Residues Involved in the Catalysis, Base Specificity, and Cytotoxicity of Ribonuclease from Rana catesbeiana Based upon Mutagenesis and X-ray Crystallography*

Ying-Jen LeuDagger §, Shuenn-Shing Chern, Sui-Chi WangDagger , Ya-Yun HsiaoDagger , Imameddin Amiraslanov||, Yen-Chywan Liaw**, and You-Di LiaoDagger **

From the Dagger  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

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.


View larger version (71K):
[in this window]
[in a new window]
 
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

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.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Data collection and refinement statistics of RC-RNase·d(ACGA) complex
Values in parentheses correspond to the highest resolution shell 1.83-1.80 Å.

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/sigma >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

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).


View larger version (31K):
[in this window]
[in a new window]
 
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).

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.


View larger version (21K):
[in this window]
[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.

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.

                              
View this table:
[in this window]
[in a new window]
 
Table II
Specific activities, cell survival rate, and relative cytotoxicity of RC-RNase and its variants

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.


View larger version (51K):
[in this window]
[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.

                              
View this table:
[in this window]
[in a new window]
 
Table III
Substrate specificities of RC-RNase and its variants
Activities were determined as described in under "Experimental Procedures." Each value is the mean ± S.D. (n = 4).

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 Calpha 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.


View larger version (49K):
[in this window]
[in a new window]
 
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 alpha -helix and beta -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 this window]
[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).

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 alpha -helices at alpha 1 (3-10), alpha 2 (19-22), and alpha 4 (45-49), and two 310 helices at alpha 3 (26-28) and alpha 5 (50-52). The four beta -strands beta 4 (37-42), beta 6 (66-73), beta 7 (83-90), and beta 1 (11-12) form sheet A. The beta -sheet B contains strand beta 5 (57-61), beta 8 (92-97), and beta 9 (100-107). Two additional short strands, beta 2 (29-30) and beta 3 (33-34), form a small sheet. There is one inverse gamma -turn at 23-25. There are nine gamma -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.

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 gamma -turns (17-19 and 31-33) in the free form are changed to two gamma -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).

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.


View larger version (41K):
[in this window]
[in a new window]
 
Fig. 7.   Stereo diagrams of RC-RNase and oligonucleotide complex. A, ribbon diagram of RC-RNase with d(CG), the alpha -helix, beta -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 sigma ). The image was generated by SwissPDBviewer (50), Turbo-Frodo, and Photoshop (Adobe Systems, Inc.). <E indicates pyroglutamate.

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 (Nzeta ) 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.

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 Oepsilon oxygen of Pyr-1 binds to the Lys-9 side chain amine (Nzeta ) (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

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 beta -sheets, and three alpha -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.

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.

                              
View this table:
[in this window]
[in a new window]
 
Table IV
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 Nzeta 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.

    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.

