The Atomic Resolution Crystal Structure of Atratoxin Determined by Single Wavelength Anomalous Diffraction Phasing
2004; Elsevier BV; Volume: 279; Issue: 37 Linguagem: Inglês
10.1074/jbc.m403863200
ISSN1083-351X
AutoresX. Lou, Qun Liu, Xiongying Tu, Jing Wang, Maikun Teng, Liwen Niu, David J. Schuller, Qingqiu Huang, Quan Hao,
Tópico(s)Ion-surface interactions and analysis
ResumoBy using single wavelength anomalous diffraction phasing based on the anomalous signal from copper atoms, the crystal structure of atratoxin was determined at the resolution of 1.5 Å and was refined to an ultrahigh resolution of 0.87 Å. The ultrahigh resolution electron density maps allowed the modeling of 38 amino acid residues in alternate conformations and the location of 322 of 870 possible hydrogen atoms. To get accurate information at the atomic level, atratoxin-b (an analog of atratoxin with reduced toxicity) was also refined to an atomic resolution of 0.92 Å. By the sequence and structural comparison of these two atratoxins, Arg33 and Arg36 were identified to be critical to their varied toxicity. The effect of copper ions on the distribution of hydrogen atoms in atratoxin was discussed, and the interactions between copper ions and protein residues were analyzed based on a statistical method, revealing a novel pentahedral copper-binding motif. By using single wavelength anomalous diffraction phasing based on the anomalous signal from copper atoms, the crystal structure of atratoxin was determined at the resolution of 1.5 Å and was refined to an ultrahigh resolution of 0.87 Å. The ultrahigh resolution electron density maps allowed the modeling of 38 amino acid residues in alternate conformations and the location of 322 of 870 possible hydrogen atoms. To get accurate information at the atomic level, atratoxin-b (an analog of atratoxin with reduced toxicity) was also refined to an atomic resolution of 0.92 Å. By the sequence and structural comparison of these two atratoxins, Arg33 and Arg36 were identified to be critical to their varied toxicity. The effect of copper ions on the distribution of hydrogen atoms in atratoxin was discussed, and the interactions between copper ions and protein residues were analyzed based on a statistical method, revealing a novel pentahedral copper-binding motif. α-Neurotoxins are small three-finger proteins, which can specifically bind to acetylcholine receptors (AChRs) 1The abbreviations used are: AChRs, acetylcholine receptors; AChBP, acetylcholine-binding protein; r.m.s., root mean square; RACE, rapid amplification of cDNA ends; Ea, erabutoxin-A; SAD, single wavelength anomalous diffraction; PDB, Protein Data Bank.1The abbreviations used are: AChRs, acetylcholine receptors; AChBP, acetylcholine-binding protein; r.m.s., root mean square; RACE, rapid amplification of cDNA ends; Ea, erabutoxin-A; SAD, single wavelength anomalous diffraction; PDB, Protein Data Bank. on the postsynaptic membrane and hence inhibit signal transmission across the synapse (1Changeux J.P. Devillers-Thiery A. Chemouilli P. Science. 1984; 225: 1335-1345Crossref PubMed Scopus (437) Google Scholar). Two groups of neurotoxins have been characterized in various snake venoms as follows: short chain α-neurotoxin with 60-62 amino acids and four disulfide bonds; and long chain α-neurotoxin with 65-74 amino acids and an additional disulfide bond at the C terminus. Crystal and NMR structures for both groups of α-neurotoxins indicate that they share high structural homology, with three finger-like loops protruding from the palm-like core (2Low B.W. Preston H.S. Sato A. Rosen L.S. Searl J.E. Rudko A.D. Richardson J.S. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 2991-2994Crossref PubMed Scopus (245) Google Scholar, 3Tsernoglou D. Petsko G.A. FEBS Lett. 1976; 68: 1-4Crossref PubMed Scopus (200) Google Scholar). Because of the potential pharmacological significance for this protein family (4Le Du M.H. Ricciardi A. Khayati M. Menez R. Boulain J.C. Menez A. Ducancel F. J. Mol. Biol. 2000; 296: 1017-1026Crossref PubMed Scopus (15) Google Scholar), the binding activity of α-neurotoxins with their acetylcholine receptors has been intensively investigated based on the crystal structure of an acetylcholine-binding protein (AChBP) (5Brejc K. van Dijk W.J. Klaassen R.V. Schuurmans M. van Der Oost J. Smit A.B. Sixma T.K. Nature. 