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Originally published In Press as doi:10.1074/jbc.M412900200 on January 4, 2005

J. Biol. Chem., Vol. 280, Issue 11, 10524-10529, March 18, 2005
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Crystal Structures and Amidolytic Activities of Two Glycosylated Snake Venom Serine Proteinases*

Zhongliang Zhu{ddagger}§, Zhi Liang{ddagger}§, Tianyi Zhang§, Zhiqiang Zhu{ddagger}§, Weihua Xu§, Maikun Teng{ddagger}§, and Liwen Niu{ddagger}§

From the {ddagger}Hefei National Laboratory for Physical Sciences at Microscale and Key Laboratory of Structural Biology, Chinese Academy of Sciences and §Department of Molecular and Cell Biology, School of Life Sciences, University of Science and Technology of China, 96 Jinzhai Road, Hefei, Anhui, 230026, The People's Republic of China

Received for publication, November 15, 2004 , and in revised form, January 4, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We deduced that Agkistrodon actus venom serine proteinases I and II, previously isolated from the venom of A. acutus (Zhu, Z., Gong, P., Teng, M., and Niu, L. (2003) Acta Crystallogr. Sect. D Biol. Crystallogr. 59, 547–550), are encoded by two almost identical genes, with only the single substitution Asp for Asn at residue 62. Amidolytic assays indicated that they possess slightly different enzymatic properties. Crystal structures of A. actus venom serine proteinases I and II were determined at resolution of 2.0 and 2.1 Å with the identification of trisaccharide (NAG301-FUC302-NAG303) and monosaccharide (NAG301) residues in them, respectively. The substrate binding sites S3 of the two proteinases appear much shallower than that of Trimeresurus stejnegeri venom plasminogen activator despite the overall structural similarity. Based on structural analysis, we showed that these Asn35-linked oligosaccharides collide spatially with some inhibitors, such as soybean trypsin inhibitor, and would therefore hinder their inhibitory binding. Difference of the carbohydrates in both the proteinases might also lead to their altered catalytic activities.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The information derived from the three-dimensional structures of snake venom serine proteinases (SV-SPs)1 remains of concern, because of the medical interest of venomosalivary serine proteinases. Most biochemical properties, biological roles, and structure of SV-SPs are mainly characterized by using TSV-PA, a plasminogen activator from Trimeresurus stejnegeri venom, with the structure of SV-SPs as the only available model (2–7). Wide variance of the sequences and enzymatic properties of these proteinases, which is the feature of SV-SPs (8, 9), requires more structural models to annotate the detailed relationship. In particular, although the carbohydrates in native TSV-PA were documented to have little influence on its amidolytic activity and plasminogen activation property (3, 4), most of SV-SPs contain carbohydrates at variant glycosylation sites, which tempts us to explore whether all of these proteinases match TSV-PA.

We have reported previously the purification, partial characterization, and crystallization of two glycosylated SV-SPs, AaV-SP-I and AaV-SP-II from the venom of Agkistrodon acutus (1). Both proteinases have the activity of esterolysis and fibrin-(ogen)olysis and share the same N-terminal amino acid sequence. The proteolytic and esterolytic activities of the proteinases could not be inhibited by some natural serine proteinase inhibitors (e.g. soybean trypsin inhibitor (SBTI)). Both proteinases have a postulated glycosylated site at Asn35, but the proportions of the carbohydrates within the two proteinases are different (i.e. ~9% for AaV-SP-I and ~4% for AaV-SP-II, respectively). In this study, we analyzed the entire amino acid sequences of the two proteinases and determined their three-dimensional structures and the linked oligosaccharide components. Structural comparison revealed that oligosaccharides in the two proteinases provide spatial hindrance toward the binding of some natural inhibitors and might be involved in the enzyme-inhibitor interactions as well as the alteration of their catalytic activities.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Sequence Analysis
Sequencing N-terminal Amino Acid Residues—Sequencing N-terminal amino acids was preformed as reported previously (1).

