Structural Basis for New Pattern of Conserved Amino Acid Residues Related to Chitin-binding in the Antifungal Peptide from the Coconut Rhinoceros Beetle Oryctes rhinoceros
2003; Elsevier BV; Volume: 278; Issue: 25 Linguagem: Inglês
10.1074/jbc.m301025200
ISSN1083-351X
AutoresHikaru Hemmi, Jun Ishibashi, Tetsuya Tomie, Minoru Yamakawa,
Tópico(s)Biochemical and Structural Characterization
ResumoScarabaecin isolated from hemolymph of the coconut rhinoceros beetle Oryctes rhinoceros is a 36-residue polypeptide that has antifungal activity. The solution structure of scarabaecin has been determined from twodimensional 1H NMR spectroscopic data and hybrid distance geometry-simulated annealing protocol calculation. Based on 492 interproton and 10 hydrogen-bonding distance restraints and 36 dihedral angle restraints, we obtained 20 structures. The average backbone root-mean-square deviation for residues 4–35 is 0.728 ± 0.217 Å from the mean structure. The solution structure consists of a two-stranded antiparallel β-sheet connected by a type-I β-turn after a short helical turn. All secondary structures and a conserved disulfide bond are located in the C-terminal half of the peptide, residues 18–36. Overall folding is stabilized by a combination of a disulfide bond, seven hydrogen bonds, and numerous hydrophobic interactions. The structural motif of the C-terminal half shares a significant tertiary structural similarity with chitin-binding domains of plant and invertebrate chitin-binding proteins, even though scarabaecin has no overall sequence similarity to other peptide/polypeptides including chitin-binding proteins. The length of its primary structure, the number of disulfide bonds, and the pattern of conserved functional residues binding to chitin in scarabaecin differ from those of chitin-binding proteins in other invertebrates and plants, suggesting that scarabaecin does not share a common ancestor with them. These results are thought to provide further strong experimental evidence to the hypothesis that chitin-binding proteins of invertebrates and plants are correlated by a convergent evolution process. Scarabaecin isolated from hemolymph of the coconut rhinoceros beetle Oryctes rhinoceros is a 36-residue polypeptide that has antifungal activity. The solution structure of scarabaecin has been determined from twodimensional 1H NMR spectroscopic data and hybrid distance geometry-simulated annealing protocol calculation. Based on 492 interproton and 10 hydrogen-bonding distance restraints and 36 dihedral angle restraints, we obtained 20 structures. The average backbone root-mean-square deviation for residues 4–35 is 0.728 ± 0.217 Å from the mean structure. The solution structure consists of a two-stranded antiparallel β-sheet connected by a type-I β-turn after a short helical turn. All secondary structures and a conserved disulfide bond are located in the C-terminal half of the peptide, residues 18–36. Overall folding is stabilized by a combination of a disulfide bond, seven hydrogen bonds, and numerous hydrophobic interactions. The structural motif of the C-terminal half shares a significant tertiary structural similarity with chitin-binding domains of plant and invertebrate chitin-binding proteins, even though scarabaecin has no overall sequence similarity to other peptide/polypeptides including chitin-binding proteins. The length of its primary structure, the number of disulfide bonds, and the pattern of conserved functional residues binding to chitin in scarabaecin differ from those of chitin-binding proteins in other invertebrates and plants, suggesting that scarabaecin does not share a common ancestor with them. These results are thought to provide further strong experimental evidence to the hypothesis that chitin-binding proteins of invertebrates and plants are correlated by a convergent evolution process. A 36-residue peptide named scarabaecin isolated from hemolymph of the coconut rhinoceros beetle Oryctes rhinoceros has been found to show strong antifungal activity against phytopathogenic fungi. 1T. Tomie, J. Ishibashi, S. Furukawa, S. Kobayashi, R. Sawahata, A. Asaoka, M. Tagawa, and M. Yamakawa, submitted for publication. 