively) or base specificity (Fig. 4). Our results from x-ray crystallography show that the side chain rotation of Val-37 only provides a better hydrophobic contact with the cytosine at the B1 site without affecting hydrogen bonds involved in the catalytic activity and base specificity.
From the aspect of B2 base specificity, mammalian ribonucleases prefer adenine, whereas frog ribonucleases prefer guanine. In RNase A, residues Asn-71 and Glu-111 contribute to the B2 base specificity and substrate binding through hydrogen bonds (34). However, the Asn-71-equivalent residue and all of the residues corresponding to the loop between Cys-65 and Cys-72 of RNase A are absent in all known frog ribonucleases. In RC-RNase, the possible Asn-71-equivalent residue is Asn-57 or Asn-59, but they are distant from the B2 substrate in the complex and not involved in base recognition (Figs. 4A and 7C). The equivalent residue of RNase A Glu-111 is Glu-97 in RC-RNase, which is conserved in the ribonuclease superfamily, bound directly to guanine, and involved in B2 base specificity (Figs. 4A and 7C). In this study, we found that a nearby residue, Lys-95, is bound directly to Glu-97 and guanine and is involved in base specificity (Figs. 4A and 7C). In contrast, the equivalent residue of RC-RNase Lys-95 in mammalian ribonucleases is Ala, which preferentially recognizes pyrimidine-adenosine at the B2 site. Therefore, it is concluded that Lys-95 and Glu-97 are the key residues of RC-RNase which determine B2 base specificity for guanine in contrast to Asn-71 and Glu-111 in RNase A which determine for adenine.
The catalytic residues His-10, Lys-35, and His-103 of RC-RNase are
conserved in all known members of the RNase A superfamily (Fig. 1).
However, Pyr-1 is found mainly in frog ribonucleases and not in most
mammalian ribonucleases. In mammals, Pyr-1 is only found in human RNase
4 and RNase 5 (angiogenin) (Fig. 1), but it is structurally flexible
and is not involved in catalytic activity (44, 45). In frog
ribonucleases, Pyr-1 has been found to be crucial for the catalytic
activity and cytotoxicity of onconase through two hydrogen bonds with
Lys-9 N and Val-96 carbonyl oxygen (11, 12, 21). In the present
study, we found that Pyr-1 contributes three hydrogen bonds linking
Lys-9, Val-102, and guanine in the RC-RNase·d(ACGA) complex (Fig. 7,
C and D), and it participates in both catalytic
activity and base specificity.
The Asn-44 of RNase A, equivalent to Asn-38 of RC-RNase, is conserved in the ribonuclease superfamily and is involved in the formation of a binding pocket for substrate as predicted by computer modeling (46). In RC-RNase, the N38A mutation caused a marked decrease in catalytic activity (1.0% remained) and cytotoxicity (1.5% remained), whereas the N38Q mutation caused less reduction in both properties (14.6 and 78.2% left, respectively) (Table II). However, their base specificities did not change (data not shown). Similarly, Val-118 of RNase A is also conserved in the RNase A superfamily, and the mutation of its equivalent residue Val-102 in RC-RNase causes differential reduction of catalytic activity and cytotoxicity, e.g. 3.4 and 2% left in RC-V102D, 19.1 and 84.4% left in RC-V102A, respectively (Table II). The base specificity of these two mutants did not change. It is suggested that N38A and V102D mutations may cause severe disruption of hydrogen bonds that are essential for catalysis and cytotoxicity because the secondary structures of these mutants are similar to that of native RC-RNase. RC-K9Q drastically lost most of its catalytic activity and cytotoxicity (1.9 and 9.2% remained, respectively), whereas the RC-K9A only lost its catalytic activity (2.2% remained) but not cytotoxicity (66.7% remained). It is suspected that the side chain of Lys-9 may play an important role in the cytotoxicity in addition to catalytic activity.
In general, the cytotoxicity of RC-RNase is correlated, but not
proportional to, the reduction of catalytic activity and is not
correlated with base specificity. In comparing the relative catalytic
activity of the seven ribonucleases from bullfrog, they exhibit
differential catalytic activity that varies from 1 to 105-fold, but they possess similar cytotoxicities toward
HeLa cells (19). The results indicate that cytotoxicity of bullfrog
ribonucleases is not proportional to their catalytic activities. The
results in Table II show that ~10% of catalytic activity is
sufficient to retain ~80% of cytotoxicity. Therefore, it is
suggested that minimum catalytic activity is sufficient for
cytotoxicity, whereas other residues may be required to bind specific
receptors for ribonuclease entry or to bind specific substrates inside
the cell (47). Our finding that the mutation K9A, N38A, K95A, and V102D reduce the cytotoxicity of RC-RNase toward HeLa cells, in conjunction with previous study that showed that replacement of an acidic residue
with a basic residue enhances the entry of RC-RNase and its
cytotoxicity toward murine leukemia P388 cells (48), suggests that
positively charged residues are crucial for the cytotoxicity as well as
the catalytic activity of RC-RNase.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Carmay Lin, Chinpan Chen, and Victor Lee Weaver for critical reading of the manuscript; research assistant Tsun-Ai Yu for the CD spectrum analysis; and the Core Facilities for Proteomic Research, Institute of Biological Chemistry, Academia Sinica, for mass spectrum analysis.
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FOOTNOTES |
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* This work was supported in part by grants from the Academia Sinica (to Y.-C. L. and Y. D. L.) and by Republic of China National Science Council Grant NSC 89-2311-B-001-083 (to Y. D. L.).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.
The atomic coordinates and the structure factors (code 1M07) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
Postdoctoral fellow of and supported by the Academia Sinica.
** To whom correspondence may be addressed. Tel.: 886-2-2789-9167; Fax: 886-2-2782-9142; E-mail: ydliao@ibms.sinica.edu.tw (to Y.-D. L.) or Tel.: 886-2-27899199; Fax: 886-2-27826085; E-mail: mbycliaw@ccvax.sinica.edu.tw (to Y.-C. L.).
Published, JBC Papers in Press, December 23, 2002, DOI 10.1074/jbc.M206701200
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ABBREVIATIONS |
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The abbreviations used are: Pyr-1, N-terminal pyroglutamate; HPLC, high performance liquid chromatography; MES, 4-morpholineethanesulfonic acid.
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