RT-PCR—Venom glands were obtained from snakes living in the southern mountains in Anhui province of China and stored in liquid nitrogen. Total RNA was extracted according to the protocol by using a total RNA isolating kit (Promega). Two primers for RT-PCR were designed based on the conserved sequence in various SV-SPs cDNAs, especially those of DAV-PA, acutobin, and tler7 (GenBankTM accession numbers AF159058 [GenBank] , AF159057 [GenBank] , and AF362127 [GenBank] ). The sense primer 5'-AACTGGTCATTGGAGGTA/GA-3' corresponds to the conserved propeptide and the N-terminal residues 16VIGGD/E20 (the amino acid residue numbering is according to sequence of chymotrypsinogen). The antisense primer 5'-CGGGGGGCAAGTC/TGCATC-3' corresponds to the C-terminal residues (245bDATCPP245g) of DAV-PA and DFA1 (GenBankTM accession number AF089847 [GenBank] ). RT-PCR was carried out for 30 cycles programmed for 1 min at 95 °C, 1 min at 55 °C, and 1.5 min at 72 °C.

Trypsin Digestion and Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry Assay—Both proteinases (0.2 mg) were dissolved in a 50-µl solution of 0.1 M ammonium bicarbonate, pH 7.5, and incubated with 1.5 µl of trypsin (4.3 mg/ml; treated with L-(tosylamido-2-phenyl) ethyl chloromethyl ketone) at 37 °C for 20 h. The digested mixtures were treated with 6 M guanidine hydrochloride and 1% (v/v) 2-mercaptoethanol at 50 °C for 30 min and then subjected to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry assay in a Bruker Biflex III mass spectrometer. The reflector-positive mode was calibrated by standard peptide mixture. All mass values were reported as monoisotopic mass [M+H]+.

Amidolytic Assay and Kinetic Analysis
The amidolytic activity was measured using chromogenic substrates containing modified oligopeptide with p-nitroanilide (p-NA) group. 50 µl of proteinases (20 µg/ml) were added into 450-µl solutions of various concentrations of substrates in 50 mM Tris-HCl, pH 7.8. The releasing of p-NA was then monitored continuously at 405 nm. The amount of substrates hydrolyzed was calculated from the increased absorbance at 405 nm with a molar extinction coefficient of 10,000 M-1·cm-1 for free p-NA (3). Evaluation of the kinetic parameters Km and kcat was performed using double-reciprocal Lineweaver-Burk plots (initial velocity versus substrate concentration).

Structure Determination and Refinement
Protein crystallization, data collection, and reduction are described elsewhere (1). The crystal structures were solved by molecular replacement using AMoRe (10). The structure of TSV-PA (Protein Data Bank code 1BQY [PDB] ) was used as a model for calculating the cross-rotation and translation function against the data set of AaV-SP-I. A unique solution was obtained. Initial model of AaV-SP-II was then determined using the refined structure of AaV-SP-I as a search model. All refinement steps were performed using the software package CNS (11), and the program O was used for manual rebuilding (12). Both of the refined models consist of all 234 amino acid residues of mature proteins. Three saccharide residues and two sulfate ions were identified in AaV-SP-I, and one saccharide residue and one sulfate ion in AaV-SP-II. The crystallographic R-factor and Rfree were 18.1% and 20.8% for AaV-SP-I and 16.5% and 21.1% for AaV-SP-II, respectively. Data collection and refinement statistics are shown in Table I. Figures were generated with the use of ESPript (13), O (12), and PyMol (14).