1T. Tomie, J. Ishibashi, S. Furukawa, S. Kobayashi, R. Sawahata, A. Asaoka, M. Tagawa, and M. Yamakawa, submitted for publication. Scarabaecin also has chitin-binding activity. 1T. Tomie, J. Ishibashi, S. Furukawa, S. Kobayashi, R. Sawahata, A. Asaoka, M. Tagawa, and M. Yamakawa, submitted for publication. Antimicrobial activity was initially identified for chitinbinding proteins isolated from plants (2Broekaert W.F. Marien 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 (272) Google Scholar, 3Koo 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 (125) Google Scholar), which commonly consist of single or multiple copies of the chitin-binding domain. The plant chitin-binding domain consists mostly of 30–43 residues, including eight cysteines, three aromatic residues, and glycines, and is frequently referred to as a hevein domain (4Beintema J.J. FEBS Lett. 1994; 350: 159-163Crossref PubMed Scopus (163) Google Scholar). This domain is indispensable to antimicrobial activity and shows significant conservation in the primary sequence (>40%) and in the tertiary structure (5Andersen N.H. Cao B. Rodriguez-Romero A. Arreguin B. Biochemistry. 1993; 32: 1407-1422Crossref PubMed Scopus (114) Google Scholar, 6Martins 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, 7Wright C.S. J. Mol. Biol. 1990; 215: 635-651Crossref PubMed Scopus (145) Google Scholar, 8Weaver J.L. Prestegard J.H. Biochemistry. 1998; 37: 116-128Crossref PubMed Scopus (24) Google Scholar). Although chitin is an important structural component of invertebrates, fungi, and bacteria, chitin-binding proteins of invertebrates have only recently been characterized (9Kawabata 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 (123) Google Scholar, 10Elvin 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 (157) Google Scholar, 11Shen Z. Jacobs-Lorena M. J. Biol. Chem. 1997; 272: 28895-28900Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 12Shen Z. Jacobs-Lorena M. J. Biol. Chem. 1998; 273: 17665-17670Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 13Watanabe T. Kono M. Aida K. Nagasawa H. Biochim. Biophys. Acta. 1998; 1382: 181-185Crossref PubMed Scopus (50) Google Scholar). The invertebrate chitin-binding domain is assumed to consist of about 65 residues (14Shen Z. Jacobs-Lorena M. J. Mol. Evol. 1999; 48: 341-347Crossref PubMed Scopus (133) Google Scholar) involving a high percentage of cysteine and aromatic residues similar to the plant chitin-binding domain. However, scarabaecin has only two cysteine residues in the sequence and shows no sequence similarity to other chitin-binding proteins in plants and invertebrates. Moreover, a computer-aided homology search of scarabaecin indicated that this antifungal peptide has no significant sequence similarity to peptides/polypeptides so far reported, suggesting that a novel immune peptide occurs in the coleopteran insect. 1T. Tomie, J. Ishibashi, S. Furukawa, S. Kobayashi, R. Sawahata, A. Asaoka, M. Tagawa, and M. Yamakawa, submitted for publication. The solution structure of tachycitin, a 73-residue polypeptide from the horseshoe crab that has chitin-binding and antimicrobial activity (9Kawabata 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 (123) Google Scholar), was determined by NMR spectroscopy (15Suetake T. Tsuda S. Kawabata S. Miura K. Iwanaga S. Hikichi K. Nitta K. Kawano K. J. Biol. Chem. 2000; 275: 17929-17932Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar), which provided the first 3D 2The abbreviations used are: 3D, three-dimensional; 2D, two-dimensional; DQF, double-quantum-filtered; COSY, correlated spectroscopy; TOCSY, total correlation spectroscopy; NOE, nuclear Overhauser effect; WGA, wheat germ agglutinin; Ac-AMP2, Amaranthus caudatus antimicrobial protein 2; UDA, Urtica dioica agglutinin; r.m.s.d., root-mean-square deviation. 