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Chitin-binding Proteins in Invertebrates and Plants Comprise a Common Chitin-binding Structural Motif

2000; Elsevier BV; Volume: 275; Issue: 24 Linguagem: Inglês

10.1074/jbc.c000184200

1083-351X

T. Suetake, Sakae Tsuda, Shun-ichiro Kawabata, Kazunori Miura, Sadaaki Iwanaga, Kunio Hikichi, Katsutoshi Nitta, Keiichi Kawano,

Lipid Membrane Structure and Behavior

Tachycitin, a 73-residue polypeptide having antimicrobial activity is present in the hemocyte of horseshoe crab (Tachypleus tridentatus). The first three-dimensional structure of invertebrate chitin-binding protein was determined for tachycitin using two-dimensional nuclear magnetic resonance spectroscopy. The measurements indicate that the structure of tachycitin is largely divided into N- and C-terminal domains; the former comprises a three-stranded β-sheet and the latter a two-stranded β-sheet following a short helical turn. The latter structural motif shares a significant tertiary structural similarity with the chitin-binding domain of plant chitin-binding protein. This result is thought to provide faithful experimental evidence to the recent hypothesis that chitin-binding proteins of invertebrates and plants are correlated by a convergent evolution process. Tachycitin, a 73-residue polypeptide having antimicrobial activity is present in the hemocyte of horseshoe crab (Tachypleus tridentatus). The first three-dimensional structure of invertebrate chitin-binding protein was determined for tachycitin using two-dimensional nuclear magnetic resonance spectroscopy. The measurements indicate that the structure of tachycitin is largely divided into N- and C-terminal domains; the former comprises a three-stranded β-sheet and the latter a two-stranded β-sheet following a short helical turn. The latter structural motif shares a significant tertiary structural similarity with the chitin-binding domain of plant chitin-binding protein. This result is thought to provide faithful experimental evidence to the recent hypothesis that chitin-binding proteins of invertebrates and plants are correlated by a convergent evolution process. three-dimensional 2,2,3,3-tetradeutero-3-(trimethylsilyl) propionic acid sodium salt double-quantum-filtered correlated spectroscopy total correlation spectroscopy nuclear Overhauser effect NOE spectroscopy simulated annealing root mean square deviation An invertebrate chitin-binding protein named tachycitin is recently found to be a member of the primordial elements of innate immune defense against bacterial and fungal infections (1.Kawabata S. Nagayama R. Hirata M. Shigenaga T. Agarwala K.L. Saito T. Cho J. Nakajima H. Takagi T. Iwanaga S. J. Biochem. 1996; 120: 1253-1260Crossref PubMed Scopus (125) Google Scholar, 2.Kawano K. Yoneya T. Miyata T. Yoshikawa K. Tokunaga F. Terada Y. Iwanaga S. J. Biol. Chem. 1990; 265: 15365-15367Abstract Full Text PDF PubMed Google Scholar, 3.Beisel H.G. Kawabata S. Iwanaga S. Huber R. Bode W. EMBO J. 1999; 18: 2313-2322Crossref PubMed Scopus (145) Google Scholar, 4.Hoess A. Watson S. Siber G.R. Liddington R. EMBO J. 1993; 12: 3351-3356Crossref PubMed Scopus (227) Google Scholar, 5.Iwanaga S. Kawabata S. Muta T. J. Biochem. 1998; 123: 1-15Crossref PubMed Scopus (255) Google Scholar). The antimicrobial activity is initially identified for chitin-binding proteins extracted from plants (6.Broekaert W.F. Mariën W. Terras F.R. De Bolle M.F. Proost P. Van Damme J. Dillen L. Claeys M. Rees S.B. Vanderleyden J. Cammue B.P. Biochemistry. 1992; 31: 4308-4314Crossref PubMed Scopus (276) Google Scholar, 7.Koo J.C. Lee S.Y. Chun H.J. Cheong Y.H. Choi J.S. Kawabata S. Miyagi M. Tsunasawa S. Ha K.S. Bae D.W. Han C.D. Lee B.L. Cho M.J. Biochim. Biophys. Acta. 1998; 1382: 80-90Crossref PubMed Scopus (126) Google Scholar), which commonly comprise single or multiple copies of the chitin-binding domain. The plant chitin-binding domain is mostly composed of 30–43 residues including eight cysteines, three aromatic residues, and glycines and is frequently referred to as a hevein domain (8.Beintema J.J. FEBS Lett. 1994; 350: 159-163Crossref PubMed Scopus (165) Google Scholar). It has been well demonstrated that this domain is indispensable for the antimicrobial activity and exhibits a significant conservation in primary sequence (>40%) and in three-dimensional (3D)1 structure (9.Andersen N.H. Cao B. Rodriguez-Romero A. Arreguin B. Biochemistry. 