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  		TABLE I
Summary of crystallographic refinement

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Sequence Analysis—Our previous studies showed that the first 24 N-terminal residues of the two proteinases are identical (1). Here, the complete amino acid sequences of the two proteinases were identified by RT-PCR and mass spectra of the tryptic peptides (Fig. 1). Sequencing four RT-PCR clones rendered two kinds of nucleotide sequences with only a difference of one nucleotide, corresponding to the 62nd amino acid. The codon GAT (encoding Asp62) in two sequences was substituted for AAT (encoding Asn62) in the other two. Based on the difference between the pI values of the two proteinases (i.e. 5.3 and 5.7 for AaV-SP-I and AaV-SP-II, respectively (data not shown)), it was deduced that the nucleotide sequences encoding Asp62 and Asn62 correspond to those of AaV-SP-I and -II, respectively (Fig. 1A). This deduction was consistent with the result of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Both spectra of the tryptic digestions matched the deduced mass values very well, within a tolerance of ±0.5 Da. The fragments with specific tryptic cleavage sites and the corresponding values of monoisotopic masses are shown in Fig. 1, B and C.



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  		FIG. 1.
The cDNA and deduced amino acid sequences of AaV-SP-I and -II as well as the tryptic peptides of the two proteins. A, cDNA and deduced amino acid sequences of the AaV-SPs. The amino acid numbering is according to chymotrypsinogen. The residues sequenced by Edman degradation in Ref. 1 are shaded. Underlining indicates the fragments obtained from specifically tryptic cleavage, and the star (*) represents the different codon and corresponding amino acid between AaV-SP-I and -II. B and C, a summary of tryptic peptides obtained from AaV-SP-I and -II, respectively, analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. DM, detected mass (in daltons) by mass spectrometry; CM, calculated mass (in daltons) of peptide sequence deduced from cDNA; Tol, a tolerance of the detected mass from the calculated mass. Slash (/) indicates the specific cleavage sites of trypsin at NH2- and COOH-terminals. The residues involved in the specific cleavage sites and cleaved from the peptides are grayed.

 
AaV-SPs display ~40% sequence identity with non-snake venom serine proteinases, such as porcine trypsin and chymotrypsin. They share 72% sequence identity with TSV-PA (major variance of the sequences located in residues 169~179) and ~60% sequence identity with other SV-SPs (Fig. 2A). Residue 35 in AaV-SPs is asparagine, constituting a potential glycosylation site. The amino acid sequence of AaV-SP-II is found to be the same as that of DAV-PA from the venom of the same species Deinagkistrodon acutus (or Agkistrodon acutus; Ref. 15). As expected, the cDNA sequences of AaV-SP-I and -II differ by only two and one nucleotides with that of DAV-PA (GenBank accession number AAF76378 [GenBank] , respectively. The variance among the sequences of DAV-PA and AaV-SPs may result from the different living regions of the snakes (i.e. the former case is in Fujian province and the latter is in Anhui province of China). Because the publication on DAV-PA reported only sequence and species, no properties of the protein encoded by gene DAV-PA were provided. For consistency of presentation, we keep the names of the two proteinases as AaV-SP-I and -II here.



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  		FIG. 2.
Sequence and structure alignments. A, sequence alignment between TSV-PA, AaV-SP-I and -II. Identical residues are highlighted in red. B, stereoview superimposition of the C{alpha} atoms of AaV-SP-I (yellow), AaV-SP-II (red), and TSV-PA (blue). The surface loops, 37 loop, 174 loop, and 99 loop differ among the three proteinases, and the NH2- and COOH-terminals are labeled.

 
Amidolytic Activity—Both AaV-SP-I and -II possess amidolytic activity against several chromogenic substrates. The kinetic parameters Km, kcat and kcat/Km for those substrates are summarized in Table II. They both have significant preference for arginine at P1 site. The enzymatic activities for substrate N-p-tosyl-Gly-Pro-Lys-p-NA are nearly 100-fold lower than those for substrate N-p-tosyl-Gly-Pro-Arg-p-NA. Moreover, among the assayed substrates, N-p-tosyl-Gly-Pro-Arg-p-NA is the most efficient substrate for both proteinases. The similarity of substrate preference between them is in agreement with their high sequence identity and their nearly identical three-dimensional structures (see below). However, we also found that AaV-SP-I seemed to have higher values of kcat/Km than AaV-SP-II, for all of the four assayed substrates. The difference of the kcat/Km values of the two proteinases is caused mainly by the variance of kcat (Table II), which implied the structural variation between the two proteins at active site.