2The abbreviations used are: 3D, three-dimensional; 2D, two-dimensional; DQF, double-quantum-filtered; COSY, correlated spectroscopy; TOCSY, total correlation spectroscopy; NOE, nuclear Overhauser effect; WGA, wheat germ agglutinin; Ac-AMP2, Amaranthus caudatus antimicrobial protein 2; UDA, Urtica dioica agglutinin; r.m.s.d., root-mean-square deviation. structural information on invertebrate chitin-binding protein. A comparison of the tertiary structure of tachycitin with that of a plant chitin-binding protein, hevein, showed that tachycitin shared a remarkable local structural similarity with hevein (15Suetake T. Tsuda S. Kawabata S. Miura K. Iwanaga S. Hikichi K. Nitta K. Kawano K. J. Biol. Chem. 2000; 275: 17929-17932Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). The local structure consists of an antiparallel β-sheet and a helical turn, and the secondary structures are constructed in both proteins in highly similar manners. Because the conserved structural motif was identified as an essential chitin-binding domain for hevein, the structural domain of tachycitin was assumed to serve as an essential chitin-binding site. The residues of cysteine, proline, and glycine, all of which may significantly influence structural construction, were well conserved in the structural motif of both proteins. Conservation of polar and hydrophobic residues was identified for putative chitin-binding residues (15Suetake T. Tsuda S. Kawabata S. Miura K. Iwanaga S. Hikichi K. Nitta K. Kawano K. J. Biol. Chem. 2000; 275: 17929-17932Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). The tertiary structure of tachycitin and 3D structure-based sequence alignment between chitin-binding proteins in invertebrates and plants for the region corresponding to Cys-40–Gly-60 of tachycitin as the conserved structural motif are thought to provide faithful evidence for the recent hypothesis (14Shen Z. Jacobs-Lorena M. J. Mol. Evol. 1999; 48: 341-347Crossref PubMed Scopus (133) Google Scholar) that chitin-binding proteins of invertebrates and plants are correlated by a convergent evolution process (15Suetake T. Tsuda S. Kawabata S. Miura K. Iwanaga S. Hikichi K. Nitta K. Kawano K. J. Biol. Chem. 2000; 275: 17929-17932Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). The residues of cysteine, proline, and glycine in the amino acid sequence of scarabaecin were well conserved by comparing amino acid sequences focusing on putative chitin-binding domains among scarabaecin, tachycitin, hevein, and other invertebrate and plant chitin-binding proteins.1 Conservation of polar and hydrophobic residues was further identified for putative chitinbinding residues.1 Thus, it is worth determining the tertiary structure of scarabaecin and comparing it with those of tachycitin and hevein to clarify that scarabaecin has a conserved structural motif as a chitin-binding site, even though this peptide has no overall sequence similarity to other peptides/polypeptides, including chitin-binding proteins. In this study, we determined the 3D structure of scarabaecin in solution by 2D 1H NMR spectroscopy and distance geometrysimulated annealing calculation. The solution structure showed that the backbone conformation of the conserved structural motif as a putative chitin-binding site in scarabaecin was very similar to those in hevein and tachycitin. The length of the primary sequence and the number of disulfide bonds in scarabaecin, however, differ from those in hevein and tachycitin, indicating a new pattern of conserved amino acid residues at the chitin-binding site. These results provide additional evidence to support the convergent evolution proposed for horseshoe crab and plant chitin-binding proteins (15Suetake T. Tsuda S. Kawabata S. Miura K. Iwanaga S. Hikichi K. Nitta K. Kawano K. J. Biol. Chem. 2000; 275: 17929-17932Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Finally, we discuss conserved functional residues crucial for binding to saccharides and the relationship between the conserved structural motif and antimicrobial specificity. Sample Preparation—The peptide scarabaecin was synthesized by solid-phase methodology with Fmoc (N-(9-fluorenyl)methoxycarbonyl)-amino acids and formed a disulfide bond between Cys-18 and Cys-29 using 2,2′-bispyridyl disulfide as described elsewhere.1 The resulting peptide was purified by reversed-phase high pressure liquid chromatography, and the molecular mass and amino acid sequence were determined using a matrix-assist laser desorption ionization time-of-flight mass spectrometer and a protein sequencer.1 NMR Spectroscopy—A NMR sample was prepared by dissolving the synthetic peptide in 500 μl of 90% H2O/10%D2O or 99.96% D2O solution. The final peptide concentration was ∼3 mm and pH was 2.4. All NMR spectra were obtained on Bruker Avance500 and Avance800 spectrometers with quadrature detection in the phase-sensitive mode by time proportional phase incrementation (TPPI) (16Marion D. Wüthrich K. Biochem. Biophys. Res. Commun. 