1993; 32: 1407-1422Crossref PubMed Scopus (114) Google Scholar, 10.Martins J.C. Maes D. Loris R. Pepermans H.A. Wyns L. Willem R. Verheyden P. J. Mol. Biol. 1996; 258: 322-333Crossref PubMed Scopus (74) Google Scholar, 11.Wright C.S. J. Mol. Biol. 1990; 215: 635-651Crossref PubMed Scopus (146) Google Scholar, 12.Weaver J.L. Prestegard J.H. Biochemistry. 1998; 37: 116-128Crossref PubMed Scopus (24) Google Scholar). Although this advanced knowledge has been provided for the plant chitin-binding proteins, less is known for the invertebrate chitin-binding proteins including tachycitin (1.Kawabata S. Nagayama R. Hirata M. Shigenaga T. Agarwala K.L. Saito T. Cho J. Nakajima H. Takagi T. Iwanaga S. J. Biochem. 1996; 120: 1253-1260Crossref PubMed Scopus (125) Google Scholar, 13.Elvin C.M. Vuocolo T. Pearson R.D. East I.J. Riding G.A. Eisemann C.H. Tellam R.L. J. Biol. Chem. 1996; 271: 8925-8935Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar, 14.Shen Z. Jacobs-Lorena M. J. Biol. Chem. 1997; 272: 28895-28900Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 15.Shen Z. Jacobs-Lorena M. J. Biol. Chem. 1998; 273: 17665-17670Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 16.Watanabe T. Kono M. Aida K. Nagasawa H. Biochim. Biophys. Acta. 1998; 1382: 181-185Crossref PubMed Scopus (51) Google Scholar). Kawabataet al. (1.Kawabata S. Nagayama R. Hirata M. Shigenaga T. Agarwala K.L. Saito T. Cho J. Nakajima H. Takagi T. Iwanaga S. J. Biochem. 1996; 120: 1253-1260Crossref PubMed Scopus (125) Google Scholar) identified that tachycitin is a 73-residues chitin-binding protein having antimicrobial activity. They also revealed that tachycitin consists of five intramolecular disulfide bridges; the connected Cys pairs are 6–33, 12–30, 24–61, 25–68, and 40–53. For invertebrates, the chitin-binding domain was assumed to comprise about 65 residues (17.Shen Z. Jacobs-Lorena M. J. Mol. Evol. 1999; 48: 341-347Crossref PubMed Scopus (136) Google Scholar) involving a high percentage of cysteine and aromatic residues in a similar manner to the plant chitin-binding domain. On the basis of such similarity between plant and invertebrate chitin-binding proteins, Shen and Jacobs-Lorena (17.Shen Z. Jacobs-Lorena M. J. Mol. Evol. 1999; 48: 341-347Crossref PubMed Scopus (136) Google Scholar) proposed a hypothesis that they are correlated by a rare evolutional process, convergent evolution, i.e. proteins from different origins develop to construct the same active site structure to acquire the same function. However, complete lack of 3D-structural information of invertebrate chitin-binding protein obscures the evolutional relationship between invertebrate and plant chitin-binding proteins. The present study determines the solution structure of tachycitin using NMR spectroscopy, which provides the first 3D structural information of invertebrate chitin-binding protein. An invertebrate chitin-binding protein, tachycitin, was isolated from hemocyte debris of horseshoe crab (Tachypleus tridentatus) as described previously (1.Kawabata S. Nagayama R. Hirata M. Shigenaga T. Agarwala K.L. Saito T. Cho J. Nakajima H. Takagi T. Iwanaga S. J. Biochem. 