Skip to main content
Download PDF

A biophysical elucidation for less toxicity of Agglutinin than Abrin-a from the Seeds of Abrus Precatorius in consequence of crystal structure

Journal of Biomedical Science volume 17, Article number: 34 (2010) Cite this article

Abstract

X-ray crystal structure determination of agglutinin from abrus precatorius in Taiwan is presented. The crystal structure of agglutinin, a type II ribosome-inactivating protein (RIP) from the seeds of Abrus precatorius in Taiwan, has been determined from a novel crystalline form by the molecular replacement method using the coordinates of abrin-a as the template. The structure has space group P41212 with Z = 8, and been refined at 2.6 Å to R-factor of 20.4%. The root-mean-square deviations of bond lengths and angles from the standard values are 0.009 Å and 1.3°. Primary, secondary, tertiary and quaternary structures of agglutinin have been described and compared with those of abrin-a to a certain extent. In subsequent docking research, we found that Asn200 of abrin-a may form a critical hydrogen bond with G4323 of 28SRNA, while corresponding Pro199 of agglutinin is a kink hydrophobic residue bound with the cleft in a more compact complementary relationship. This may explain the lower toxicity of agglutinin than abrin-a, despite of similarity in secondary structure and the activity cleft of two RIPs.

Background

Ribosome inactivating proteins (RIPs) are enzymes that can inactivate ribosomes. The molecular mechanism of inhibitory effect on protein synthesis has been shown that RIPs act as a RNA N-glycosidase hydrolyzing the C-N glycosidic bond of the adenosine residue at position 4324 in rat 28S rRNA [1, 2]. They can cleave the synthetic RNA structure having a short double-helical stem and a loop containing a centered GAGA sequence, the first A being the cleavage site [3]. The depurination inactivates the ribosomes for binding to elongation factor 2 catalyzing GTP hydrolysis and translocation of peptidyl-tRNA to the P site [4], with a consequence inhibiting the protein synthesis. There are three categories of RIPs according to the physical composition and characteristics. Most commonly RIPs are type I RIPs, only single polypeptide chain proteins composed of the toxophoric A subunit with a molecular mass around 30 kDa [5–8] such as curcin [9] and trichomislin [10]. Some are type II RIPs consisting of two types of polypeptide subunits, A chain of homologous and functionally similar to type I RIPs and B chain with a galactose-specific lectin domain that binds to cell surfaces, such as ricin [11] abrin and abrus agglutinin (AAG) [12]. A chain and B chain are from one gene and link through disulfide bond after post-translation modification [13]. Type III RIPs are derived from inactive pro-protein and activated after proteolysis [14]. The mature type III RIPs are two polypeptide subunits acting as an N-glycosidase jointly.

Various RIPs can be isolated from the same plants [15, 16]. Some type II RIPs have been isolated from the beans of the tropical and subtropical leguminous plant Abrus precatorius, jequirity. They are lectins and have an inhibitory effect on the growth of experimental animal tumors [17, 18]. They can be classified as abrins and AAG by oligomerization. Abrins are potent toxic heterodimeric glycoproteins with an LD50 of 20 μg/kg body weight; while AAG is a relatively less toxic heterotetrameric glycoprotein of which the LD50 is 5 mg/kg body weight [12]. But their therapeutics indexes are similar [18].

The primary structures of abrin-a and AAG were determined [19–21]. AAG had high homology to the extremely toxic ABRa, with 44 (8.0%) similar amino acid residues and 382 (69.8%) invariant amino acid residues. In the A chain of AAG, the 13 amino acid residues with catalytic function among RIPs were completely conserved [21]. The cDNAs of the RIPs isolated from Abrus precatorius have been cloned and their A chains were expressed in Escherichia. coli [21–23]. The amino acid residues at proposed active sites and Pro199 of AAG, which corresponding to Asn200 of abrin-a, were analyzed with site-directed mutagenesis for studying the structure and function of these RIPs [21, 23, 24]. And the results showed that Pro199 in A- (or C-) chain of AAG impair the activity of protein synthesis inhibition because of steric hindrance [21]. According to the biochemical experiments, the mutation of Asn200 on abrin a-chain to Pro200 dramatically decreases the activity than other kind of mutation, including those residues without side-chain, such as Gly [23, 24]. These peculiar results motivate us to crystallize AAG, and make comparison with abrin, since both contains almost identical active pocket, and most important of all, different at Asn200 (the corresponding residue is on AAG Pro199). Bagaria et al., [25] reported a 3.5 Å X-ray crystal structure, and proposed the less toxic nature is because of the fewer interactions involved with the substrate adenine.