2001; 411: 269-276Crossref PubMed Scopus (1564) Google Scholar). These studies have shown that both long chain α-neurotoxin and short chain α-neurotoxin have binding activity with AChBP. The complex structural studies for α-bungarotoxin (a long chain α-neurotoxin) and its putative binding peptide in AChBP indicate that the second loop of α-bungarotoxin could insert into the interface of two adjacent subunits of the AChBP homopentamer to realize its binding activity (6Harel M. Kasher R. Nicolas A. Guss J.M. Balass M. Fridkin M. Smit A.B. Brejc K. Sixma T.K. Katchalski-Katzir E. Sussman J.L. Fuchs S. 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No crystal structures to date explain such functional differences clearly, especially at atomic resolution. In this study, we present atomic resolution crystal structures of two pharmacologically important proteins, atratoxin and atratoxin-b (a homolog of atratoxin but with lower toxicity) from the Naja atra venom. The atomic resolution crystal structure of atratoxin-b was refined to 0.92 Å from the previously reported (10Lou X.H. Tu X.Y. Pan G.Q. Xu C.Y. Fan R. Lu W.H. Deng W.H. Rao P.F. Teng M.K. Niu L.W. Acta Crystallogr. Sect. D Biol. Crystallogr. 2003; 59: 1038-1042Crossref PubMed Scopus (6) Google Scholar) structure at 1.5 Å resolution determined by molecular replacement. However, the atratoxin structure could not be solved by molecule replacement because there are two planar atratoxin molecules in the crystallographic asymmetric unit. By using erabutoxin A (PDB code 5EBX, the same model used to phase the atratoxin-b structure) as the search model, it was not possible to identify solutions because of the high R factors (∼53%), low correlation coefficients (∼19%), and no clear contrast between possible solutions and noise. Considering that the protein was crystallized in the presence of copper ions, anomalous diffraction data to 1.5 Å resolution at the copper K-edge (λ = 1.379 Å) were collected in addition to an atomic resolution (0.87 Å) data set collected at a wavelength of 0.916 Å. By using a SAD phasing method developed jointly by the Institute of Physics, Chinese Academy of Sciences, and MacCHESS, Cornell University (11Fan H.F. Han F.S. Qian J.Z. Acta Crystallogr. Sect. A. 1984; 40: 495-498Crossref Scopus (14) Google Scholar, 12Hao Q. Gu Y.X. Zheng C.D. Fan H.F. J. Appl. Cryst. 2000; 33: 980-981Crossref Scopus (61) Google Scholar, 13Hao Q. Gu Y.X. Yao J.X. Zheng C.D. Fan H.F. J. Appl. Crystallogr. 2003; 36: 1274-1276Crossref Scopus (9) Google Scholar), the crystal structure of atratoxin was determined at 1.5 Å resolution and finally refined to an atomic resolution of 0.87 Å. This resolution is higher than any structure published to date on postsynaptic neurotoxins (14Corfield P.W. Lee T.J. Low B.W. J. Biol. Chem. 1989; 264: 9239-9242Abstract Full Text PDF PubMed Google Scholar, 15Kimball M.R. Sato A. Richardson J.S. Rosen L.S. Low B.W. Biochem. Biophys. Res. Commun. 1979; 88: 950-959Crossref PubMed Scopus (76) Google Scholar, 16Smith J.L. Corfield P.W. Hendrickson W.A. Low B.W. Acta Crystallogr. Sect. A. 1988; 44: 357-368Crossref PubMed Scopus (89) Google Scholar, 17Bourne P.E. Sato A. Corfield P.W. Rosen L.S. Birken S. Low B.W. Eur. J. Biochem. 1985; 153: 521-527Crossref PubMed Scopus (52) Google Scholar, 18Saludjian P. Prange T. Navaza J. Menez R. Guilloteau J.P. Rieskautt M. Ducruix A. Acta Crystallogr. Sect. B Struct. 