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  		TABLE II
Kinetic parameters: comparison of AaV-SP-I and -II Data have deviations of ~5% according to the mean of three assays.

 
Overall Crystal Structure—The overall structures of the two proteinases display a typical fold of trypsin-like serine proteinases. Both structures contain a C-terminal extension of seven residues, a common feature among SV-SPs, and have the same backbone as TSV-PA (Fig. 2, A and B). Similar to the structure of TSV-PA, a salt bridge between the C-terminal Pro245g and the amino group of Lys101 and a disulfide bridge between Cys245e and Cys91 result in a defined conformation of C-terminal extension. An internal salt bridge between the {alpha}-amino group of N-terminal Val and the carboxyl group of Asp194 is involved in the formation of active site. With a common cis Pro219 at this site, the S1 pockets of both proteinases are similar to that of TSV-PA and glandular kallikrein (Ref. 16; Protein Data Bank code 2PKA [PDB] ). As expected from the 72% sequence identity with TSV-PA, superimposing optimal 228 of all 234 C{alpha} atoms of AaV-SP-I and -II on those of TSV-PA rendered low root-mean-square deviations of 0.81 Å and 0.73 Å, respectively. Consistent with the sequence variance, the structural variances mainly localize at the 37 loop, the 174 loop, and the 99 loop (Fig. 2, A and B).

One significant feature of AaV-SPs is the N-glycosylated 37 loop, which contains an Asn35-X-Ser37 sequence, a typical motif for glycosylation. The Asn35-linked carbohydrates were identified in both structures. One monosaccharide NAG301 molecule in AaV-SP-II and one trisaccharide NAG301-FUC302-NAG303 molecule in AaV-SP-I were clearly displayed in the 2Fo-Fc maps (Fig. 3, A and B), which confirmed our previous proposition (1). The existence of a core-fucosylated at C6 of NAG301 as proved by the electron density map of AaV-SP-I was also in good agreement with the fact that most N-linked oligosaccharides of SV-SPs possess fucose (17, 18). Sidechains of Asn35 of both AaV-SPs and the linked carbohydrates protrude into the space between the 60 loop and the 37 loop. However, orientation of the carbohydrates is different. NAG301 of AaV-SP-II is nearer the 60 loop than that of AaV-SP-I, and the bond angle of the glycosidic bond between Asn35 and NAG301 of AaV-SP-I has a shift of about 20° compared with that of AaV-SP-II (Fig. 3C). The distinct orientation of carbohydrates in AaV-SP-I is also concomitant with the additional interactions between FUC302 and other residues. As shown in Fig. 3C, there are two hydrogen bonds, one between the O3 atom of FUC302 and the O atom of His57, and the other between the O4 atom of FUC302 and the O atom of Asp59. In addition, FUC302 even partially occupies the S1' site, implying that it might have an influence on the macromolecular substrate-enzyme interaction. Furthermore, because the 60 loop and the 37 loop are involved in the formation of the binding site toward some natural inhibitors, such as SBTI, it is possible that the protruded carbohydrates have an ability to influence the inhibitor binding. In fact, when the structures of the two AaV-SPs were superimposed on the structure of trypsin-SBTI complex (Ref. 19; Protein Data Bank code 1AVW [PDB] ), the NAG301 of AaV-SP-II collided with the side-chain of Phe502 of SBTI (Fig. 4A) and the trisaccharide NAG301-FUC302-NAG303 of AaV-SP-I occupied even the space encompassed by the residues 502–503, 564–566, and 510–511 of SBTI (Fig. 4B), resulting in broadly steric hindrance to bind SBTI. These hindrances of the carbohydrates of AaV-SPs for inhibitor binding agree with our previous experimental results that the proteolytic and esterolytic activities of AaV-SPs could not be inhibited by SBTI (1).



