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Crystal Structures of Allosamidin Derivatives in Complex with Human Macrophage Chitinase

2003; Elsevier BV; Volume: 278; Issue: 22 Linguagem: Inglês

10.1074/jbc.m300362200

ISSN

1083-351X

Autores

Francesco Rao, Douglas R. Houston, Rolf G. Boot, Johannes M. F. G. Aerts, Shohei Sakuda, Daan M. F. van Aalten,

Tópico(s)

Invertebrate Immune Response Mechanisms

Resumo

The pseudotrisaccharide allosamidin is a potent family 18 chitinase inhibitor with demonstrated biological activity against insects, fungi, and the Plasmodium falciparum life cycle. The synthesis and biological properties of several derivatives have been reported. The structural interactions of allosamidin with several family 18 chitinases have been determined by x-ray crystallography previously. Here, a high resolution structure of chitotriosidase, the human macrophage chitinase, in complex with allosamidin is presented. In addition, complexes of the allosamidin derivatives demethylallosamidin, methylallosamidin, and glucoallosamidin B are described, together with their inhibitory properties. Similar to other chitinases, inhibition of the human chitinase by allosamidin derivatives lacking a methyl group is 10-fold stronger, and smaller effects are observed for the methyl and C3 epimer derivatives. The structures explain the effects on inhibition in terms of altered hydrogen bonding and hydrophobic interactions, together with displaced water molecules. The data reported here represent a first step toward structure-based design of specific allosamidin derivatives. The pseudotrisaccharide allosamidin is a potent family 18 chitinase inhibitor with demonstrated biological activity against insects, fungi, and the Plasmodium falciparum life cycle. The synthesis and biological properties of several derivatives have been reported. The structural interactions of allosamidin with several family 18 chitinases have been determined by x-ray crystallography previously. Here, a high resolution structure of chitotriosidase, the human macrophage chitinase, in complex with allosamidin is presented. In addition, complexes of the allosamidin derivatives demethylallosamidin, methylallosamidin, and glucoallosamidin B are described, together with their inhibitory properties. Similar to other chitinases, inhibition of the human chitinase by allosamidin derivatives lacking a methyl group is 10-fold stronger, and smaller effects are observed for the methyl and C3 epimer derivatives. The structures explain the effects on inhibition in terms of altered hydrogen bonding and hydrophobic interactions, together with displaced water molecules. The data reported here represent a first step toward structure-based design of specific allosamidin derivatives. Family 18 chitinases hydrolyze chitin, a polymer of β-(1,4)-linked N-acetylglucosamine. Chitin is not found in humans but plays a key role in the life cycles of several classes of human pathogens, such as fungi (1Kuranda M.J. Robbins P.W. J. Biol. Chem. 1991; 266: 19758-19767Abstract Full Text PDF PubMed Google Scholar), nematodes (2Wu Y. Egerton G. Underwood A.P. Sakuda S. Bianco A.E. J. Biol. Chem. 2001; 276: 42557-42564Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), protozoan parasites (3Vinetz J.M. Dave S.K. Specht C.A. Brameld K.A. Xu B. Hayward R. Fidock D.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14061-14066Crossref PubMed Scopus (94) Google Scholar), and insects (4Cohen E. Arch. Insect Biochem. Physiol. 1993; 22: 245-261Crossref PubMed Scopus (121) Google Scholar). Several chitinase inhibitors with biological activity have been identified, such as allosamidin (5Sakuda S. Isogai A. Matsumoto S. Suzuki A. Koseki K. Tetrahedron Lett. 1986; 27: 2475-2478Crossref Scopus (220) Google Scholar), styloguanidines (6Kato T. Shizuri Y. Izumida H. Yokoyama A. Endo M. Tetrahedron Lett. 1995; 36: 2133-2136Crossref Scopus (117) Google Scholar), and the cyclic peptides CI-4 (7Izumida H. Imamura N. Sano H. J. Antibiot. 