Bagaria et al., [25] assigned their low resolution of AAG crystal to belong to the space group of P42212, instead of our present and previous P41212 [26], to analyze the crystal structure based on a mixture of indigenous and alien data. They crystallized their Indian AAG material in a condition similar to, but different from ours [25, 26]. Strange to us, they did not determine their own Indian AAG amino acid sequence, but adopted the Taiwanese primary structure [21, 25]. Indian AAG molecular packing may be different from our Taiwanese that could manifest itself some way in different space group. Although they published the controversial paper of 60 kDa structure in advance [25], this detail worthwhile work of more complicated and precise 120 kDa heterotetramer agglutinin structure spurs the continuous study of our last research [26].

Methods

Purification

AAG was isolated from the kernels of Abrus precatorius seeds by chromatographies on a Sepharose 6B column and a Sephadex G-100 column as described previously [12]. The flow rate of chromatography was 20 ml/hr and protein concentration was determined by the bicinchonic acid method [27]. The kernels of 200 g were soaked in 5% cold acetic acid of 1 L overnight and homogenized. After centrifuging at 10,000 g at 4°C for 15 mins, the supernatant was collected for subsequently subjecting to the ammonium sulfate fraction between 35 and 90 and then centrifuging at 10,000 g at 4°C for 20 mins. The precipitate was collected for dialysis against cold 10 mM sodium phosphate buffer, pH 8 at 4°C. The dialysis buffer was changed every 8 hrs for more than 2 days. The supernatant of dialysate was centrifuged at 17,800 g at 4°C for 20 mins and then applied on a Sepharose 6B affinity column (3.0 × 50 cm) pre-equilibrated and washed with 10 mM sodium phosphate buffer, pH 8. The eluent constiting of abrins and AAG were obtained with the elution buffer, the wash buffer containing 100 mM D-galactose. Then the precipitate was obtained from the eluent subjected to 90% ammonium sulfate and dialyzed and centrifuged as mentioned above. The supernatant was loaded onto gel filtration on Sephadex G-100 column (2.2 × 100 cm) with 10 mM sodium phosphate buffer, pH 8. Two major peaks can be observed and the fractions of AAG, corresponding to the first peak, were pooled and lyophilized.

Crystallization

The formula for crystallization was described in our previous paper [26]. Crystals suitable for X-ray analysis were obtained by the sitting drop vapor-diffusion method at room temperature (297 (2) K) [28]. 8 μl of protein solution at a concentration of 10 mg/ml prepared from lyophilized protein was mixed with 8 μl of reservoir solution containing PEG 8000; the precipitant condition was 0.1 M Tris pH 7.5 with 6.5% PEG 8000 plus 1% sodium azide and crystals appeared after nearly four months.

Data Collection

X-ray Data were collected with a crystal of dimensions 0.30 × 0.30 × 0.25 mm that was mounted in a cryo-loop manufactured by Hampton Research. After immersed in the cryo-protectant of 20% glycerol and 80% mother liquor for several seconds, the cryo-loop was mounted on goniometer head inside liquid nitrogen stream at 100 K. X-ray diffraction was measured with CCD (ADSC Quantum-Q4R CCD Area Detector), on 1 D synchrotron radiation X-ray (SPring-8 Taiwan Contract Beam-line BL12B2 of NSRRC). The crystal-to-detector distance was 215 mm. The space group and unit-cell parameters were determined from the well resolved diffraction spots. The data were processed using the programs HKL2000 [29]. The agglutinin crystal belongs to the tetragonal system, with unit-cell parameters a = b = 137.05, c = 214.42 Å, V = 4.0275 × 106 Å3, Z = 8. A 99.1% complete dataset to 2.47 Å resolution of 73,976 unique reflections was collected with averaged Rsym of 7.2%, averaged χ2 of 1.153, averaged I/σ of 11.89, and redundancy of 4.1.

Determination of space group and initial phase

The systematic absences, l = 4n + 1, 2, 3 for 00l reflections, and h = 2n + 1 for h00 reflections, indicate that there are two possible space groups, namely P41212 or P43212. The ambiguity of space group was solved together with the initial phase problem by molecular replacement method using version 1.1 of CNS program [30] with the coordinates of abrin-a [31] as model. An X-ray diffraction data shell from 4 to 15 Å was used for the calculation of the cross rotation function with CNS program [32]. The highest two were corresponding to a rotation of the model by the rotation angel of θ1 = 37.9E, θ2 = 39.6E, θ3 = 342.1E, and θ1 = 358.1E, θ2 = -0.5E, θ3 = 2.4E in the space group of P41212. After translation searches with CNS program [33] according to these two rotation angles, the initial model of AB- and CD-chains of agglutinin was established.