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  		FIG. 3.
The saccharides of AaV-SP-I and -II. A and B, the electron density (2Fo-Fc) maps of NAG301-FUC302-NAG303 and NAG301 of AaV-SP-I and AaV-SP-II are contoured at the 1.0 {sigma} level. C, superimposed C{alpha} atoms of segments formed by residues 30–70 for comparing the saccharide residues between AaV-SP-I (cyan) and -II (yellow). The His57 and the Asp59 of AaV-SP-I as well as saccharides of the two AaV-SPs are shown with a stick model. The two hydrogen bonds in AaV-SP-I, one between FUC302 and His57 and the other between FUC302 and Asp59, are labeled.

 



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  		FIG. 4.
Superimposition of AaV-SP-II (A) and AaV-SP-I (B) with trypsin-SBTI complex for showing the collision between the side-chains of some residues of SBTI and carbohydrates of AaV-SPs. Both AaV-SPs are shown in cyan, trypsin in yellow, and SBTI in magenta. NAG301, FUC302, and NAG303 of AaV-SPs and Phe502, Val503, Ile564, and Phe566 of SBTI are shown with a stick model.

 
174 Loop and 99 Loop—Conformation of the 174 loop in either AaV-SP differs from that of TSV-PA as well as those of non-snake venom serine proteinases, such as bovine trypsin (Ref. 20; Protein Data Bank code 1MTS [PDB] ), chymotrypsin (Ref. 21; Protein Data Bank code 1CHO [PDB] ), and human thrombin (Ref. 22; Protein Data Bank code 1DX5 [PDB] ). First, the segments of residues 175–178 in AaV-SPs protrude to the surface and get closer to the 99 loop compared with TSV-PA (Fig. 5). As the 174 loop and 99 loop participate in the formation of the substrate binding sites S2–S4, the different architectures and relative positions between the two loops influence the substrate specificity. Second, AaV-SPs have a hydrogen bond between the side chains of Glu173 and Tyr215 (Fig. 5), which is substituted by a residue Trp in TSV-PA. This hydrogen bond pulls the side chain of Tyr215 into the substrate-binding site S3 (Fig. 5), and makes the S3 site shallower than that of TSV-PA. It is consistent with their amidolytic activities. Both AaV-SPs show the highest efficiency for the substrate with P3 site occupied by small residue (i.e. N-p-tosyl-Gly-Pro-Arg-p-NA), whereas TSV-PA has a preference for bulky residue at P3 site (i.e. D-Phe-L-Pip-Arg-p-NA (Ref. 3; Table II)). Finally, because AaV-SPs possess the non-charged residue Leu at site 174, they lack the corresponding salt bridge between the side chains of Asp97 and Arg174 in TSV-PA.



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  		FIG. 5.
Comparison of the 174 loop and 99 loop in AaV-SP-I (cyan) AaV-SP-II (yellow), and TSV-PA (silver-gray) shows the architectural features of substrate binding sites S2~S4 of the three proteins. The key residues (215, 178, 173, 174, 97, 98, 99) for substrate-binding are shown with stick model. The hydrogen bonds between Tyr215 and Glu173 of the two AaV-SPs and the hydrogen bond between Glu97 and Tyr178 of AaV-SP-I are shown. The amino acid residues of AaV-SPs and TSV-PA are displayed in black and blue. The schematic substrate-binding sites 2, 3, and 4 are indicated with a light green ellipse.