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The comparison of these two atomic resolution structures shows clearly the essential residues and their configurations involved in the binding of atratoxin to its target, which could be of importance in protein engineering (24Vita C. Roumestand C. Toma F. Menez A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6404-6408Crossref PubMed Scopus (146) Google Scholar); the correlation between thermal motions of atratoxin at atomic resolution and the dynamic behavior of its NMR structures was revealed by the comparison of the anisotropy of atratoxin and the r.m.s. deviation of multiple NMR structures. In addition, the co-crystallization of copper in the crystal of atratoxin not only aids the phasing of the structure from one wavelength anomalous diffraction data but also causes the dimerization of atratoxin molecules in crystals and could affect hydrogen distributions in the atratoxin. Furthermore, the toxicity of atratoxin was analyzed under the condition of Cu2+ binding, providing an alternative way to study the toxicity of the protein. Determination of Copper Content in Crystals—In order to determine the ratio between the copper ions and the atratoxin molecule, four crystals from the crystallization drops were dissolved into 120 μl of 0.1 m HCl solutions. The A280 value of the sample solution was obtained by using a DU640 nucleic acid and protein analyzer (Beckman Coulter). The concentration of the protein was calculated from the standard curve of the proteins. The concentration of copper ions was examined by using an inductively coupled plasma atomic emission spectrometer (Thermo Jarrell Ash Corp.). The sample was nitrated with nitric acid before loading onto the inductively coupled plasma atomic emission spectrometer. The Multimeric State of Atratoxin in Copper Solution—To study the effect of copper ions on the multimeric state of atratoxin, 20-μl protein samples were loaded onto a Protein-Pak™ Diol (OH) 100-μm 7.8 × 300-mm column, mounted on a Waters 600 system, pre-equilibrated with 0.02 m NaAc-HAc buffer, pH 4.6 (containing 0.15 m NaCl), and then eluted with the same solution. Various ratios of atratoxin to copper ions were achieved by using incremental protein concentrations of 1, 10, and 50 mg/ml. The monitor wavelength was 280 nm, and the flow rate was 0.5 ml/min. Protein Sequence and Gene Cloning—The sequence of atratoxin was determined by the Edman degradation method as implemented on an Applied Biosystems Procise 491 protein sequencer. The N-terminal and C-terminal amino acid residues were sequenced by using protein from dissolved crystals of atratoxin. The first strand cDNA synthesis and final amplification of two genes were obtained by using the access reverse transcriptase-PCR method. Typically, venom gland total RNA was isolated from the venom gland (from the southern mountain region, Anhui Province, China) with SV Total RNA Isolation System (Promega) according to the manufacturer's protocol. Compared with other neurotoxins, the conservative sequence was obtained and used to design two primers as follows: sense, 5′-AGGATACACCCTGGAATGTC-3′ (N terminus), and antisense, 5′-ATTGTTGCATCTGTCTGTTGTGC-3′ (C terminus). PCR products were subcloned into the pGEM-T vector (Promega) and sequenced (Takara, Japan). According to the sequence, the 3′-RACE amplification was performed with the following primers: 5′-CACTACAGGTTGTTCAGGT-3′ for atratoxin and 5′-AAAGTGAAGCCCGGTGTTAATC-3′ for atratoxin-b. The 5′-RACE amplification was performed with the following primers: atratoxin, 5′-(P)-TTTCAATGCCGTT-3′, 5′-GGCGTGATCACCGTGGATA-3′, 5′-GCAATTGGTCTCCCCACC-3′, 5′-AGGGGATGTGGTTGCCCTTC-3′, and 5′-GCGATGATTGTTGGTTGTGAC-3′; atratoxin-b, 5′-(P)-GATTAACACCGGG-3′, 5′-GGTCTGATCACCGTGGAAC-3′, 5′-GGTCTCCCCTGAACAAGT-3′, 5′-CGAAAGGGGATGTGGTTGCC-3′, and 5′-GCGATGATTGTTGGTTGTGAC-3′. 5′- and 3′-RACE were used to determine the nucleotide sequence of the 5′ and 3′ end cDNAs with the Full Race Core Set cDNA amplification kit (Takara, Japan). Crystallization and Data Collection—Atratoxin was purified and crystallized using procedures reported previously (25Tu X.Y. Huang Q.Q. Lou X.H. Teng M.K. Niu L.W. Acta Crystallogr. Sect. D Biol. Crystallogr. 