Crystallographic Refinement

Structural refinement were performed in the following iteration steps: rigid body refinement [34], simulated annealing [35] of residue coordinates, group B factor refinement [34], density modification [36], manual manipulation using O program [37], and energy minimization [38]. The crystal data and R factor are listed in Table 1. The final R factor using all reflections in the resolution range 2.6 to 30 Å is 20.4%, while Rfree using randomly selected 10% reflections which were excluded from refinement is 23.6%. The Ramachandran plot including A-, B-, C-, and D-chains is acceptable as shown in Table 1.

Table 1 Crystal data and refinement statistics for AAG.
Full size table

Docking

The program SPHGEN identifies the active site, and other sites of interest, and generates the sphere centers that fill the site. It has been described in the original paper [39]. The program GRID generates the scoring grids [40, 41]. Within the DOCK suite of programs, the program DOCK matches spheres (generated by SPHGEN) with ligand atoms and uses scoring grids (from GRID) to evaluate ligand orientations [38, 39]. Program DOCK also minimizes energy based scores [42]. Parameters used in DOCK were modified from the paper of protein docking and complementary principle [43].

The atomic coordinates of the refined agglutinin structure and the reflection data have been deposited with the Protein Data Bank in Japan. The accession numbers for these atomic coordinates are (PDB ID) 2ZR1and (RCSB ID) RCSB028317.

Results and Discussion

As shown in figure 1, the AAG AB-chains are very similar to the abrin-a molecule, the structure of which has been described in detail [31]. A conserved disulfide bond between Cys246 of A (or C)-chain and Cys8 of B (or D)-chain holds the two chains tightly as shown in figure 1. An asymmetric unit of AAG crystal contains four peptide chains, AB- and non-crystallographical-symmetric related CD-chains, as shown in figure 1. The two heterodimers AB and CD are bonded together through hydrogen bonds by using the water molecules between them as intermediate bridges. They are identical except two N-acetylglucosamines (NAGs) are found in AB-chains, and one in CD-chains. An AAG molecule is a tetramer, consisting of AB (or CD) and symmetry-related A'B' (or C'D').

Figure 1
figure 1

Comparison of AAG with abrin-a (green) molecule. The α-carbon backbone of abrin-a AB-chains are superimposed on that of the AAG molecule using least-squares analysis. A P41212 asymmetric unit of AAG contains an AB-chain and a CD-chain. Disulphide bonds are plotted as big yellow balls. This figure was generated by O program (Jones et al., 1991).

Full size image

Structure of the AAG A(or C)-chain

The AAG A(or C)-chain was divided into three folding domains γ1,γ2, and γ3 by reference to the description of the abrin-a A-chain [31], and to the CATH database [44]. Figure 2 shows the sequence and secondary structures, while figure 3 shows the cartoon of the three domains. Domain γ1 (figure 3(a)), composed of residues 1 to 111, consists of two β-sheets and two α-helices. The former β-sheets include six strands of adefgh (sheet 1) and two strands of bc (sheet 2), while the latter α-helices include helix A of residues 13 to 27, and helix B of residues 91 to 96. The strands and helices alternate in the order aAbcdefgBh. In sheet 1, the first strand, a, of the β-sheet 1 and the last strand, h, lie parallel to the neighboring strands, d and g, respectively. The four central strands of the β-sheet 1, d to g, are anti-parallel. In sheet 2, strands b and c are anti-parallel. The main differences between domains γ1 of AAG and abrin-a occurred in N-terminal. The N-terminal of the AAG A-chain is one residue shorter than that of the abrin-a A-chain and the first five terminal residues are different. Domain γ2, residues 112 to 195, is dominated by five helices (figure 3(b)), C to G. Helix C, composed of residues 112 to 119, D, residues 120 to 141, E, residues 147 to 166, F, residues 168 to 180, and G, residues 188 to 194. Helix C is 3 residues longer than that of abrin-a, due to replacement of Thr114 and Arg118 in abrin-a by Asp113 and Lys117 in AAG. Other secondary structures in domain 2 are almost conserved in abrin-a and AAG. Domain γ3 (figure 3(c)), composed of residues 198 to 250, contains two helices, H, residues 197 to 206 and I, residues 234 to 238, and a β-sheet of two anti-parallel strands, i and j, situated in the order HijI, and a random coil in the C terminal part. The last 8 residues in the C terminal of A-chain are severely disordered, and we could not determine their structures by X-ray diffraction.

Figure 2
figure 2

AAG A (or C)-chain sequence & secondary structures. The symbol of "arrow" represents a β-strand, "spiral" represents an α-helix, "dot" represents missing residues, and the alphabets a, b, A, etc, denote the corresponding secondary structures in figure 3.

Full size image
Figure 3
figure 3

Three domains of AAG A (or C)-chain: (a) domain γ1, (b) domain γ2, (c) domain γ3. These figures were generated by O program (Jones et al., 1991).