1996; 49: 76-80Crossref PubMed Scopus (61) Google Scholar, 8Izumida H. Nishijima M. T. T. A. N. Sano H. J. Antibiot. 1996; 49: 829-831Crossref PubMed Scopus (39) Google Scholar, 9Houston D.R. Eggleston I. Synstad B. Eijsink V.G.H. van Aalten D.M.F. Biochem. J. 2002; 368: 23-27Crossref PubMed Scopus (52) Google Scholar), argifin (10Shiomi K. Arai N. Iwai Y. Turberg A. Koelbl H. Omura S. Tetrahedron Lett. 2000; 41: 2141-2143Crossref Scopus (49) Google Scholar), and argadin (11Arai N. Shiomi K. Yamaguchi Y. Masuma R. Iwai Y. Turberg A. Koelbl H. Omura S. Chem. Pharm. Bull. 2000; 48: 1442-1446Crossref PubMed Scopus (104) Google Scholar, 12Houston D.R. Shiomi K. Arai N. Omura S. Peter M.G. Turberg A. Synstad B. Eijsink V.G.H. van Aalten D.M.F. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9127-9132Crossref PubMed Scopus (96) Google Scholar). Allosamidin (see Fig. 1) is a pseudotrisaccharide isolated from Streptomyces cultures (5Sakuda S. Isogai A. Matsumoto S. Suzuki A. Koseki K. Tetrahedron Lett. 1986; 27: 2475-2478Crossref Scopus (220) Google Scholar). It consists of two N-acetylallosamine sugars, linked to a novel moiety termed allosamizoline, which contains a cyclopentitol group, coupled to an oxazoline that carries a dimethyl amine (Fig. 1 and Table I). The inhibitor has been shown to inhibit all family 18 chitinases, with Ki in the nm to μm range (13Sakuda S. Chitin Enzymology. 2. Atec Edizioni, Grottammare, Italy1996: 203-212Google Scholar, 14Berecibar A. Grandjean C. Siriwardena A. Chem. Rev. 1999; 99: 779-844Crossref PubMed Scopus (315) Google Scholar). It inhibits cell separation in fungi (1Kuranda M.J. Robbins P.W. J. Biol. Chem. 1991; 266: 19758-19767Abstract Full Text PDF PubMed Google Scholar, 15Sakuda S. Nishimoto Y. Ohi M. Watanabe M. Takayama S. Isogai A. Yamada Y. Agr. Biol. Chem. Tokyo. 1990; 54: 1333-1335Crossref Scopus (1) Google Scholar), transmission of the malaria parasite Plasmodium falciparum (3Vinetz J.M. Dave S.K. Specht C.A. Brameld K.A. Xu B. Hayward R. Fidock D.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14061-14066Crossref PubMed Scopus (94) Google Scholar, 16Vinetz J.M. Valenzuela J.G. Specht C.A. Aravind L. Langer R.C. Ribeiro J.M.C. Kaslow D.C. J. Biol. Chem. 2000; 275: 10331-10341Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 17Tsai Y.-L. Hayward R.E. Langer R.C. Fidock D.A. Vinetz J.M. Infect. Immun. 2001; 69: 4048-4054Crossref PubMed Scopus (90) Google Scholar), and insect development (13Sakuda S. Chitin Enzymology. 2. Atec Edizioni, Grottammare, Italy1996: 203-212Google Scholar). Several natural allosamidin derivatives have been isolated and characterized (reviewed in Refs. 13Sakuda S. Chitin Enzymology. 2. Atec Edizioni, Grottammare, Italy1996: 203-212Google Scholar and 14Berecibar A. Grandjean C. Siriwardena A. Chem. Rev. 1999; 99: 779-844Crossref PubMed Scopus (315) Google Scholar), and the total synthesis of the inhibitor has been achieved through several strategies (14Berecibar A. Grandjean C. Siriwardena A. Chem. Rev. 1999; 99: 779-844Crossref PubMed Scopus (315) Google Scholar).Table ISubstitutions of allosamidin and its derivativesR1R2R3R4Allosamidin (ALLO)CH3HOHHDemethylallosamidin (DEME)HHOHHMethylallosamidin (METH)CH3CH3OHHGlucoallosamidin B (GLCB)HCH3HOH Open table in a new tab The structure of allosamidin in complex with family 18 chitinases has been solved for hevamine (18Terwisscha van Scheltinga A.C. Armand S. Kalk K.H. Isogai A. Henrissat B. Dijkstra B.W. Biochemistry. 1995; 34: 15619-15623Crossref PubMed Scopus (313) Google Scholar), chitinase B from Serratia marcescens (19van Aalten D.M.F. Komander D. Synstad B. Gåseidnes S. Peter M.G. Eijsink V.G.