2002; 58: 839-842Crossref PubMed Scopus (5) Google Scholar). Crystals of ∼0.4 × 0.3 × 0.3 mm were soaked in a NaCl solution (pH 4.6) containing 10% ethanol, 10 mm CuCl2, and 25% (v/v) glycerol for about 20 s and then subjected to cryogenic flash freezing at a temperature of 100 K. Both SAD data and atomic resolution data were collected under the same cryoprotection conditions. The anomalous data set was collected at the MacCHESS F2 station. The x-ray wavelength was optimized to 1.379 Å (equivalent to 8.98 keV) to collect the strongest anomalous signal from copper as indicated by fluorescence scanning. A total of 360 frames was recorded with an oscillation angle of 1° from one crystal. Shortly afterward, the atomic resolution data set to 0.87 Å was collected at MacCHESS F1 station, at a fixed wavelength of 0.916 Å. Data processing for both anomalous data and atomic resolution data was performed with the HKL2000 package (26Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref Scopus (38253) Google Scholar), and the statistics of data collection and processing are summarized in Table I. The Rmerge values for anomalous data and atomic resolution data were 14.0 and 6.2%, respectively, indicating that using a shorter wavelength, which usually means less absorption of x-ray to the crystal, may give better crystallographic statistics.Table ICrystallographic data and structure refinement statistics for two neurotoxinsData collectionSAD-atratoxinaThe data set of atratoxin was collected for SAD phasing.AtratoxinAtratoxin-bSpace groupC2221C2221P41212Unit cell dimensions a, b, c (Å)46.72, 47.29, 89.8546.81, 47.29, 90.0248.46, 48.46, 44.13Resolution ranges (Å)bThe values for the data in the highest resolution shell are shown in parentheses.20-1.50 (1.53-1.50)30-0.87 (0.90-0.87 Å)30-0.92 (0.94-0.92)Wavelength (Å)1.3790.920.92Temperature (K)100100100No. observations191,227443,936474,033No. unique reflections16,06976,11636,852Overall completeness (%)cCompleteness is the ratio of the number of observed reflections to the number of possible reflections.98.4 (99.0)92.8 (38.8)99.4 (92.0)Ratio of mean I/(I)26.67 (4.50)57.16 (3.55)81.02 (6.16)Rmerge (%)dRmerge=∑hkl∑j|I(hkl)j−〈I(hkl)〉|/∑hkl∑jI(hkl)j. I(hkl)j is the observed intensity of the jth reflection, and 〈I(hkl)〉 is the mean intensity of reflection hkl.14.0 (28.1)6.2 (42.6)7.1 (26.1)Structure refinement and validation Resolution limits for refinement (Å)10-0.8730-0.92Ratio of observations to parameters5.646.24R factor (%)11.9611.42Rfree factor (%)15.2913.29No. amino acid residues12461No. copper ions5.54 (12 sites)No. sodium ions2No. chlorine ions1No. ethanol molecules2No. water molecules248122No. TRS molecules1No. sulfate ion1Root mean square (bond lengths) (Å)0.0150.014Root mean square (bond angles) (°)3.12.4a The data set of atratoxin was collected for SAD phasing.b The values for the data in the highest resolution shell are shown in parentheses.c Completeness is the ratio of the number of observed reflections to the number of possible reflections.d Rmerge=∑hkl∑j|I(hkl)j−〈I(hkl)〉|/∑hkl∑jI(hkl)j. I(hkl)j is the observed intensity of the jth reflection, and 〈I(hkl)〉 is the mean intensity of reflection hkl. Open table in a new tab Atratoxin-b was purified and crystallized as reported before (10Lou X.H. Tu X.Y. Pan G.Q. Xu C.Y. Fan R. Lu W.H. Deng W.H. Rao P.F. Teng M.K. Niu L.W. Acta Crystallogr. Sect. D Biol. Crystallogr. 2003; 59: 1038-1042Crossref PubMed Scopus (6) Google Scholar). Crystals of ∼0.3 × 0.3 × 0.2 mm were soaked in crystallization mother liquid with 25% (v/v) glycerol for 20 s and then subjected to flash freezing at the temperature of 100 K for cryoprotection. The atomic resolution data of 0.92 Å were collected and processed at MacCHESS F1 station using a similar procedure as described above. Crystallographic statistics for atratoxin-b data are also listed in Table I. Phasing with SAD Data for Atratoxin—To determine the structure of atratoxin from the anomalous scattering of copper atoms, a novel three-step phasing protocol was adopted, enabling the structure to be solved in few hours. The first step was to find substructures of these anomalous scatterers by a direct methods implementation (13Hao Q. Gu Y.X. Yao J.X. Zheng C.D. Fan H.F. J. Appl. Crystallogr. 