Full size image

Structure of the AAG B (or D)-chain

The overall folding of the AAG B (or D)-chain and the abrin-a B-chain is very similar, as shown in figure 1, and the disulfide bond connecting A- and B-chains is conserved. The α-carbon traces of their N terminal, residues 1 to 12 differ significantly. The first four residues in the AAG B (or D)-chain are severely disordered, and we could not determine their structures by X-ray diffraction. The AAG B-chain is composed of two homologous domains, δ1 and δ2, mainly formed by β-sheets and loops. Figure 4 shows the sequence and secondary structures, while figure 5 shows the cartoon of the two domains. Domain δ1 (figure 5(a)), composed of residues 5 to 140, consists of five anti-parallel β-sheets, one 4-stranded (of ijkl), one 3-stranded (of aef), and three 2-stranded (strands bm, cd, and gh respectively), and one α-helix of residues 90 to 94. The strands and helices alternate in the order abcdefghAijklm. Domain δ2 (figure 5(b)), composed of residues 141 to 267, consists of four anti-parallel β-sheets, including two 4-stranded (strands ynqr and uvwx respectively), and two 2-stranded (strands op and st) sheets.

Each domain of δ1 and δ2 contains two intra-domain disulfide bonds (Cys25-Cys44, Cys68-Cys85, Cys156-169, and Cys195-Cys212), which are conserved in abrin-a. Two NAGs are found in B-chain, but only one presents in D chain. The NAGs are bound to B-Asn100 (figure. 6), B-Asn140, and D-Asn140 respectively. The bond length between NAG and Asn140 is 1.45 Å.

Figure 4
figure 4

AAG B (or D)-chain sequence & secondary structures. The symbol of "arrow" represents a β-strand, "spiral" represents an α-helix, "dot" represents missing residues, and the alphabets a, b, A, etc, denote the corresponding secondary structures in figure 5.

Full size image
Figure 5
figure 5

Two domains of AAG B (or D)-chain: (a) domain δ1, and (b) domain δ2. These figures were generated by O program (Jones et al., 1991).

Full size image
Figure 6
figure 6

Electron density of the NAG (red) near B 100Asn using the (2Fo - Fc) map contoured at 2.0 F. This figure was generated by O program (Jones et al., 1991).

Full size image

Structure of Active site

The active site is exactly the cleft formed by the intersection of all 3 domains in AAG A (or C)-chain. The location of the active site region of the AAG A (or C)-chain is shown in figure 7(a), and enlarged in figure 7(b). Five invariant residues (Tyr73, Tyr112, Glu163, Arg166 and Trp197) and five conserved residues (Asn71, Arg123, Gln159, Glu194 and Asn195) are located in the active site cleft. The alignment of the amino acid sequences shows that all five invariant residues in the active site of abrin-a are absolutely conserved throughout the wide range of ribosome-inactivating proteins [19, 45]. The similarity of active site structures between abrin-a and AAG shows in figure 7(b) that they may work in the same way, but could not explain the less than half biochemical activity of AAG. We try to answer this question by the 28SRNA docking study.

Figure 7
figure 7

Three domains of AAG A (or C)-chain are drawn as ribbons. (a) Gray purple and green indicate domain γ1 and green indicate domain γ1 γ2 γ2 and γ3 respectively. Active site residues are drawn in red. (b) Active Site comparison of abrin-a (red) and γ3 respectively. Active site residues are drawn in red. (b) Active Site comparison of abrin-a (red) AAG A-chain (black) AAG A-chain (black) and AAG C-chain (blue). These figures were generated by O program (Jones et al. and AAG C-chain (blue). These figures were generated by O program (Jones et al. 1991) and UCSF Chimera [32].

Full size image

Quaternary Structure of AAG

An AAG molecule is a hetero-tetramer (as shown in figure 8) contains two subunits, ABA'B' (or CDC'D'), stabilized by mainly hydrophilic and little hydrophobic forces. The two subunits are in equivalent positions of the space group P41212. The transformation from AB to A'B' is (x, y, z) to (1-y, 1-x, 0.5-z), while CD to C'D' is (x, y, z) to (y, x, 1-z). The hydrophilic interaction is dominated by inter-subunit hydrogen bonds, as listed in table 2. These hydrogen bonds belong to residues of domains γ2 and γ2'. Since the γ2 domain is almost made up with α-helices, which hydrophobic side-chains are buried inside, hydrophobic forces contribute little to the stabilization of quaternary structure of AAG. The total buried surface area is 9360 for ABA'B' and 9460 for CDC'D' interfaces. The gain in hydrophobic energy is -68 KCal/Mol for ABA'B' and -72 KCal/Mol for CDC'D'. The buried surface and hydrophobic energy are calculated by Protein interfaces, surfaces and assemblies service PISA at European Bioinformatics Institute http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html[46].