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8979-8984Crossref PubMed Scopus (407) Google Scholar), and chitinase 1 from Coccidioides immitis (20Bortone K. Monzingo A.F. Ernst S. Robertus J.D. J. Mol. Biol. 2002; 320: 293-302Crossref PubMed Scopus (70) Google Scholar). A preliminary soaking study has also been reported for the human chitinase (21Fusetti F. von Moeller H. Houston D. Rozeboom H.J. Dijkstra B.W. Boot R.G. Aerts J.M.F. van Aalten D.M.F. J. Biol. Chem. 2002; 277: 25537-25544Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). The inhibitor appears to bind from the -3 to -1 subsites, with the allosamizoline occupying the -1 subsite. Several hydrogen bonds and stacking interactions with aromatic residues appear to be responsible for the tight binding of allosamidin to the family 18 chitinases (19van Aalten D.M.F. Komander D. Synstad B. Gåseidnes S. Peter M.G. Eijsink V.G.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8979-8984Crossref PubMed Scopus (407) Google Scholar, 20Bortone K. Monzingo A.F. Ernst S. Robertus J.D. J. Mol. Biol. 2002; 320: 293-302Crossref PubMed Scopus (70) Google Scholar, 22Terwisscha van Scheltinga A.C. Kalk K.H. Beintema J.J. Dijkstra B.W. Structure. 1994; 2: 1181-1189Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). Allosamidin is thought to resemble the structure of a reaction intermediate that is unique among the glycoside hydrolases (18Terwisscha van Scheltinga A.C. Armand S. Kalk K.H. Isogai A. Henrissat B. Dijkstra B.W. Biochemistry. 1995; 34: 15619-15623Crossref PubMed Scopus (313) Google Scholar). Retaining glycoside hydrolases mostly function through a double displacement mechanism that involves a catalytic acid and a nucleophile and proceeds through a covalent enzyme-substrate intermediate (such as shown recently (23Vocadlo D.J. Davies G.J. Laine R. Withers S.G. Nature. 2001; 412: 835-838Crossref PubMed Scopus (539) Google Scholar) for lysozyme). In family 18 chitinases, however, a suitable nucleophile is missing in the protein, and instead the reaction proceeds through nucleophilic attack of the N-acetyl group on the substrate itself, resulting in an oxazoline intermediate (18Terwisscha van Scheltinga A.C. Armand S. Kalk K.H. Isogai A. Henrissat B. Dijkstra B.W. Biochemistry. 1995; 34: 15619-15623Crossref PubMed Scopus (313) Google Scholar, 19van Aalten D.M.F. Komander D. Synstad B. Gåseidnes S. Peter M.G. Eijsink V.G.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8979-8984Crossref PubMed Scopus (407) Google Scholar, 24Tews I. Terwisscha van Scheltinga A.C. Perrakis A. Wilson K.S. Dijkstra B.W. J. Am. Chem. Soc. 1997; 119: 7954-7959Crossref Scopus (277) Google Scholar, 25Brameld K.A. Goddard W.A. J. Am. Chem. Soc. 1998; 120: 3571-3580Crossref Scopus (87) Google Scholar) that is stabilized by the conserved Asp neighboring the catalytic Glu in the characteristic DXXDXDXE sequence motif (Fig. 2). It is this intermediate that is mimicked by allosamidin (Fig. 1). The inhibitor is hydrolytically stable, because it lacks the pyranose oxygen. Allosamidin is a broad-spectrum inhibitor, inhibiting all characterized family 18 chitinases. If allosamidin is to be used as a pharmacophore for development of novel compounds with activity against human pathogens, it is also necessary to take into account the human macrophage chitinase identified recently (26Hollak C.E.M. van Weely S. van Oers M.H.J. Aerts J.M.F.G. J. Clin. Invest. 1994; 93: 1288-1292Crossref PubMed Scopus (780) Google Scholar, 27Renkema G.H. Boot R.G. Muijsers A.O. Donker-Koopman W.E. Aerts J.M.F.G. J. Biol. Chem. 1995; 270: 2198-2202Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar, 28Renkema G.H. Boot R.G. Strijland A. Donker-Koopman W.E. van den Berg M. Muijsers A.O. Aerts J.M.F.G. Eur. J. Biochem. 