1983; 113: 967-974Crossref PubMed Scopus (3514) Google Scholar) and States-TPPI (17Marion D. Ikura M. Tschudin R. Bax A. J. Magn. Reson. 1989; 85: 393-399Google Scholar). The following spectra were recorded at 20 °C, 25 °C, 30 °C, and 35 °C with 15 ppm spectral widths in the t1 and t2 dimensions: 2D double quantum-filtered correlated spectroscopy (DQF-COSY) (18Rance M. Sorense O.W. Bodenhausen G. Wagner G. Ernst R.R. Wuthrich K. Biochem. Biophys. Res. Commun. 1983; 117: 479-485Crossref PubMed Scopus (2593) Google Scholar), recorded with 512 and 2048 complex points in the t1 and t2 dimensions; 2D homonuclear total correlated spectroscopy (TOCSY) (19Davis D.G. Bax A. J. Am. Chem. Soc. 1985; 107: 2820-2821Crossref Scopus (1070) Google Scholar) with DIPSI-2 mixing sequence, recorded with mixing times of 35, 60, and 80 ms, 512 and 2048 complex points in the t1 and t2 dimensions; 2D nuclear Overhauser effect (NOE) spectroscopy (20Kumar A. Ernst R.R. Wüthrich K. Biochem. Biophys. Res. Commun. 1980; 95: 1-6Crossref PubMed Scopus (2007) Google Scholar), recorded with mixing times of 60, 100, 200, and 400 ms, 512 and 2046 complex points in the t1 and t2 dimensions; and 2D rotating frame NOE spectroscopy (21Griesinger C. Ernst R.R. J. Magn. Reson. 1987; 75: 261-271Google Scholar), recorded with mixing time of 100 ms, 512 and 1024 complex points in the t1 and t2 dimensions. The high digital resolution DQF-COSY and exclusive 2D scalar COSY (22Griesinger C. Sorensen O.W. Ernst R.R. J. Magn. Reson. 1987; 75: 474-492Google Scholar) spectra were recorded using 800 and 4096 complex points in the t1 and t2 dimensions. Water suppression was performed using WATERGATE sequence (23Piotto M. Saudek V. Sklenar V. J. Biomol. NMR. 1992; 2: 661-666Crossref PubMed Scopus (3487) Google Scholar, 24Sklenar V. Piotto M. Leppik R. Saudek V. J. Magn. Reson. Ser. A. 1993; 102: 241-245Crossref Scopus (1105) Google Scholar). 2D 1H-13C heteronuclear single quantum correlation spectroscopy (25Bodenhausen G. Ruben D.J. Chem. Phys. Lett. 1980; 69: 185Crossref Scopus (2407) Google Scholar) was recorded with 1024 × 128 complex points for 15 ppm in the 1H dimension and 100 ppm in the 13C dimension at a natural isotope abundance. Slowly exchanging amide protons were determined by lyophilizing the protein from an H2O solution, dissolving the protein in D2O, and collecting sequential 2-hour 2D TOCSY spectra. All NMR spectra were processed using XWINNMR (Bruker) and Felix2000 (MSI, San Diego, CA). Peak-picking and assignment were performed with Sparky program (UCSF; www.cgl.ucsf.edu/home/sparky/). Before Fourier transformation, the shifted sine bell window function was applied to the t1 and t2 dimensions. All 1H dimensions were referenced to internal 2,2-dimethyl-2-silapentane-5-sulfonate. 13C dimensions were indirectly referenced to 2,2-dimethyl-2-silapentane-5-sulfonate at 25 °C (26Wishart D.S. Bigam C.G. Yao J. Abildgaard F. Dyson H.J. Oldfield E. Markley J.L. Sykes B.D. J. Biomol. NMR. 1995; 6: 135-140Crossref PubMed Scopus (2045) Google Scholar). Structure Calculations—NOE-derived distance restraints were classified into three ranges, 1.8–2.7 Å, 1.8–3.5 Å, and 1.8–5.0 Å, according to the relative NOE intensities. Upper distance limits for NOEs involving methyl protons and nonstereospecifically assigned methylene protons were corrected appropriately for center averaging (27Wüthrich K. Billetter M. Braun W. J. Mol. Biol. 1983; 169: 949-961Crossref PubMed Scopus (1005) Google Scholar). In addition, a distance of 0.5 Å was added to the upper distance limits only for NOEs involving methyl proton (28Clore G.M. Gronenborn A.M. Nilges M. Ryan C.A. Biochemistry. 