1996; 120: 1253-1260Crossref PubMed Scopus (125) Google Scholar) and used without further purification. The NMR samples were prepared by dissolving tachycitin in either 0.3 ml of D2O or H2O containing 10% D2O to give a final concentration of 1–2 mm, whose pH values were adjusted to be 4.0–6.5 by addition of DCl and/or NaOD. The NMR experiments were performed on JEOL JNM-Alpha 500 and 600 spectrometers operating at temperatures of 15, 20, 30, and 40 °C. The two-dimensional experiments, DQF-COSY, TOCSY (mixing time = 75, 85 ms), and NOESY (mixing time = 75, 250 ms), were acquired with low-power (20 Hz) presaturation on the water. The temperature coefficient (−Δδ/ΔT, ppb K−1) was estimated from the temperature dependence (15–40 °C) of the chemical shift of the HN resonance. The chemical shifts were referenced to the internal standard, TSP (0.00 ppm). Interproton distance restraints were derived from NOE cross-peaks in the NOESY spectra (mixing time = 75 ms), calibrated the peak intensities with known distances (2.2 Å for Hα(i)-HN(i + 1) of β-sheet and 1.75 Å for Hβ-Hβ′), and were used as inputs for 3D structural calculations of tachycitin. The NOEs were classified into strong, medium, and weak, corresponding to three distance restraints with an upper limit of 2.7, 3.5, and 5.0 Å, respectively. The upper distance limit was corrected for methyl and methylene protons that were not assigned stereospecifically. The 35 dihedral φ angle restraints were obtained by measuring3 J NH-Hα coupling constants; theφ angle restraint of −60 ± 30° was used for the residues having3 J HN-Hα coupling constants less than 6 Hz, and that of −120 ± 30° was used for the residues having 3 J HN-Hαconstants larger than 8 Hz. Hydrogen bond distance restraints were applied between nitrogen and oxygen atoms (2.4–3.5 Å) and Hn and oxygen atoms (1.5–2.5 Å) for regular secondary structures. The hydrogen bonding was assumed for the residues 18, 27–29, 31, 34, 36, 38, 45, 47, 52, 54, and 59, which show low temperature coefficients ( 0.2 Å)0 Dihedral (>2 degrees)0RMSD (Å) Well defined regionaWell defined region includes residues 6–68.Backbone atoms (heavy atoms)1.15 ± 0.21 (1.95 ± 0.24) β-Sheet regionsbThe β-sheet region includes residues 17–19, 26–31, 34–39, 45–47, and 52–54.Backbone atoms (heavy atoms)0.72 ± 0.15 (1.73 ± 0.34)Energies (kcal/mol) F total125.93 ± 8.75 F bonds3.92 ± 0.76 F angles93.08 ± 5.01 F impropers14.95 ± 1.41 F van der Waals (Frepel)cThe force constant for the van der Waals energy calculation was 4.0 kcal mol−1 Å−4.9.52 ± 2.84 F NOEdForce constants for the calculation of NOE and dihedral energies were 50 kcal mol−1 Å−2 and 200 kcal mol−1 rad−2, respectively.4.20 ± 1.35 F dihedraldForce constants for the calculation of NOE and dihedral energies were 50 kcal mol−1 Å−2 and 200 kcal mol−1 rad−2, respectively.0.26 ± 0.16RMSD from experimental restraints NOE distance restraints (Å)0.0089 ± 0.0014 Dihedral angle restraints (degrees)0.3339 ± 0.1036RMSD from ideal covalent geometry Bonds (Å)0.001833 ± 0.000180 Angles (degrees)0.5424 ± 0.0146 Impropers0.3912 ± 0.0182φ and ψ in core and allowed regions (%) 97.3a Well defined region includes residues 6–68.b The β-sheet region includes residues 17–19, 26–31, 34–39, 45–47, and 52–54.c The force constant for the van der Waals energy calculation was 4.0 kcal mol−1 Å−4.d Force constants for the calculation of NOE and dihedral energies were 50 kcal mol−1 Å−2 and 200 kcal mol−1 rad−2, respectively. Open table in a new tab It was revealed that tachycitin shares a remarkable local structural similarity with a plant chitin-binding protein named hevein. Comparison between our determined structure of tachycitin (Fig.2 A) and a previously reported structure of hevein (9.Andersen N.H. Cao B. Rodriguez-Romero A. Arreguin B. Biochemistry. 1993; 32: 1407-1422Crossref PubMed Scopus (114) Google Scholar) (Fig. 2 B) clearly shows that an antiparallel β-sheet (colored in blue) and a helical turn (colored in red) are constructed in both proteins in highly similar manners. In addition, formation of a disulfide bridge (between Cys-40 and Cys-53) connecting the middle of β5 and the C terminus of β3 for tachycitin (colored in green, Fig. 2 A) is similarly identified in hevein (Fig. 2 B). The structural similarity further includes the loop regions, e.g. a hairpin loop structure involved in the antiparallel β-sheet (colored in orange). It should be noted that the hairpin loop of tachycitin (Asn-47–Val-52) comprises six residues with βααγαLβ conformation whereas the corresponding loop of hevein comprises five residues with βαγαLβ conformation. Kawabata et al. (1.Kawabata S. Nagayama R. Hirata M. Shigenaga T. Agarwala K.L. Saito T. Cho J. Nakajima H. Takagi T. Iwanaga S. J. Biochem. 