 
Despite having almost the same sequence, AaV-SP-I and -II show a largely conformational variance in 99 loop with a maximal displacement of ~1.3 Å (Fig. 5). In TSV-PA, it has been suggested that Asp97 might alter its conformation to bind the substrate, because the salt bridge between the side-chains of Arg174 and Asp97 observed in the crystal structure would occlude the S4 site (2). We found that Glu97 consistently forms a hydrogen bond with the side chain of Tyr178 in AaV-SP-I, whereas the side chain of Glu97 was parallel to the phenyl ring of Tyr178 in AaV-SP-II (Fig. 5), indicating high flexibility of the residue 97. In addition, S2 site of AaV-SPs is different from that of TSV-PA. The S2 site in TSV-PA is defined by residues Val99 and Glu98. However, in AaV-SPs, it is mainly limited by residue Val98, because the Leu99 of AaV-SPs is oriented away from the site.

Comparison between AaV-SP-I and AaV-SP-II—The structures of AaV-SP-I and -II display high similarity, with a 0.34-Å root-mean-square deviation of superimposed C{alpha} atoms, which is consistent with their similar enzymatic properties. Besides the 99 loop, another significant difference between the two proteinases is located in the glycosylated 37 loop, which possibly correlates with their different carbohydrate constitutions. Although the base (residue 189–195) and the back (residue 225–228) of the S1 pocket in the two AaV-SPs exhibit apparent similarity, the entrances (residue 213–220) of S1 pocket in them are of some difference, which is possibly caused by the flexibility of side chains of the His218 and Pro219. Side-chain of His218 in TSV-PA was also considered flexible for its ill defined density. In addition, as a main component of the S2 site, side chains of Val98 have different conformation between the AaV-SPs (Fig. 5). Finally, the architectures of the catalytic triads vary a little in the two proteinases. The distance of the hydrogen bond between O atom of Ser195 and N {epsilon}2 atom of His is 2.9 Å, whereas the corresponding distance between Ser195 and His57 in AaV-SP-II is 3.2 Å. All of these variances between the two proteinases in the active site perhaps correlate with the variant enzymatic properties more or less, as shown in Table II.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Most SV-SPs are insensitive to some natural serine proteinase inhibitors, such as SBTI, hirudin, and heparin, although they show thrombin- or trypsin-like activities (9). Our previous experiments indicated that some natural inhibitors, such as SBTI and heparin, could not inhibit the esterolytic activities of AaV-SPs (1). In TSV-PA, the side chain of Phe193 was documented to play a crucial role for its insensitivity toward bovine pancreatic trypsin inhibitor (5). However, most SV-SPs have a distinct Gly193 and retain the resistance against inhibitors. Recent research showed that the 37 loop of TSV-PA could also influence the inactivation by some inhibitors (e.g.plasminogen activator inhibitor 1), indicating a key role of the 37 loop in the interaction between enzyme and inhibitors (7). It is interesting that structural superimposition of the AaV-SPs and the trypsin-SBTI complex indicates that the bulky residues of saccharide in the glycosylated 37 loop would hinder AaV-SPs from binding the inhibitor. The bulky ring of NAG301 of AaV-SP-II clashes with the residue Phe502 of SBTI, and the NAG301 and FUC302 of AaV-SP-I collide extensively with residues 503, 564, and 566 of SBTI (Fig. 4, A and B). Therefore, the glycosylation of the 37 loop in AaV-SPs is at least one reason for their resistance against the inhibition of SBTI. It is the first structural evidence for the function of the carbohydrates in SV-SPs and may help us to understand the biological roles of the Asn35-linked carbohydrates in other SV-SPs. Agkistrodon contortrix contortrix venom protein C, a SV-SP possessing a glycosylated 37 loop and used in clinical assays (23, 24), is sensitive to anti-thrombin III/heparin complex but not to SBTI (25). Therefore, the carbohydrates may not influence the heparin-mediated inhibition. The recombinant AaV-SPs would help further delineate the role of the glycosylated 37 loop. However, the Phe193 in TSV-PA and the glycosylated 37 loop in AaV-SPs are not common features for SV-SPs. It would be interesting to investigate the relationship between the distinct glycosylation forms and the resistance against inhibitors in other SV-SPs.