Figure 8
figure 8

Ribbon presentation of AAG quaternary structure: Red residues indicate the active site location. Purple and green residues constitute inter-subunit hydrogen bonds. Domain γ2s are drawn in brown. This figure was generated by O program (Jones et al., 1991).

Full size image
Table 2 Hydrogen bonds between inter-subunit with symmetry-related AA' and CC' chains.
Full size table

Docking of 28SRNA to AAG and Abrin-a

As pointed out in the mutagenesis study [21], Pro199 in A- (or C-) chain of AAG impair the activity of protein synthesis inhibition. Bagaria et al., [25] suggested that the less toxic nature is because of the fewer interactions involved with the substrate adenine. From our docking study, we found Asn200 of abrin-a may form a critical hydrogen bond with G4323 of 28SRNA, while corresponding Pro199 of agglutinin is a non-extended residue bound with the cleft in a more compact complementary relationship as shown in figure 9(c). This may explain the lower toxicity of agglutinin, despite of similarity in secondary structure and the activity cleft of two RIPs. The docking model of 28SRNA was artificially deformed at the ribose sugar of A4324 from the X-ray crystal structure 430D [47], as shown in figure 9(a), so that A4324 of 28SRNA can overlap with the adenine of the ricin-adenine complex, 1IFS [48]. Figure 9(b) shows deformed model fit well in the active clefts of both abrin-a and AAG. Figures 9(a), 9(b) and 9(c) were generated with UCSF Chimera [49].

Figure 9
figure 9

(a) The docking model (blue) and the original 28SRNA (yellow). A4324 was artificially manipulated. (b) The docking of 28SRNA (blue) on abrin-a A-chain (purple) and AAG A-chain (yellow). (c) G4323 (red) of 28SRNA docking model (blue), is hydrogen-bonded with Asn2000 of abrin-a A-chain (purple), but has no interaction with corresponding Pro199 of AAG A-chain (yellow).

Full size image

We want to explore whether the difference in the critical residue, bring any change in structure. However, we could not observe significant main-chain distortion due to the difference in the 200 residue on higher resolution structure. Hence, the reason for the lower toxicity of agglutinin than abrin-a might be due to the deformation from inactive to active state of abrin depends on the meta-stable huge helix, composed of helix h and helix g. The mutation of Asn200 to Pro200 destroys the mechanism.

Conclusion

We have successfully solved the structure of 120 kDa heterotetramer agglutinin AB and CD chains, and reduced the R-factor to 20.4% at 2.6 Å resolution data. Ten disulfide bonds, three N-acetylglucosamines, and 169 water molecules were found in the successive (2Fo-Fc) map. 22 hydrogen bonds between A (or C)-chain and symmetry-related A' (or C') were found. Water molecules were not found in the Bagaria's paper and no subsequent hydrogen bond lengths were listed based on their lower resolution structure [25]. Docking study revealed that due to Pro199, agglutinin is unable to form a critical hydrogen bond with G4323 of 28SRNA, which is found in the docking result of abrin-a. This may explain the lower toxicity of agglutinin than abrin-a, despite of similarity in secondary structure and the activity cleft of two RIPs.

Abbreviations

RIP:

ribosome-inactivating protein

AAG:

agglutinin

NAG:

N-acetylglucosamine

Fo:

observed structure factor

Fc:

calculated structure factor

PDB:

protein data bank.

References

  1. Endo Y, Misui K, Motizuki K, Tsurugi K: The mechanism of action of ricin and related toxic lectins on eukaryotic ribosomes. The site and the characteristics of the modification in 28 S ribosomal RNA caused by the toxins. J Biol Chem. 1987, 262: 5908-5912.

    CAS  PubMed  Google Scholar 

  2. Endo Y, Tsurugi K: RNA N-glycosidase activity of ricin A-chain Mechanism of action of the toxic lectin ricin on eukaryotic ribosomes. J Biol Chem. 1987, 262: 8128-8130.

    CAS  PubMed  Google Scholar 

  3. Endo Y, Gluck A, Wool IG: Ribosomal RNA identity elements for ricin A-chain recognition and catalysis. J Mol Biol. 1991, 221: 193-207. 10.1016/0022-2836(91)80214-F.

    Article  CAS  PubMed  Google Scholar 

  4. Jimenez A, Vazquez DC: Plant and Fungal Protein and Glycoprotein Toxins Inhibiting Eukaryote Protein Synthesis. Annu Rev Microbiol. 1985, 39: 649-672. 10.1146/annurev.mi.39.100185.003245.

    Article  CAS  PubMed  Google Scholar 

  5. Barbieri L, Stirpe F: Ribosome-inactivating proteins from plants: properties and possible uses. Cancer Surv. 1982, 1: 489-520.