1997; 244: 279-285Crossref PubMed Scopus (151) Google Scholar). This enzyme has endochitinase activity against chitin azure and colloidal chitin (27Renkema G.H. Boot R.G. Muijsers A.O. Donker-Koopman W.E. Aerts J.M.F.G. J. Biol. Chem. 1995; 270: 2198-2202Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar, 29Boot R.G. Blommaart E.F.C. Swart E. van der Vlugt K.G. Bijl N. Moe C. Place A. Aerts J.M.F.G. J. Biol. Chem. 2001; 276: 6770-6778Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar) and has been shown to be able to degrade chitin from the Candida albicans cell wall (29Boot R.G. Blommaart E.F.C. Swart E. van der Vlugt K.G. Bijl N. Moe C. Place A. Aerts J.M.F.G. J. Biol. Chem. 2001; 276: 6770-6778Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar). Furthermore, 6% of the human population is homozygous for an inactivated form of the gene (26Hollak C.E.M. van Weely S. van Oers M.H.J. Aerts J.M.F.G. J. Clin. Invest. 1994; 93: 1288-1292Crossref PubMed Scopus (780) Google Scholar, 30Boot R.G. Renkema G.H. Verhoek M. Strijland A. Bliek J. de Meulemeester T.M.A.M.O. Mannens M.M.A.M. Aerts J.M.F.G. J. Biol. Chem. 1998; 273: 25680-25685Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar), which preliminary studies have associated with an increased susceptibility to nematodal infections (31Choi E.H. Zimmerman P.A. Foster C.B. Zhu S. Kumaraswami V. Nutman T.B. Chanock S.J. Genes Immun. 2001; 2: 248-253Crossref PubMed Scopus (129) Google Scholar). It has therefore been proposed that the human chitinase plays a role in defense against chitinous pathogens (29Boot R.G. Blommaart E.F.C. Swart E. van der Vlugt K.G. Bijl N. Moe C. Place A. Aerts J.M.F.G. J. Biol. Chem. 2001; 276: 6770-6778Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar, 30Boot R.G. Renkema G.H. Verhoek M. Strijland A. Bliek J. de Meulemeester T.M.A.M.O. Mannens M.M.A.M. Aerts J.M.F.G. J. Biol. Chem. 1998; 273: 25680-25685Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar). Thus, it would be necessary to design allosamidin derivatives with specific activity against chitinases from pathogens but only weak inhibition of the human chitinase. Several allosamidin derivatives are already available (13Sakuda S. Chitin Enzymology. 2. Atec Edizioni, Grottammare, Italy1996: 203-212Google Scholar, 14Berecibar A. Grandjean C. Siriwardena A. Chem. Rev. 1999; 99: 779-844Crossref PubMed Scopus (315) Google Scholar). Although complexes of family 18 chitinases with allosamidin itself have been characterized (18Terwisscha van Scheltinga A.C. Armand S. Kalk K.H. Isogai A. Henrissat B. Dijkstra B.W. Biochemistry. 1995; 34: 15619-15623Crossref PubMed Scopus (313) Google Scholar, 19van Aalten D.M.F. Komander D. Synstad B. Gåseidnes S. Peter M.G. Eijsink V.G.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8979-8984Crossref PubMed Scopus (407) Google Scholar, 20Bortone K. Monzingo A.F. Ernst S. Robertus J.D. J. Mol. Biol. 2002; 320: 293-302Crossref PubMed Scopus (70) Google Scholar), none of its derivatives have been analyzed structurally in the context of a chitinase. As a first step toward the design of specific allosamidins, we describe here the crystal structures of the human chitinase complexed with allosamidin (ALLO) 1The abbreviations used are: ALLO, allosamidin; DEME, demethylallosamidin; METH, methylallosamidin; GLCB, glucoallosamidin B; HCHT, human chitinase; MES, 4-morpholineethanesulfonic acid. and three derivatives, demethylallosamidin (DEME), methylallosamidin (METH), and glucoallosamidin B (GLCB) (Fig. 1). We also report the inhibitory properties of these derivatives against human chitinase, which, together with the structures, suggest that development of a specific, yet still potent, allosamidin-based chitinase inhibitor should be possible. Structure Determination—Human chitinase (HCHT) was isolated as described previously (21Fusetti F. von Moeller H. Houston D. Rozeboom H.J. Dijkstra B.W. Boot R.G. Aerts J.M.F. van Aalten D.M.F. J. Biol. Chem. 2002; 277: 