The substrate binding sites S3 of AaV-SPs are shallower than that of TSV-PA, which mainly results from a substitution at residue 215. Most non-snake-venom serine proteinases, such as bovine trypsin (20), chymotrypsin (21), and human thrombin (22), possess a Trp at residue 215. However, SV-SPs have a variance at this site. Two of the most frequent residues in SV-SPs are Trp and Tyr, as presented in TSV-PA and AaV-SPs, respectively. The distinct architecture of site S3 of AaV-SPs could explain the relatively high efficiency for the substrate with small residue at P3 site. Because tyrosine is the major residue at 215 in SV-SPs from venom of Agkistrodon acutus (15), it could also help us to understand the structural features of site S3 in other SV-SPs.

Our assays showed that the two AaV-SPs had different enzymatic properties, although they differ only at residue 62 at the 60 loop and the component of carbohydrates. Site-directed mutagenesis of TSV-PA showed that substitution of 60SNNFQ64 of TSV-PA by a segment 60RRFMR64 of batroxobin, another SV-SP from Bothrops atrox (26), did not significantly alter the catalytic parameters of TSV-PA (4), so the variance of amidolytic activity between AaV-SPs, perhaps, is not caused only by the single substitution of residue 62. On the other hand, the carbohydrates in the two AaV-SPs, NAG301-FUC302-NAG303 in AaV-SP-I and NAG301 in AaV-SP-II, are close to the active site (Fig. 3C). The distinct saccharides would have different influence on the structure of active site and consequently result in the alteration of amidolytic activity.


   FOOTNOTES
 
The atomic coordinates and structure factors (codes 1OP0 and 1OP2) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* This work was supported by research grants from the National Natural Science Foundation of China (Grants 30025012 and 30121001), the 973 Plan of the Ministry of Science and Technology of China (Grants G199905603 and 2002BA711A13), and the Chinese Academy of Sciences (Grants KSCX1-SW-17 and STZ01-29). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: School of Life Sciences, University of Science and Technology of China, 96 Jinzhai Rd., Hefei, Anhui, 230026, The People's Republic of China. Tel.: 086-551-3606334; Fax: 086-551-3603046; E-mail: lwniu{at}ustc.edu.cn.

1 The abbreviations used are: SV-SP, snake venom serine proteinase; TSV-PA, Trimeresurus stejnegeri venom plasminogen activator; AaV-SP, Agkistrodon actus venom serine proteinase; RT, reverse transcription; DAV-PA Deinagkistrodon acutus venom plasminogen activator; NAG, N-acetyl-D-glucosamine; FUC, fucose; SBTI, soybean trypsin inhibitor; p-NA, p-nitroanilide. Back