2003; 36: 1274-1276Crossref Scopus (9) Google Scholar). The second step was to find the correct hand for the substructure without trying both possibilities in the following steps. The last step was to resolve phase ambiguity arising from SAD (11Fan H.F. Han F.S. Qian J.Z. Acta Crystallogr. Sect. A. 1984; 40: 495-498Crossref Scopus (14) Google Scholar, 12Hao Q. Gu Y.X. Zheng C.D. Fan H.F. J. Appl. Cryst. 2000; 33: 980-981Crossref Scopus (61) Google Scholar). Find Heavy Atoms Substructures with the Program SAPI—The copper anomalous scattering sites were located by the direct methods program SAPI (13Hao Q. Gu Y.X. Yao J.X. Zheng C.D. Fan H.F. J. Appl. Crystallogr. 2003; 36: 1274-1276Crossref Scopus (9) Google Scholar), using the magnitudes of the anomalous differences, for reflections up to 2.5 Å resolution. Three potential copper sites were found by SAPI in about half a minute with default running parameters. There was a clear gap between three copper atoms peaks and other peaks in terms of peak height. Two rounds of Karle-recycle calculation made the substructure converge to an R factor of 31.3%, strongly indicating the correctness of the solution. Select the Correct Hand with the Program ABS—If the arrangement of anomalous scatterers is non-centrosymmetric then it is also necessary to find their absolute configuration (hand). This is usually done by going through all phasing procedures with two possible configurations and manually choosing one that gives right-hand α-helices in the electron density map. The ABS program, based on the Ps function method (27Woolfson M.M. Yao J. Acta Cryst. Sect. D. Biol. Crystallogr. 1994; 50: 7-10Crossref PubMed Google Scholar, 28Hao Q. J. Appl. Crystallogr. 2004; 37: 498-499Crossref Scopus (66) Google Scholar), has been written to determine the absolute configuration at a much earlier stage, i.e. immediately after the heavy atoms sites are found by a direct methods or a Patterson procedure. For these three copper coordinates, hand was correctly assigned by using anomalous data at about 3.0 Å resolution cut off. The calculation of ABS is very fast and can usually finish in less than a minute. Resolve the Phase Ambiguity (SAD Phasing) and Improve Phases—Initial phases for all reflections to 1.5 Å resolution were generated by using the program OASIS (11Fan H.F. Han F.S. Qian J.Z. Acta Crystallogr. Sect. A. 1984; 40: 495-498Crossref Scopus (14) Google Scholar). The average figure of merit of phases was 0.54. Density modification using the CCP4 program DM (29Number Collaborative Computational Project Acta Crystallogr. Sect. D. Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19668) Google Scholar) was then applied to the resulting phase set, and the average FOM was gradually improved to 0.65. Both electron density maps calculated using phases after OASIS and after OASIS/DM are traceable for the protein main chain. However, the OASIS/DM map gives better protein solvent contrast and clearer density. Model Building and Refinement of Atratoxin—The main chain was built automatically by the program ARP/wARP (30Perrakis A. Sixma T.K. Wilson K.S. Lamzin V.S. Acta Crystallogr. Sect. D. Biol. Crystallogr. 1997; 53: 448-455Crossref PubMed Scopus (479) Google Scholar) using diffraction data from 12 to 1.5 Å. The initial R factor was 41.2%. After 50 cycles of free atom unrestrained refinements, the connectivity index increased steadily from 0.62 to 0.90, and the main chain peptide was successfully traced. In total, 110 residues, pertaining to two chains, were found by the program. Except for a few disordered residues at the molecular surface and the loop area, it was very easy to recognize and to build almost all side chains based on both 2Fo - Fc and Fo - Fc maps using the program O (31Jones T.A. Zou J.Y. Cowan S.K. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (12999) Google Scholar). Residues with poor electron density were assigned according to the cDNA sequence. Three peaks that appear on the difference map at the contour of 8.0σ corresponded to the anomalous scatterer peaks found by SAPI and were modeled as copper atoms. The crystallographic R factor of the model built at this stage (including the dummy free atoms) dropped to 18.5%. Further refinement with REFMAC (32Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D. Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13712) Google Scholar) was performed with cross-validation by using 5% of the observed reflections. By using the high resolution data set in the resolution range 10-1.2 Å, the model was first refined to convergence with an R factor of 22.0% and an Rfree factor of 24.0% after solvent building. Then the diffraction data were transformed to SHELX format, and the 5% validation reflections were kept. The refinement continued with SHELX97-2 (33Sheldrick G.M. Schneider T.R. Methods Enzymol. 1997; 277: 319-343Crossref PubMed Scopus (1867) Google Scholar) using conjugate gradient least squares minimization against an intensity-based residual target function that included distance, planarity, and chiral restraints. A bulk solvent correction (SWAT) was carried out. New ordered solvent molecules were found in the difference Fourier map. 2Fo - Fc and Fo - Fc electron density maps were calculated after each step, and the model (protein and other molecules) was checked and rebuilt. The R and Rfree factors calculated after the first run, with isotropic B factors, were 21.68 and 24.63%, respectively. Then the anisotropic displacement parameters were introduced into the refinement, reducing the R and Rfree factors to 17.20 and 20.70%, respectively. At this stage, clear electron density was observed near several side chains, which were subsequently modeled in two conformations by the inspection of both Fo - Fc and 2Fo - Fc maps. In relation to the side chain disorder, the main chain was also found to adopt two conformations in residues 31 and 32 of molecule A and residues 34 and 35 of molecule B. Two ethanol molecules were added in the subsequent refinement based on the Fourier difference map. Hydrogen atoms were added to the model according to geometrical criteria, decreasing the R and Rfree factors by 0.5 and 0.9% respectively. In the post-refinement, nine partial occupancy copper ions and two sodium ions were added, and the structure was refined to convergence again, leading to a final R factor of 11.96% Rfree of 15.29% for all data in the resolution range 10-0.87 Å. Structural Refinement of Atratoxin-b—For atratoxin-b, the 1.55-Å crystal structure of the same protein (PDB code 1onj) was chosen as the starting model. The refinement continued with SHELX97-2 (33Sheldrick G.M. Schneider T.R. Methods Enzymol. 1997; 277: 319-343Crossref PubMed Scopus (1867) Google Scholar) by using similar procedures as described for atratoxin. The final R factor and Rfree factor of atratoxin-b after adding riding hydrogen atoms were 11.42 and 13.29%, respectively. Eight residues were modeled as having alternative conformations in the final structure. Other structural statistics are listed in Table I. Protein Sequence and Gene Cloning—The N-terminal sequence of intact atratoxin, determined by protein sequencing, is LECHNQQSSQTPTTTGCSGGETNCYKKR (this is a correction to the previously reported result (25Tu X.Y. Huang Q.Q. Lou X.H. Teng M.K. Niu L.W. Acta Crystallogr. Sect. D Biol. Crystallogr. 2002; 58: 839-842Crossref PubMed Scopus (5) Google Scholar)). The C-terminal fragment sequence of the enzymatically digested fragments of atratoxin is NGIEINCCTTDRCNN. By reverse transcriptase-PCR utilizing the N-terminal and C-terminal sequence primers, two genes (atratoxin and atratoxin-b) were obtained (Fig. 1). Based on the gene sequences, the primers for 3′- and 5′-RACE were designed, and amplification was performed. The cloned atratoxin cDNA is 470 bp in length, encoding a product of 83 amino acid residues. Sequence alignment reveals that the peptide sequence of atratoxin is identical to that of cobrotoxin (34Yu C. Bhaskaran R. Chuang L.C. Yang C.C. Biochemistry. 