    FOOTNOTES

* 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

    ABBREVIATIONS

The abbreviations used are: Pyr-1, N-terminal pyroglutamate; HPLC, high performance liquid chromatography; MES, 4-morpholineethanesulfonic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Durack, D. T., Ackerman, S. J., Loegering, D. A., and Gleich, G. J. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 5165-5169[Abstract]
2. McClaren, D. J., McKean, J. R., Olsson, I., Venge, P., and Kay, A. B. (1981) Parasite Immunol. 3, 359-373[Medline] [Order article via Infotrieve]
3. Fett, J. W., Strydom, D. J., Lobb, R. R., Alderman, E. M., Bethune, J. L., Riordan, J. F., and Vallee, B. L. (1985) Biochemistry 24, 5480-5486[Medline] [Order article via Infotrieve]
4. Youle, R. J., and D'Alessio, G. (1997) in Ribonuclease: Structure and Functions (D'Alessio, G. , and Riodan, J. F., eds) , pp. 491-514, Academic Press, New York
5. Irie, M., Nitta, K., and Nonaka, T. (1998) Cell. Mol. Life Sci. 54, 775-784[CrossRef][Medline] [Order article via Infotrieve]
6. Huang, H. C., Wang, S. C., Leu, Y. J., Lu, S. C., and Liao, Y. D. (1998) J. Biol. Chem. 273, 6395-6401[Abstract/Free Full Text]
7. Shapiro, R., and Vallee, B. L. (1989) Biochemistry 28, 7401-7408[Medline] [Order article via Infotrieve]
8. D'Alessio, G., Di, Donato, A., Parente, A., and Piccoli, R. (1991) Trends Biochem. Sci. 16, 104-106[CrossRef][Medline] [Order article via Infotrieve]
9. Ardelt, W., Mikulski, S. M., and Shogen, K. (1991) J. Biol. Chem. 266, 245-251[Abstract/Free Full Text]
10. Sorrentino, S., Glitz, D. G., Hamann, K. J., Loegering, D. A., Checkel, J. L., and Gleich, G. J. (1992) J. Biol. Chem. 267, 14859-14865[Abstract/Free Full Text]
11. Boix, E., Wu, Y., Vasandani, V. M., Saxena, S. K., Ardelt, W., Ladner, J., and Youle, R. J. (1996) J. Mol. Biol. 257, 992-1007[CrossRef][Medline] [Order article via Infotrieve]
12. Newton, D. L., Boque, L., Wlodawer, A., Huang, C. Y., and Rybak, S. M. (1998) Biochemistry 37, 5173-5183[CrossRef][Medline] [Order article via Infotrieve]
13. Schmid, F. X., Grafl, R., Wrba, A., and Beintema, J. J. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 872-876[Abstract]
14. Cuchillo, C. M., Vilanova, M., and Nogues, M. V. (1997) in Ribonuclease: Structure and Functions (D'Alessio, G. , and Riodan, J. F., eds) , pp. 272-304, Academic Press, New York
15. Nogues, M. V., Vilanova, M., and Cuchillo, C. M. (1995) Biochim. Biophys. Acta 1253, 16-24[Medline] [Order article via Infotrieve]
16. McPherson, A., Brayer, G., Cascio, D., and Williams, R. (1986) Science 232, 765-768[Medline] [Order article via Infotrieve]
17. Fontecilla-Camps, J. C., de Llorens, R., le Du, M. H., and Cuchillo, C. M. (1994) J. Biol. Chem. 269, 21526-21531[Abstract/Free Full Text]
18. Raines, R. T. (1998) Chem. Rev. 98, 1045-1066[CrossRef][Medline] [Order article via Infotrieve]
19. Liao, Y. D., Huang, H. C., Leu, Y. J., Wei, C. W., Tang, P. C., and Wang, S. C. (2000) Nucleic Acids Res. 28, 4097-4104[Abstract/Free Full Text]
20. Nitta, K., Ozaki, K., Ishikawa, M., Furusawa, S., Hosono, M., Kawauchi, H., Sasaki, K., Takayanagi, Y., Tsuiki, S., and Hakomori, S. (1994) Cancer Res. 54, 920-927[Abstract]
21. Mosimann, S. C., Ardelt, W., and James, M. N. (1994) J. Mol. Biol. 236, 1141-1153[CrossRef][Medline] [Order article via Infotrieve]
22. Liao, Y. D. (1995) Mol. Biol. Rep. 20, 149-154
23. Shapiro, R., Riordan, J. F., and Vallee, B. L. (1986) Biochemistry 25, 3527-3532[Medline] [Order article via Infotrieve]
24. Russo, N., Acharya, K. R., Vallee, B. L., and Shapiro, R. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 804-808[Abstract/Free Full Text]