    Google Scholar 

  6. Stirpe F, Barbieri L, Battelli MG, Soria M, Lappi DA: Ribosome-inactivating proteins leads to increased fungal protection in transgenic tobacco plants. Bio-Technology. 1992, 10: 405-412.

    Article  CAS  PubMed  Google Scholar 

  7. Hartley MR, Lord JM: Cytotoxic ribosome-inactivating lectins from plants. Biochim Biophys Acta. 2004, 1701: 1-14.

    Article  CAS  PubMed  Google Scholar 

  8. Olsnes S: The history of ricin abrin and related toxins. Toxicon. 2004, 44: 361-370. 10.1016/j.toxicon.2004.05.003.

    Article  CAS  PubMed  Google Scholar 

  9. Lin J, Chen Y, Xu Y, Yan F, Tang L, Chen F: Cloning and expression of curcin a ribosome inactivating protein from the seeds of jatropha curcas. Acta Botanica Sinica. 2003, 45: 858-863.

    CAS  Google Scholar 

  10. Mi SL, An CC, Wang Y, Chen JY, Che NY, Gao Y, Chen ZL: Trichomislin a novel ribosome-inactivating protein a novel ribosome-inactivating protein induces apoptosis that involves mitochondria and caspase-3. Archives of Biochemistry and Biophysics. 2005, 434: 258-265. 10.1016/j.abb.2004.11.009.

    Article  CAS  PubMed  Google Scholar 

  11. Olsnes S, Phil A: Different biological properties of the two constituent peptide chains of ricin a toxic protein inhibiting protein synthesis. Biochemistry. 1973, 12: 3121-3126. 10.1021/bi00740a028.

    Article  CAS  PubMed  Google Scholar 

  12. Lin JY, Lee TC, Hsu ST, Tung TC: Isolation of four isotoxic proteins and one agglutinin from jequiriti bean (Abrus precatorius). Toxicon. 1981, 19: 41-51. 10.1016/0041-0101(81)90116-1.

    Article  CAS  PubMed  Google Scholar 

  13. Lord JM: Synthesis and intracellular transport of lectin and storage protein precursors in endosperm from castor bean. Eur J Biochem. 1985, 146: 403-409. 10.1111/j.1432-1033.1985.tb08666.x.

    Article  CAS  PubMed  Google Scholar 

  14. Mundy J, Leah R, Boston R, Endo Y, Stirpe F: Genes encoding ribosome-inactivating proteins. Plant Mol Biol Rep. 1994, 12: 60-62. 10.1007/BF02671573.

    Article  Google Scholar 

  15. Leah R, Tommerup H, Svendsen I, Mundy J: Biochemical and molecular characterization of three barley seed proteins with antifungal properties. J Biol Chem. 1991, 266: 1564-1573.

    CAS  PubMed  Google Scholar 

  16. Desvoyes B, Poyet JL, Schlick JL, Adami P, Jouvenot M, Dulieu P: Identification of a biological inactive complex form of pokeweed antiviral protein. FEBS Lett. 1997, 410: 303-308. 10.1016/S0014-5793(97)00648-0.

    Article  CAS  PubMed  Google Scholar 

  17. Olsnes S, Phil A: In Receptors and Recognition Series: The Specificity and Action of Animal. Bacterial and Plant Toxins. Edited by: Cuatrecasas P. 1982, Chapman and Hall, London, 31-131.

    Google Scholar 

  18. Lin JY, Li JS, Tung TC: Lectin Derivatives of Methotrexate and Chlorambucil as Chemotherapeutic Agents. J Natl Cancer Inst. 1981, 66: 523-528.

    CAS  PubMed  Google Scholar 

  19. Funatsu G, Taguchi Y, Kamenosno M, Yanaka M: The complete amino acid sequence of the A-chain of abrin-a a toxic protein from the seeds of Abrus precatorius. Agric Biol Chem. 1988, 52: 1095-1097.

    Article  CAS  Google Scholar 

  20. Chen YL, Chow LP, Tsugita A, Lin JY: The complete primary structure of abrin-a B chain. FEBS Lett. 1992, 309: 115-118. 10.1016/0014-5793(92)81076-X.

    Article  CAS  PubMed  Google Scholar 

  21. Liu CL, Tsai CC, Lin SC, Wang LI, Hsu CI, Hwang MJ, Lin JY: Primary Structure and Function Analysis of the Abrus precatorius Agglutinin A Chain by Site-directed Mutagenesis. J Biol Chem. 2000, 275: 1897-1901. 10.1074/jbc.275.3.1897.

    Article  CAS  PubMed  Google Scholar 

  22. Hung CH, Lee MC, Lee TC, Lin JY: Primary Structure of Three Distinct Isoabrins Determined by cDNA Sequencing: Conservation and Significance. J Mol Biol. 1993, 229: 263-267. 10.1006/jmbi.1993.1029.