1996; 120: 1253-1260Crossref PubMed Scopus (125) Google Scholar) reported that the N-terminal 5–28 region of tachycitin shows sequence similarity with the N-terminal 2–21 region of hevein. However, such similarity is not identified by the present study; the secondary structural arrangement, as well as the disulfide-bond patterns, appears to be quite different for the suggested regions. The structural similarity between segment Cys-40–Gly-60 of tachycitin and segment Cys-12–Ser-32 of hevein, both comprising the antiparallel β-sheet (β4 and β5), was examined by looking at the superimpositions of the segments (Fig.3). The structural motif shown in Fig. 3has been found in several plant chitin-binding proteins (10.Martins J.C. Maes D. Loris R. Pepermans H.A. Wyns L. Willem R. Verheyden P. J. Mol. Biol. 1996; 258: 322-333Crossref PubMed Scopus (74) Google Scholar, 11.Wright C.S. J. Mol. Biol. 1990; 215: 635-651Crossref PubMed Scopus (146) Google Scholar, 12.Weaver J.L. Prestegard J.H. Biochemistry. 1998; 37: 116-128Crossref PubMed Scopus (24) Google Scholar) (Fig.4). For hevein, segment Cys-12–Ser-32 was identified as an essential chitin-binding domain (24.Asensio J.L. Canada F.J. Bruix M. Rodriguez-Romero A. Jimenez-Barbero J. Eur. J. Biochem. 1995; 230: 621-633Crossref PubMed Scopus (111) Google Scholar). It appears that arrangements of the two structural motifs shown in Fig. 3 are significantly consistent with each other (backbone RMSD = ∼1.5 Å). The aromatic side-chain groups of Trp-21 and Trp-23 of hevein (Fig. 3) are known to bind specifically to chitin-derived oligosaccharides through hydrophobic interactions (24.Asensio J.L. Canada F.J. Bruix M. Rodriguez-Romero A. Jimenez-Barbero J. Eur. J. Biochem. 1995; 230: 621-633Crossref PubMed Scopus (111) Google Scholar, 25.Asensio J.L. Canada F.J. Bruix M. Gonzalez C. Khiar N. Rodriguez-Romero A. Jimenez-Barbero J. Glycobiology. 1998; 8: 569-577Crossref PubMed Scopus (68) Google Scholar). This binding is further strengthened by a hydrogen bonding with Ser-19 of hevein (25.Asensio J.L. Canada F.J. Bruix M. Gonzalez C. Khiar N. Rodriguez-Romero A. Jimenez-Barbero J. Glycobiology. 1998; 8: 569-577Crossref PubMed Scopus (68) Google Scholar). As shown in Fig. 3, the residues of Asn-47, Tyr-49, and Val-52 of tachycitin are located at perfectly corresponding positions to the residues of Ser-19, Trp-21, and Trp-23 of hevein. Therefore, one could assume that the region shown in Fig. 3 comprising an antiparallel β-sheet and a helical turn (β4, β5, and α1; Fig. 2 A) in the C-domain of tachycitin serves as an essential chitin-binding site, which protrudes the side-chains of the putative functional residues, Asn-47, Tyr-49, and Val-52. Overall, it could be assumed that the N-terminal region comprising β1–β3 of tachycitin (colored in gray in Fig.2 A) behaves as a stable domain so as to locate the C-terminal domain chitin-binding site proper for its function.Figure 43D structure-based sequence alignment of several chitin-binding proteins in invertebrates and plants with regard to the region corresponding to Cys-40–Gly-60 of tachycitin.Invertebrates are as follows: T. tridentatustachycitin (Tachycitin), Anopheles gambiaechitinase (Ag-chit) (14.Shen Z. Jacobs-Lorena M. J. Biol. Chem. 1997; 272: 28895-28900Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar), Penaeus japonicachitinase 1 (Pj-chit1) (27.Watanabe T. Kono M. Aida K. Nagasawa H. Mol. Mar. Biol. Biotechnol. 