25. Otwinowski, Z., and Minor, W. (1997) Methods Enzymol. 276, 307-326
26. Navaza, J. (1994) Acta Crystallogr. A 50, 157-163[CrossRef]
27. Grosse-Kunstleve, R. W., and Brunger, A. T. (1999) Acta Crystallogr. D 55, 1568-1577[CrossRef][Medline] [Order article via Infotrieve]
28. Jones, T. A., Zou, J. Y., Cowan, S. W., and Kjeldgaard. (1991) Acta Crystallogr. A 47, 110-119[CrossRef][Medline] [Order article via Infotrieve]
29. Laskowski, R. A., Moss, D. S., and Thornton, J. M. (1993) J. Mol. Biol. 231, 1049-1067[CrossRef][Medline] [Order article via Infotrieve]
30. Kleywegt, G. J., and Jones, T. A. (1997) Methods Enzymol. 277, 208-230
31. Kraulis, P. J. (1991) J. Appl. Crystallogr. 24, 946-950[CrossRef]
32. Bernstein, F. C., Koetzle, T. F., Williams, G. J., Meyer, E. F., Jr., Brice, M. D., Rodgers, J. R., Kennard, O., Shimanouchi, T., and Tasumi, M. (1977) J. Mol. Biol. 112, 535-542[Medline] [Order article via Infotrieve]
33. Hutchinson, E. G., and Thornton, J. M. (1996) Protein Sci. 5, 212-220[Abstract/Free Full Text]
34. Tarragona-Fiol, A., Eggelte, H. J., Harbron, S., Sanchez, E., Taylorson, C. J., Ward, J. M., and Rabin, B. R. (1993) Protein Eng. 6, 901-906[Abstract]
35. Chang, C. F., Chen, C., Chen, Y. C., Hom, K., Huang, R. F., and Huang, T. H. (1998) J. Mol. Biol. 283, 231-244[CrossRef][Medline] [Order article via Infotrieve]
36. Aguilar, C. F., Thomas, P. J., Moss, D. S., Mills, A., and Palmer, R. A. (1991) Biochim. Biophys. Acta 1118, 6-20[Medline] [Order article via Infotrieve]
37. Vitagliano, L., Merlino, A., Zagari, A., and Mazzarella, L. (2000) Protein Sci. 9, 1217-1225[Abstract]
38. Nogues, M. V., Moussaoui, M., Boix, E., Vilanova, M., Ribo, M., and Cuchillo, C. M. (1998) Cell. Mol. Life Sci. 54, 766-774[CrossRef][Medline] [Order article via Infotrieve]
39. Sorrentino, S., and Libonati, M. (1994) Arch. Biochem. Biophys. 312, 340-348[CrossRef][Medline] [Order article via Infotrieve]
40. Shapiro, R., Fett, J. W., Strydom, D. J., and Vallee, B. L. (1986) Biochemistry 25, 7255-7264[Medline] [Order article via Infotrieve]
41. Ardelt, W., Lee, H.-S., Randolph, G., Viera, A., Mikulski, S. M., and Shogen, K. (1994) Protein Sci. 3 (Suppl. 1), 137
42. Mosimann, S. C., Newton, D. L., Youle, R. J., and James, M. N. (1996) J. Mol. Biol. 260, 540-552[CrossRef][Medline] [Order article via Infotrieve]
43. Acharya, K. R., Shapiro, R., Allen, S. C., Riordan, J. F., and Vallee, B. L. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2915-2919[Abstract]
44. Terzyan, S. S., Peracaula, R., de Llorens, R., Tsushima, Y., Yamada, H., Seno, M., Gomis-Ruth, F. X., and Coll, M. (1999) J. Mol. Biol. 285, 205-214[CrossRef][Medline] [Order article via Infotrieve]
45. Shapiro, R., Harper, J. W., Fox, E. A., Jansen, H. W., Hein, F., and Uhlmann, E. (1988) Anal. Biochem. 175, 450-461[Medline] [Order article via Infotrieve]
46. Seshadri, K., Balaji, P. V., Rao, V. S., and Vishveshwara, S. (1993) J. Biomol. Struct. Dyn. 11, 395-415[Medline] [Order article via Infotrieve]
47. Leland, P. A., and Raines, R. T. (2001) Chem. Biol. 8, 405-413[CrossRef][Medline] [Order article via Infotrieve]
48. Ogawa, Y., Iwama, M., Ohgi, K., Tsuji, T., Irie, M., Itagaki, T., Kobayashi, H., and Inokuchi, N. (2002) Biol. Pharm. Bull. 25, 722-727[CrossRef][Medline] [Order article via Infotrieve]
49. Smith, R. F., Wiese, B. A., Wojzynski, M. K., Davason, D. B., and Worley, K. C. (1996) Genome Res. 6, 454-462[Abstract]
50. Guex, N., and Peitsch, M. C. (1997) Electrophoresis 18, 2714-2723[Medline] [Order article via Infotrieve]


Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.