25537-25544Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). As reported earlier, soaking of HCHT crystals with ALLO and its derivatives resulted in severe cracking (21Fusetti F. von Moeller H. Houston D. Rozeboom H.J. Dijkstra B.W. Boot R.G. Aerts J.M.F. van Aalten D.M.F. J. Biol. Chem. 2002; 277: 25537-25544Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). To overcome these problems, HCHT was co-crystallized with ALLO and its derivatives DEME, METH, and GLCB (Fig. 1). The complexes were formed through addition of 10 mm allosamidin derivative to the protein, which was at a concentration of 8 mg/ml. Crystals were then grown by vapor diffusion using 1 μl of protein-inhibitor complex and 1 μl of mother liquor consisting of 25% polyethylene glycol, 550 monomethyl ether, 0.01 m ZnSO4, and 0.1 m MES, pH 6.5, equilibrated against a reservoir containing 1 ml of mother liquor. Crystals appeared after 2 days and grew to a maximum size of 0.2 × 0.1 × 0.1 mm. The crystals were cryoprotected in a solution of mother liquor containing 3 m Li2SO4 and then frozen in a nitrogen cryostream for data collection. Data were collected on beamline ID14-EH2 at the European Synchrotron Radiation Facility (Grenoble, France) and beamline X11 at the Deutsches Elektronen Synchrotron (the Deutsches Elektronen Synchrotron, Hamburg, Germany), and processed with the HKL suite of programs (32Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38777) Google Scholar) (Table II). The HCHT·ALLO structure was solved by molecular replacement with AMoRe (33Navaza J. Acta Crystallogr. Sect. A. 1994; 50: 157-163Crossref Scopus (5038) Google Scholar) (search model, the native HCHT structure (21Fusetti F. von Moeller H. Houston D. Rozeboom H.J. Dijkstra B.W. Boot R.G. Aerts J.M.F. van Aalten D.M.F. J. Biol. Chem. 2002; 277: 25537-25544Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar); top solution, r = 0.344; correlation coefficient, 0.694) and was used as a starting structure for the refinement of the other complexes. Refinement was performed with CNS (34Brunger A.T. Adams P.D. Clore G.M. Gros P. Grosse-Kunstleve R.W. Jiang J.-S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (17024) Google Scholar) interspersed with model building in O (35Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13055) Google Scholar). Topologies for the allosamidins were obtained from the PRODRG server (36van Aalten D.M.F. Bywater R. Findlay J.B.C. Hendlich M. Hooft R.W.W. Vriend G. J. Comput. Aided Mol. Des. 1996; 10: 255-262Crossref PubMed Scopus (599) Google Scholar). The inhibitors were not included until defined by unbiased |Fo| - |Fc|,ϕcalc maps (Fig. 3).Table IIDetails of data collection and structure refinementALLODEMEMETHGLCBCell dimensions (Å)a = 94.86a = 93.83a = 93.99a = 94.14b = 94.86b = 93.83b = 93.99b = 94.14c = 83.48c = 86.90c = 87.09c = 88.43Resolution range (Å)25-1.85 (2.92-1.85)25-2.55 (2.64-2.55)25-2.60 (2.69-2.60)25-2.55No. observed reflections201273 (13633)63006 (6132)54650 (5358)52134 (5376)No. unique reflections32893 (3050)13215 (1283)12526 (1233)13275 (1306)Redundancy6.1 (4.5)4.8 (4.8)4.4 (4.3)3.9 (4.1)I/σI15.9 (5.2)11.3 (2.3)11.4 (2.5)11.3 (2.7)Completeness (%)99.3 (93.8)99.6 (98.9)99.9 (100.0)98.7 (100.0)Rsym (%)4.8 (31.4)10.3 (64.2)9.4 (64.5)8.3 (66.1)Rcrys (%)18.121.521.122.5Rfrec (%)19.225.725.327.5No. Rfren reflections675260246261No. protein atoms2871287728772877No. water molecules253534866No. inhibitor atoms86424443Root mean square deviation from ideal geometryBonds (Å)0.0100.0080.0110.012Angles (°)2.01.41.61.9B-factor Root mean square deviation (Å2) (bonded, main chain)1.41.51.51.6Bprotein (Å2)27.442.944.346.7Binhibitor (Å2)30.531.733.844.9 Open table in a new tab Enzymology—The IC50 values (i.e. inhibitor concentration resulting in 50% inhibition) of the allosamidin derivatives were determined using the fluorogenic substrate 4-methylumbelliferyl-β-d-N,N′,N″-triacetylchitotriose (4MU-NAG3; Sigma) in a standard assay, as described previously (26Hollak C.E.M. van Weely S. van Oers M.H.J. Aerts J.M.F.G. J. Clin. Invest. 