1996; 5: 299-303PubMed Google Scholar), Chelonus sp. chitinase (Ch-chit) (28.Krishnan A. Nair P.N. Jones D. J. Biol. Chem. 1994; 269: 20971-20976Abstract Full Text PDF PubMed Google Scholar), 44-kDa glycoprotein fromLucilia cuprina (Peritrophin-44) (13.Elvin C.M. Vuocolo T. Pearson R.D. East I.J. Riding G.A. Eisemann C.H. Tellam R.L. J. Biol. Chem. 1996; 271: 8925-8935Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar), andTrichoplusia ni intestinal mucin (Tn-IM) (29.Wang P. Granados R.R. J. Biol. Chem. 1997; 272: 16663-16669Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Plants are as follows: hevein from rubber tree (Hevein) (9.Andersen N.H. Cao B. Rodriguez-Romero A. Arreguin B. Biochemistry. 1993; 32: 1407-1422Crossref PubMed Scopus (114) Google Scholar),Amaranthus caudatus antimicrobial protein 2 (Ac-AMP2) (10.Martins J.C. Maes D. Loris R. Pepermans H.A. Wyns L. Willem R. Verheyden P. J. Mol. Biol. 1996; 258: 322-333Crossref PubMed Scopus (74) Google Scholar), and four homologous domains of wheat germ agglutinin (WGA A, -B, -C, and-D) (11). Residue numbers for each segment are indicated in parentheses. Amino acids conserved between invertebrate and plant proteins are indicated with bold letters. The chitin-binding residues in plants and the corresponding residues in invertebrates are found to be aligned (indicated by asterisks at the bottom), for which polar and hydrophobic residues are colored in red andblue, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Conservation of the chitin-binding structural motif among the chitin-binding proteins in invertebrates and plants was further examined by alignment tests of the proteins with regard to their amino acid sequences corresponding to Cys-40–Gly-60 of tachycitin (Fig. 4). The 3D structural information has been available for the plant chitin-binding proteins (9.Andersen N.H. Cao B. Rodriguez-Romero A. Arreguin B. Biochemistry. 1993; 32: 1407-1422Crossref PubMed Scopus (114) Google Scholar, 10.Martins J.C. Maes D. Loris R. Pepermans H.A. Wyns L. Willem R. Verheyden P. J. Mol. Biol. 1996; 258: 322-333Crossref PubMed Scopus (74) Google Scholar, 11.Wright C.S. J. Mol. Biol. 1990; 215: 635-651Crossref PubMed Scopus (146) Google Scholar). The information is now available for only tachycitin among the invertebrate chitin-binding proteins. It appears that the residues of Cys, Pro, and Gly, all of which have significant influence on the structural constructions, are well conserved in the chitin-binding proteins listed in Fig. 4. Conservation of polar and hydrophobic residues is further identified for the putative chitin-binding residues (e.g. Asn-47, Tyr-49, and Val-52 for tachycitin). For all plant chitin-binding proteins, the 21-residues segments listed in Fig. 4 appear to construct a closely similar 3D structure (9.Andersen N.H. Cao B. Rodriguez-Romero A. Arreguin B. Biochemistry. 1993; 32: 1407-1422Crossref PubMed Scopus (114) Google Scholar, 10.Martins J.C. Maes D. Loris R. Pepermans H.A. Wyns L. Willem R. Verheyden P. J. Mol. Biol. 1996; 258: 322-333Crossref PubMed Scopus (74) Google Scholar, 11.Wright C.S. J. Mol. Biol. 1990; 215: 635-651Crossref PubMed Scopus (146) Google Scholar) to the putative chitin-binding site of tachycitin. Further similarity in primary sequence identified between tachycitin, Ag-chit, Pj-chit1, Ch-chit, Peritrophin-44, and Tn-IM (nomenclatures described in the figure legend) assumes that these segments of the invertebrate chitin-binding proteins commonly comprise the chitin-binding structural motif as identified in tachycitin. In 1999, Shen and Jacobs-Lorena (17.Shen Z. Jacobs-Lorena M. J. Mol. Evol. 1999; 48: 341-347Crossref PubMed Scopus (136) Google Scholar) proposed a hypothesis that chitin-binding proteins in invertebrates and plants are correlated by a rare evolutional process, convergent evolution. Our present structural determination of tachycitin and the 3D structure-based sequence alignment are thought to provide faithful evidences for the proposed idea of the convergent evolution relationship between invertebrate and plant chitin-binding proteins. We are grateful to Shin-ya Ohki, Nobuyuki Matsuki, and Nobuaki Nemoto for help with NMR measurements and Ai Miura for keeping the NMR spectrometer at the optimum performance level.