   ACKNOWLEDGMENTS
 
We thank Min Guo for editing the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Zhu, Z., Gong, P., Teng, M., and Niu, L. (2003) Acta Crystallogr. Sect. D Biol. Crystallogr. 59, 547-550[Medline] [Order article via Infotrieve]
  2. Parry, M. A., Jacob, U., Huber, R., Wisner, A., Bon, C., and Bode, W. (1998) Structure 6, 1195-1206[Medline] [Order article via Infotrieve]
  3. Zhang, Y., Wisner, A., Xiong, Y., and Bon, C. (1995) J. Biol. Chem. 270, 10246-10255[Abstract/Free Full Text]
  4. Zhang, Y., Wisner, A., Maroun, R. C., Choumet, V., Xiong, Y., and Bon, C. (1997) J. Biol. Chem. 272, 20531-20537[Abstract/Free Full Text]
  5. Braud, S., Parry, M. A., Maroun, R., Bon, C., and Wisner, A. (2000) J. Biol. Chem. 275, 1823-1828[Abstract/Free Full Text]
  6. Braud, S., Le Bonniec, B. F., Bon, C., and Wisner, A. (2002) Biochemistry 41, 8478-8484[CrossRef][Medline] [Order article via Infotrieve]
  7. Henriques, E. S., Fonseca, N., and Ramos, M. J. (2004) Protein Sci. 13, 2355-2369[Abstract/Free Full Text]
  8. Braud, S., Bon, C., and Wisner, A. (2000) Biochimie (Paris) 82, 851-859
  9. Matsui, T., Fujimura, Y., and Titani, K. (2000) Biochim. Biophys. Acta 1477, 146-156[CrossRef][Medline] [Order article via Infotrieve]
  10. Navaza, J. (1994) Acta Crystallogr. Sect. A 50, 157-163[CrossRef]
  11. Brünger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gross, P., Grosse-Kunstleve, R. W., Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T., and Warren, G. L. (1998) Acta Crystallogr. Sect. D Biol. Crystallogr. 54, 905-921[CrossRef][Medline] [Order article via Infotrieve]
  12. Jones, T. A., Zou, J. Y., Cowan, S. W., and Kjeldgaard, M. (1991) Acta Crystallogr. Sect. A 47, 110-119
  13. Gouet, P., Courcelle, E., Stuart, D. I., Metoz, F. (1999) Bioinformatics 15, 305-308[Abstract/Free Full Text]
  14. DeLano, W. L. (2002) The PyMOL Molecular Graphics System, DeLano Scientific, San Carlos, CA
  15. Wang, Y. M., Wang, S. R., and Tsai, I. H. (2001) Biochem. J. 354, 161-168[CrossRef][Medline] [Order article via Infotrieve]
  16. Bode, W., Chen, Z., Bartels, K., Kutzbach, C., Schmidt-Kastner, G., and Bartunik, H. (1983) J. Mol. Biol. 164, 237-282[CrossRef][Medline] [Order article via Infotrieve]
  17. Lochnit, G., and Geyer, R. (1995) Eur. J. Biochem. 228, 805-816[Medline] [Order article via Infotrieve]
  18. Pfeiffer, G., Dabrowski, U., Dabrowski, J., Stirm, S., Strube, K. H., and Geyer, R. (1992) Eur. J. Biochem. 205, 961-978[Medline] [Order article via Infotrieve]
  19. Song, H. K., and Suh, S. W. (1998) J. Mol. Biol. 275, 347-363[CrossRef][Medline] [Order article via Infotrieve]
  20. Stubbs, M. T., Huber, R., and Bode, W. (1995) FEBS Lett. 375, 103-107[CrossRef][Medline] [Order article via Infotrieve]
  21. Fujinaga, M., Sielecki, A. R., Read, R. J., Ardelt, W., Laskowski, M., Jr., and James, M. N. (1987) J. Mol. Biol. 195, 397-418[CrossRef][Medline] [Order article via Infotrieve]
  22. Fuentes-Prior, P., Iwanaga, Y., Huber, R., Pagila, R., Rumennik, G., Seto, M., Morser, J., Light, D. R., and Bode, W. (2000) Nature 404, 518-525[CrossRef][Medline] [Order article via Infotrieve]
  23. McMullen, B. A., Fujikawa, K., and Kisiel, W. (1989) Biochemistry 28, 674-679[CrossRef][Medline] [Order article via Infotrieve]
  24. Gempeler-Messina, P. M., Volz, K., Buhler, B., and Muller, C. (2001) Haemostasis 31, 266-272[CrossRef][Medline] [Order article via Infotrieve]
  25. Kisiel, W., Kondo, S., Smith, K. J., McMullen, B. A., and Smith, L. F. (1987) J. Biol. Chem. 262, 12607-12613[Abstract/Free Full Text]
  26. Itoh, N., Tanaka, N., Mihashi, S., and Yamashina, I. (1987) J. Biol. Chem. 262, 3132-3135[Abstract/Free Full Text]

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