1987; 26: 8012-8023Crossref PubMed Scopus (264) Google Scholar) after correction for center averaging. Torsion angle restraints on the backbone ϕ-angle were derived from 3JHN-Hα coupling constants from the high digital resolution 2D DQF-COSY spectra and intraresidue and sequential NOEs. We obtained 30 ϕ angle restraints. Backbone ϕ-angles were restrained to–60° ± 30° for 3JHN-Hα < 6 Hz and–120° ± 40° for 3JHN-Hα > 8 Hz. The additional ϕ-angle restraint of 100° ± 80° was applied to residues for which the intraresidue HN-Hα NOE was clearly weaker than the NOE between HN and the Hα of the preceding residue (29Clubb R.T. Ferguson S.B. Walsh C.T. Wagner G. Biochemistry. 1994; 33: 2761-2772Crossref PubMed Scopus (87) Google Scholar). An ψ-angle restraint was used for residues in β-strand structures, as predicted from the chemical shift index (30Wishart D.S. Sykes B.D. Richards F.M. Biochemistry. 1992; 31: 1647-1651Crossref PubMed Scopus (1998) Google Scholar) and from NOE patterns characteristic of the secondary structure. Five ψ-angles in the β-sheet region were restrained to 120° ± 60°. Side-chain χ1-angles were determined by 3JHα-Hβ coupling constants from exclusive 2D scalar COSY and short-mixing TOCSY connectivities combined with NH-Hβ and Hα-Hβ NOEs (31Wagner G. Braun W. Havel T.F. Schaumman T. Go N. Wüthrich K. J. Mol. Biol. 1987; 196: 611-639Crossref PubMed Scopus (635) Google Scholar). We obtained seven χ1-angle restraints. The χ1-angle restraints were normally restricted to ±60° from staggered conformations, g+ (+60°), t (180°), or g–(–60°). Hydrogen-deuterium exchange experiments identified seven hydrogen bond donors. Corresponding hydrogen bond acceptors were determined based on NOE patterns observed for regular secondary structural regions and preliminary calculated structures without restraints regarding hydrogen bonds. Hydrogen-bond restraints were applied to N–H and C=O groups: 1.7–2.4 Å for the H–O distance and 2.7–3.4 Å for the N–O distance. Structure calculations were performed using the hybrid distance geometry-simulated annealing method using X-PLOR 3.851 (32Brünger A.T. X-PLOR Version 3.1 Manual. Yale University, New Haven, CT.1993Google Scholar). A total of 502 interproton distance restraints and 36 dihedral angle restraints were used to calculate an ensemble of structures. The structure calculation proceeded in two stages by using the standard X-PLOR protocol. In the first stage, a low-resolution structure was preliminarily determined using NOE-derived distance restraints and dihedral angle restraints, except for ψ-angle restraints. In the second stage, the same protocol was applied by adding hydrogen bond restraints and ψ-angle restraints. The force constants for the distance restraints were set to 50 kcal mol–1 Å–2 throughout all the calculations, and dihedral angle restraints were initially set to 5 kcal mol–1 rad–2 during the high-temperature dynamics and gradually increased to 200 kcal mol–1 rad–2 during the annealing stage. The final round of calculations began with 200 initial structures, and the best 20 structures were selected and analyzed with MOLMOL (33Koradi R. Billeter M. Wüthrich K. J. Mol. Graph. 1996; 14: 51-55Crossref PubMed Scopus (6454) Google Scholar), InsightII (MSI, San Diego, CA), and PROCHECK-NMR (34Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). The average coordinates of ensembles of the 20 lowest energy structures were subjected to 1000 cycles of Powell restrained minimization to improve stereochemistry and nonbonded contacts. Structure figures were generated using MOLMOL. Resonance Assignment and Secondary Structure—Essentially complete 1H resonance assignments were obtained for the peptide using spin system identification and sequential assignment (35Wüthrich K. NMR of Proteins and Nucleic Acids. John Wiley & Sons, New York.1986Crossref Google Scholar). Any ambiguous peaks caused by overlapping with water resonance or degeneration in the chemical shift were resolved by comparing NOE spectroscopy and TOCSY spectra at four different temperatures (20 °C, 25 °C, 30 °C, and 35 °C) and by combining with the 2D 1H-13C heteronuclear single quantum correlation spectrum. The fingerprint region of the DQF-COSY spectrum collected