1993; 32: 2131-2136Crossref PubMed Scopus (62) Google Scholar), but their mRNAs have only 97% identities. There is a single mutation in the third base of the codon (ACC→ACA) in the encoding regions, which still deduces the same residue (a threonine) at position 13, and the other differences lie in the 3′-untranslational regions sporadically. We refer to the protein as atratoxin throughout the paper. Overall Crystallographic Structure—The final refined model of atratoxin contains 124 amino acid residues belonging to 2 molecules, A and B, 2 ethanol molecules, 12 copper ions (some with partial occupancy), 1 chlorine ion, 2 sodium ions, and 248 water molecules in the asymmetric unit. In total, 38 amino acid residues could be modeled as multiple conformations. Both molecules are folded into three adjacent loops rich in β-pleated sheets that protrude from a small globular core made rigid by four disulfide bridges (Cys3-Cys24, Cys17-Cys41, Cys43-Cys54, and Cys55-Cys60), and displaying the characteristic "three-finger" structure with a concave bowl-shaped face commonly found in neurotoxin (Fig. 2). The main chain of each molecule is organized into two anti-parallel β-pleated sheets as follows: one consists of strand 1 (residues 2-6) and strand 2 (residues 12-16) through four backbone H-bonds, and the other is the triple-stranded anti-parallel sheet with strand 3 (residues 24-30), strand 4 (residues 36-40), and strand 5 (residues 51-55). There are lower B factors for two stable β-sheets with H-bonds between strand 3 and strand 4, and between strand 3 and strand 5, respectively. But loop II (connecting strand 3 and strand 4) and loop III (the peripheral strand region 43-50) exhibit relatively higher B factors and less stability. The packing between the two molecules in the asymmetric unit adopts a back-to-face binding pattern (Fig. 2). Cu1 plays a key role in stabilizing the dimer through strong interactions with Glu38 of molecule A and His4, Arg59, and Asn62 of molecule B (Fig. 6A). Superposition reveals that molecule A and molecule B are not identical in conformation. The r.m.s. deviation between the C-α atoms of the two-stranded β-sheet is 0.140 Å and that between the C-α atoms of the triple-stranded β-sheet is 0.356 Å. The major difference between the molecules occurs in loop II and loop III. When these two loops are excluded from the superposition calculation, the r.m.s. deviation will drop to 0.320 Å from 0.759 Å for all C-α atoms (Fig. 3A).Fig. 3Structural and anisotropic comparison of atratoxin with other neurotoxins. A, superposition of the C-α atoms of molecule A (green), molecule B (red), atratoxin-b (blue), mean NMR structure (PDB code 1cod) (brown), and erabutoxin A (black). B, anisotropy variation of the C-α atoms in atratoxin molecule A (green), atratoxin molecule B (red), and atratoxin-b (blue) as a function of residue number. C, r.m.s. deviation of C-α atoms between 11 cobrotoxin NMR structures as a function of residue number.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Comparison with the Solution Structure of Cobrotoxin—The solution NMR structure of cobrotoxin has been reported (34Yu C. Bhaskaran R. Chuang L.C. Yang C.C. Biochemistry. 1993; 32: 2131-2136Crossref PubMed Scopus (62) Google Scholar), and its protein sequence is the same as that of atratoxin. The overall folds of the two structures are similar. The r.m.s. deviations between C-α atoms of the crystal structure and 11 generated NMR structures range from 1.85 to 2.84 Å. The largest deviations in the NMR structure from the crystal structure generally involve the regions of loop I, loop II, loop III, and one turn (residues 18-22). The superposition of the backbone atoms of the x-ray structure and the mean solution structure is shown in Fig. 3A. Comparison of the backbone torsion angles between the mean solution structure and x-ray structure shows that residues 6, 9, 10, 18-20, 43, 45-48, and 58 have
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