    Article  CAS  PubMed  Google Scholar 

  23. Hung CH, Lee MC, Chen JK, Lin JY: Cloning and expression of three abrin A-chains and their mutants derived by site-specific mutagenesisin Escherichia coli. Eur J Biochem. 1994, 219: 83-87. 10.1111/j.1432-1033.1994.tb19917.x.

    Article  CAS  PubMed  Google Scholar 

  24. Chen JK, Hung CH, Liaw YC, Lin JY: Identification of amino acid residues of abrin-a A chain is essential for catalysis and reassociation with abrin-a B chain by site-directed mutagenesis. Protein Engineering. 1997, 10: 827-833. 10.1093/protein/10.7.827.

    Article  CAS  PubMed  Google Scholar 

  25. Bagaria A, Surendranath K, Ramagopal UA, Ramakumar S, Karande AA: Structure-Function Analysis and Insights into the Reduced Toxicity of Abrus precatorius Agglutinin I in Relation to Abrin. J Biol Chem. 2006, 281: 34465-34474. 10.1074/jbc.M601777200.

    Article  CAS  PubMed  Google Scholar 

  26. Panneerselvam K, Lin SC, Liu CL, Liaw YC, Lin JY, Lu TH: Crystallization of agglutinin from the seeds of Abrus precatorius. Acta Cryst. 2000, D56: 898-899.

    CAS  Google Scholar 

  27. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC: Measurement of protein using bicinchoninic acid. Anal Biochem. 1985, 150: 76-85. 10.1016/0003-2697(85)90442-7.

    Article  CAS  PubMed  Google Scholar 

  28. McPherson A: Preparation and Analysis of Protein Crystals. 1982, John Wiley & Sons New York USA, 94-96. 115

    Google Scholar 

  29. Otwinowski Z, Minor W: Processing of X-ray Diffraction Data Collected in Oscillation Mode. Methods in Enzymology. Macromolecular Crystallography part A. 1997, 276: 307-326. full_text.

    Article  CAS  Google Scholar 

  30. Brunger AT, Adams PD, Clore GM, Delano WL, Gros P, Grosse-Kunstleve RW, Jiang JS, Kuszewski J, Nilges M, Pannu NS, Read RJ, Rice LM, Simonson T, Warren GL: Crystallography & NMR system: A new software system for macromolecular structure determination. Acta Cryst. 1998, D54: 905-921.

    CAS  Google Scholar 

  31. Tahirov TH, Lu TH, Liaw YC, Chen YL, Lin JY: Crystal Structure of Abrin-a at 2.14 D. J Mol Biol. 1995, 250: 354-367. 10.1006/jmbi.1995.0382.

    Article  CAS  PubMed  Google Scholar 

  32. DeLano WL, Brunger AT: The Direct Rotation Function: Rotational Patterson Correlation Search Applied to Molecular Replacement. Acta Cryst. 1995, D51: 740-748.

    CAS  Google Scholar 

  33. Brunger AT: Extension of molecular replacement: A new search strategy based on Patterson correlation refinement. Acta Cryst. 1990, A46: 46-57.

    Article  CAS  Google Scholar 

  34. Brunger AT: The Free R Value: a Novel Statistical Quantity for Assessing the Accuracy of Crystal Structures. Nature. 1992, 355: 472-474. 10.1038/355472a0.

    Article  CAS  PubMed  Google Scholar 

  35. Brunger AT, Krukowski A, Erickson J: Slow-Cooling Protocols for Crystallographic Refinement by Simulated Annealing. Acta Cryst. 1990, A46: 585-593.

    Article  CAS  Google Scholar 

  36. Abrahams JP, Leslie AG W: Methods used in the structure determination of bovine mitochondrial F1 ATPase. Acta Cryst. 1996, D52: 30-42.

    CAS  Google Scholar 

  37. Jones TA, Zou JY, Cowan SW, Kjeldgaard M: Improved methods for the building of protein models in electron density maps and the location of errors in these models. Acta Cryst. 1991, A47: 110-119.

    Article  CAS  Google Scholar 

  38. Adams PD, Pannu NS, Read RJ, Brunger AT: Cross-validated Maximum Likelihood Enhances Crystallographic Simulated Annealing Refinement. Proc Natl Acad Sci USA. 1997, 94: 5018-5023. 10.1073/pnas.94.10.5018.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  39. Kuntz ID, Blaney JM, Oatley SJ, Langridge R, Ferrin TE: A geometric approach to macromolecule-ligand interactions. J Mol Biol. 1982, 161: 269-288. 10.1016/0022-2836(82)90153-X.