at 25 °C is shown in Fig. 1 with sequence-specific resonance assignments. In these assignments, Hα(i)-Hδ(i+1:Pro) (dαδ) or Hα(i)-Hα(i+1:Pro) (dαα) NOEs instead of dαN were used for Pro residues. All four proline residues (Pro-3, Pro-6, Pro-19, and Pro-32) showed strong dαδ NOEs, indicating that all proline residues in the peptide have a trans configuration. Finally, the resonance assignments for the backbone and side-chain 1H were completed except for the amide 1H of Glu-1. The resonance assignment was extended by determining stereospecific assignments of methylene protons to obtain high-precision NMR structures. Stereospecific assignments of β-methylene protons were obtained for 7 of 28 residues of the peptide using information on 3JHα-Hβ coupling constants from exclusive 2D scalar COSY and qualitatively estimated from short-mixing time TOCSY spectra combined with intraresidue NH-Hβ and Hα-Hβ NOEs. A qualitative analysis of short- and medium-range NOEs, 3JHα-Hβ coupling constants, and a slowly exchanging amide proton pattern was used to characterize the secondary structure of scarabaecin (Fig. 2A). The absence of consecutive Hα(i)NH(i+3) NOEs and only one small 3JHN-Hα coupling constant ( 8 Hz) and its characteristic NOE pattern: strong consecutive sequential dαN(i,i+1) and long-range interstrand dαα(i, j) cross-peaks. From these data, a two-stranded antiparallel β-sheet consisting of the two regions of residues Val-23–Ans-25 and Asp-28–Lys-30 was identified in scarabaecin (Fig. 2B). The existence of the β-sheet was supported by four slowly exchanging NH protons at positions 23, 25, 28, and 30, and chemical shift index pattern (Fig. 2A). The region Asn-25–Asp-28, linking the two β-strands and residues 23–25 and 28–30, was identified as a β-turn based on characteristic NOE patterns, weak Hα(i)-NH(i+2) connectivity, and weak NH(i)-NH(i+2) connectivity between Gly-26 and Asp-28 (Fig. 2B). The slow exchanging NH proton at position Asp-28 supports the presence of the tight turn (Fig. 2A). Tertiary Structure of Scarabaecin—The 3D structure of scarabaecin was determined based on the distance and dihedral angle restraints from the NMR data using the hybrid distance geometry-simulated annealing approach. 200 structures were calculated. Of these, 20 final structures showing the lowest energy values, no distance constraint violation of >0.5 Å, and no dihedral constraint violation of >5° were selected. The restraints used and the structural statistics for the final structures are summarized in Table I. The structures exhibited good covalent geometry and stereochemistry, as evidenced by the low r.m.s.d. values for bond, angle, and improper from idealized geometry. In a Ramachandran plot, 100% of backbone dihedral angles of the 20 structures fall in either core or allowed regions. Fig. 3A shows best-fit superposition of backbone atoms of the 20 structures of scarabaecin. The r.m.s.d.s of these structures from the average structure were 0.72 ± 0.21 Å for backbone heavy atoms in the whole sequence (excluding three N-terminal residues and one C-terminal residue, Lys-4–Phe-35) and 1.30 ± 0.23 Å for all heavy atoms in the same region, whereas the corresponding values were 0.35 ± 0.14 Å for backbone heavy atoms and 0.84 ± 0.23 Å for all heavy atoms in the C-terminal half region of residues 18–35 involving all secondary structure elements and a conserved disulfide bond. This data indicates that the C-terminal half region of residues 18–35 converges very well in the calculated structures.Table IStatistics for best 20 NMR structures of scarabaecinTotal restraints used538Total distance restraints502Intraresidue128Sequential165Medium (1 <|i - j| < 5)82Long (|i - j| ≥ 5)117Hydrogen bond (2 per bond)10Total dihedral angle restraints36ϕ25ψ4χ17Energies (kcal/mol)aThe final values of the square well NOE and dihedral angle potentials were calculated with force constants of 50 kcal mol-1 Å-2 and 200 kcal mol-1 rad-2, respectively. The force contacts for the van der Waals energy calculation was 4.0 kcal mol-1 Å-4, with van der Waals radii set to 0.75 times the values used in the CHARMM empirical energy function.Ftotal109.6 ± 5.69Fbonds6.82 ± 0.64Fangles42.94 ± 1.99Fimpropers4.95 ± 0.60Frepel17.54 ± 2.73FNOE35.16 ± 2.87Fdihedral2.16 ± 0.71r.m.s.d. from experimental restraintsNOE distance restraints (Å)0.0373 ± 0.0015Dihedral angle restraints (°)0.979 ± 0.164r.m.s.d. from ideal covalent geometryBonds (Å)0.0034 ± 0.0001Angles (°)0.513 ± 0.011Impropers0.325 ± 0.019ϕ and ψ in core and allowed regions (%)bThe program PROCHECK-NMR (38) was used for Ramachandran plot analysis.100r.m.s.d. relative to the mean structure (Å)Residues 4-350.728 ± 0.217cBackbone (N, Cα, and C′ atoms).1.303 ± 0.238dAll non-H.Residues 18-350.350 ± 0.147cBackbone (N, Cα, and C′ atoms).0.841 ± 0.230dAll non-H.a The final values of the square well NOE and dihedral angle potentials were calculated with force constants of 50 kcal mol-1 Å-2 and 200 kcal mol-1 rad-2, respectively. The force contacts for the van der Waals energy calculation was 4.0 kcal mol-1 Å-4, with van der Waals radii set to 0.75 times the values used in the CHARMM empirical energy function.b The program PROCHECK-NMR (38Parijs J.V. Broekaert W.F. Goldstein I.J. Peumans W.J. Planta. 1991; 183: 258-264Crossref PubMed Scopus (270) Google Scholar) was used for Ramachandran plot analysis.c Backbone (N, Cα, and C′ atoms).d All non-H. Open table in a new tab A ribbon representation of the restrained energy minimized average structure of scarabaecin is shown in Fig. 3, B and C. The solution structure of the peptide consists of an antiparallel β-sheet connected by a type-I β-turn (Val-23–Lys-30) after a short helical turn (Ser-31–Ala-34). The β-turn, residues 25–28, was classified as type I based on dihedral angles of Gly-26 and Phe-27, the hydrogen bonding between Asn-25 and Asp-28, and the spatially positions of carbonyl oxygen of Gly-26 and Cβ of Phe-27. All secondary structures of scarabaecin are located in the C-terminal half of the peptide (residues 18–36), not in the N-terminal half (residues 1–17). Seven hydrogen bond donors were observed in hydrogen-deuterium exchange experiments. Four hydrogen bonds (Val-23–HN–Lys-30–O, Ans-25–HN–Asp-28–O, Asp-28–HN–Asn-25–O, and Lys-30–HN–Val-23–O) were found within the secondary structures, and one hydrogen bond (Ile-12–HN–Asn-17–Oδ1) was determined from the preliminary structures; five hydrogen bonds were confirmed from the final 20 structures. Evidence for two additional hydrogen bonds (Leu-5–HN–Pro-3–C and Val-10–HN–Asp8–O) was observed in a subset of the family of structures. Within the ordered portion of the molecule, a subset of side chains forms a well-defined hydrophobic core centered on Pro-19 and the disulfide bond between Cys-18 and Cys-29. These residues have NOE contacts to side chains of Leu-5, Pro-6, Leu-11, and Trp-24. The chitin-binding domains of plants and invertebrates involve a high percentage of cysteine residues in their sequences, and cysteine residues have been elucidated in maintaining protein fold (14Shen Z. Jacobs-Lorena M. J. Mol. Evol. 1999; 48: 341-347Crossref PubMed Scopus (133) Google Scholar). However, scarabaecin has only two cysteine residues, Cys-18 and Cys-29, to form a disulfide bond. The N-terminal half of the peptide (residues 1–17) has no secondary structure or disulfide bond. Thus, the overall folding of scarabaecin is stabilized by a combination of one covalent disulfide linkage (Cys-18–Cys-29), seven hydrogen bonds, and hydrophobic side chain packing consisting of seven residues (Leu-5, Pro-6, Leu-11, Cys-18, Pro-19, Trp-24, and Cys-29) (Fig. 3C). Comparison of the Tertiary Structure of the Conserved Structural Motif in Scarabaecin with Those in Tachycitin and Hevein—So far, the plant chitin-binding domains have been well characterized by information on their primary sequences, 3D structures, and functional experiments. Less is known about invertebrate chitin-binding proteins, however. Recently, the solution structure of tachycitin was determined as the first 3D structure of invertebrate chitin-binding protein by using 2D NMR methods (15Suetake T. Tsuda S. Kawabata S. Miura K. Iwanaga S. Hikichi K. Nitta K. Kawano K. J. Biol. Chem. 2000; 275: 17929-17932Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). A
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