1994; 93: 1288-1292Crossref PubMed Scopus (780) Google Scholar). Briefly, in a final volume of 125 μl, a constant amount of enzyme was incubated with 0.022 mm substrate in McIlvain buffer (100 mm citric acid, 200 mm sodium phosphate, pH 5.2) containing 1 mg/ml bovine serum albumin, for 20 min at 37 °C in the presence of different concentrations of inhibitor. After addition of 2.5 ml of 0.3 m glycine-NaOH, pH 10.6, the fluorescence of the liberated 4MU was quantified using a PerkinElmer Life Sciences LS2 fluorimeter (excitation 445 nm, emission 366 nm). The ability of chitotriosidase to transglycosylate does not allow determination of Ki values. Overall Structures—HCHT were grown in the presence of ALLO, DEME, METH, and GLCB (Fig. 1). The crystals diffracted to 1.85, 2.55, 2.60, and 2.55 Å, respectively. The structures were solved by molecular replacement using the native HCHT structure as a search model (21Fusetti F. von Moeller H. Houston D. Rozeboom H.J. Dijkstra B.W. Boot R.G. Aerts J.M.F. van Aalten D.M.F. J. Biol. Chem. 2002; 277: 25537-25544Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar) and refined to R-factors (Rfree) of 0.181 (0.192), 0.215 (0.257), 0.211 (0.253), and 0.225 (0.275), respectively. Models for the allosamidins were only included in the refinement, when they were well defined by unbiased Fo - Fc,ϕcalc density (Fig. 3). Analysis of Ramachandran plots calculated with PROCHECK (37Laskowski R.A. McArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar) reveal that there is only one residue (Asp-328) in a disallowed conformation, yet electron density for this residue is well defined. The allosamidins bind in a groove on the chitinase, occupying subsites -3 to -1 (Figs. 3 and 4). In the HCHT·ALLO complex, a second, disordered, allosamidin molecule (average B-factors 40.1 Å2, compared with 20.8 Å2 for the first molecule) is observed to bind to the protein, approximately occupying the +1 to +3 subsites. It is possible that this represents a weaker binding interaction and only occurs because of the high concentrations (10 mm) of allosamidin in the mother liquor. Subsequent comparisons and discussions will focus on the ordered allosamidin molecule only. Three chitinase·allosamidin complexes have been reported previously for hevamine (18Terwisscha van Scheltinga A.C. Armand S. Kalk K.H. Isogai A. Henrissat B. Dijkstra B.W. Biochemistry. 1995; 34: 15619-15623Crossref PubMed Scopus (313) Google Scholar), chitinase B from S. marcescens (19van Aalten D.M.F. Komander D. Synstad B. Gåseidnes S. Peter M.G. Eijsink V.G.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8979-8984Crossref PubMed Scopus (407) Google Scholar), and chitinase 1 from C. immitis (20Bortone K. Monzingo A.F. Ernst S. Robertus J.D. J. Mol. Biol. 2002; 320: 293-302Crossref PubMed Scopus (70) Google Scholar). In the HCHT·ALLO structure, the inhibitor binds in the same location and orientation as observed in these complexes. There are no significant backbone conformational changes; the HCHT·ALLO complex superimposes with an root mean square deviation of 0.36 Å on the HCHT structure Cα atoms. The tightest interactions are formed with the allosamizoline in the -1 subsite, which is lined with residues that are conserved in family 18 chitinases from a wide range of organisms (Figs. 2 and 3). Trp-358 stacks with the hydrophobic face of the allosamizoline, similar to the interaction of this residue with the -1 boat pyranose in the chitinase B-NAG5 complex (19van Aalten D.M.F. Komander D. Synstad B. Gåseidnes S. Peter M.G. Eijsink V.G.