    Article  CAS  PubMed  Google Scholar 

  40. Shoichet BK, Bodian DL, Kuntz ID: Molecular docking using shape descriptors. J Comp Chem. 1992, 13: 380-397. 10.1002/jcc.540130311.

    Article  CAS  Google Scholar 

  41. Meng EC, Shoichet BK, Kuntz ID: Automated docking with grid-based energy evaluation. J Comp Chem. 1992, 13: 505-524. 10.1002/jcc.540130412.

    Article  CAS  Google Scholar 

  42. Meng EC, Gschwend DA, Blaney JM, Kuntz ID: Orientational sampling and rigid-body minimization in molecular docking. Proteins. 1993, 17: 266-278. 10.1002/prot.340170305.

    Article  CAS  PubMed  Google Scholar 

  43. Shoichet BK, Kuntz ID: Protein docking and complementarity. J Mol Biol. 1991, 221: 327-346. 10.1016/0022-2836(91)80222-G.

    Article  CAS  PubMed  Google Scholar 

  44. Pearl F, Todd A, Sillitoe I, Dibley M, Redfern O, Lewis T, Bennett C, Marsden R, Grant A, Lee D, Akpor A, Maibaum M, Harrison A, Dallman T, Reeves G, Diboun I, Addou S, Lise S, Johnston C, Sillero A, Thornton J, Orengo C: The CATH Domain Structure Database and related resources Gene3D and DHS provide comprehensive domain family information for genome analysis. Nucl Acids Res. 2005, D247-D251. 33 Database

  45. Rutenber E, Robertus JD: Structure of ricin B-chain at 2.5 Å resolution. Proteins Struct Func Genet. 1991, 10: 260-269. 10.1002/prot.340100310.

    Article  CAS  Google Scholar 

  46. Krissinel E, Henrick K: Inference of macromolecular assemblies from crystalline state. J Mol Biol. 2007, 372: 774-797. 10.1016/j.jmb.2007.05.022.

    Article  CAS  PubMed  Google Scholar 

  47. Correll CC, Munishkin A, Chan YL, Ren Z, Wool IG, Steitz TA: Crystal structure of the ribosomal RNA domain essential for binding elongation factors. Proc Natl Acad Sci USA. 1998, 95: 13436-13441. 10.1073/pnas.95.23.13436.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  48. Weston SA, Tucker AD, Thatcher DR, Derbyshire DJ, Pauptit RA: X-ray structure of recombinant ricin A-chain at 1.8 Å resolution. J Mol Biol. 1994, 244: 410-422. 10.1006/jmbi.1994.1739.

    Article  CAS  PubMed  Google Scholar 

  49. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE: UCSF Chimera - A Visualization System for Exploratory Research and Analysis. J Comput Chem. 2004, 25: 1605-1612. 10.1002/jcc.20084.

    Article  CAS  PubMed  Google Scholar 

  50. Laskowski RA, MacArthur MW, Moss DS, Thornton JM: PROCHECK: a program to check the stereochemical quality of protein structures. J App Cryst. 1993, 26: 283-291. 10.1107/S0021889892009944.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors thank Mr Shyh-Ming Chen for setting up the computing programs. They also thank to the National Science Council of Taiwan for financial support. They are indebted to SPring-8 and the National Synchrotron Radiation Research Center for data collection.

Author information

Authors and Affiliations

  1. Department of Physics, National Tsing Hua University, Hsinchu, 30013, Taiwan

    Jack Cheng & Tian-Huey Lu

  2. Graduate School of Biochemical Engineering, Ming Chi University of Technology, Taishan, Taipei, 24301, Taiwan

    Chao-Lin Liu

  3. Institute of Biochemistry, College of Medicine, National Taiwan University, Taipei, 10018, Taiwan

    Jung-Yaw Lin

Authors
  1. Jack Cheng
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tian-Huey Lu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Chao-Lin Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jung-Yaw Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Tian-Huey Lu.

Additional information

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

JC collected the X-ray diffraction data, analyzed the crystal structure and prepared the initial manuscript. LTH set up the laboratory of the crystal structure determination, screened the crystallization conditions and got the right one, and advised such studies. LJY and LCL purified the material of the agglutinin.

Authors’ original submitted files for images

Rights and permissions

Open Access This article is published under license to BioMed Central Ltd. This is an Open Access article is distributed under the terms of the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/2.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Reprints and permissions

About this article

Cite this article

Cheng, J., Lu, TH., Liu, CL. et al. A biophysical elucidation for less toxicity of Agglutinin than Abrin-a from the Seeds of Abrus Precatorius in consequence of crystal structure. J Biomed Sci 17, 34 (2010). https://doi.org/10.1186/1423-0127-17-34

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/1423-0127-17-34

Keywords

Comments

Journal of Biomedical Science

ISSN: 1423-0127