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8979-8984Crossref PubMed Scopus (407) Google Scholar). Tyr-27, Phe-58, Gly-98, Ala-183, Met-210, and Met-356 are the main contributors to a hydrophobic pocket, which is occupied by the two allosamizoline methyl groups (Figs. 3 and 4). The allosamizoline moiety has several hydrogen bonding interactions with the protein (see Table IV). Asp-138 stabilizes the positive charge on the oxazoline (Fig. 3) and is flipped ∼180° around χ1 compared with the native structure (21Fusetti F. von Moeller H. Houston D. Rozeboom H.J. Dijkstra B.W. Boot R.G. Aerts J.M.F. van Aalten D.M.F. J. Biol. Chem. 2002; 277: 25537-25544Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar), as also observed in all other chitinase·ALLO complexes (19van Aalten D.M.F. Komander D. Synstad B. Gåseidnes S. Peter M.G. Eijsink V.G.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8979-8984Crossref PubMed Scopus (407) Google Scholar, 20Bortone K. Monzingo A.F. Ernst S. Robertus J.D. J. Mol. Biol. 2002; 320: 293-302Crossref PubMed Scopus (70) Google Scholar, 22Terwisscha van Scheltinga A.C. Kalk K.H. Beintema J.J. Dijkstra B.W. Structure. 1994; 2: 1181-1189Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). The backbone nitrogen of Trp-99 hydrogen bonds the allosamizoline O3 (Fig. 3). On the opposite side of the inhibitor, Tyr-212 and Asp-213 hydrogen bond with the allosamizoline O7 and O6, respectively (Fig. 3).Table IVHCHT-inhibitor hydrogen bondsAtom-1 subsite-2 subsite-3 subsiteProtein/waterD-AHB2Protein/waterD-AHB2Protein/waterD-AHB2N2 ALLOAsp-138, Oδ22.730.68DEMEAsp-138, Oδ22.770.72H2O3.030.89METHAsp-138, Oδ22.670.57H2O3.120.81GLCBAsp-138, Oδ22.520.54O3 ALLOTrp-99, N3.010.85Glu-297, Oc13.030.47H2O2.600.46DEMETrp-99, N2.940.82H2O2.770.78METHTrp-39, N2.890.80GLCBTrp-99, N3.010.69H2O2.900.87O4 ALLOAsn-100, Nδ22.930.43DEMEH2O2.630.92METHAsn-100, Nδ23.540.34GLCBO5 ALLOH2O3.270.58DEMEMETHGLCBO6 ALLOAsp-213, Oδ22.490.71H2O + Asn-100, N2.59,3.130.82,0.73H2O3.210.31DEMEAsp-213, Oδ22.580.76H2O + Asn-100, N2.69,3.200.90,0.64Glu-297, Oϵ1, H2O2.42,2.640.60,0.60METHAsp-213, Oδ22.770.85H2O + Asn-100, N2.89,3.220.81,0.79GLCBAsp-13, Oδ22.830.69Asn-100, N3.020.87O7 ALLOTyr-212, Oη3.030.93H2O + Trp-358, Nc12.90,2.670.62,0.58Asn-100, Nδ2, H2O3.13,3.050.64,0.62DEMETyr-212, Oη3.180.92H2O + Trp-358, Nc12.88,2.660.69,0.53Asn-100, Nδ22.430.42METHTyr-212, Oη3.140.90H2O + Trp-358, Ne13.10,2.640.62,0.44GLCBTyr-212, Oη2.990.98Trp-358, Ne12.670.56 Open table in a new tab In the chitinase B·ALLO structure, an ordered water molecule was observed within 3.3 Å of the allosamizoline C1 carbon, and subsequent analysis of the hevamine·ALLO complex also revealed such a water molecule (19van Aalten D.M.F. Komander D. Synstad B. Gåseidnes S. Peter M.G. Eijsink V.G.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8979-8984Crossref PubMed Scopus (407) Google Scholar). A similar water molecule is also found upon inspection of the C. immitis CTS1·ALLO complex published recently (20Bortone K. Monzingo A.F. Ernst S. Robertus J.D. J. Mol. Biol. 2002; 320: 293-302Crossref PubMed Scopus (70) Google Scholar). This interaction is thought to be reminiscent of the attack of a water molecule, which hydrolyzes the oxazolinium ion reaction intermediate (19van Aalten D.M.F. Komander D. Synstad B. Gåseidnes S. Peter M.G. Eijsink V.G.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8979-8984Crossref PubMed Scopus (407) Google Scholar). However, this water molecule is not observed in the complexes with the allosamidins described here. In the HCHT·ALLO complex, the position of this water molecule is occupied by the N-acetyl group of the second disordered allosamidin molecule. The relatively low resolution diffraction data for the complexes with the allosamidin derivatives may not be sufficient to define the position of this particular water molecule. Although the allosamizoline moiety tightly binds conserved residues through hydrogen bonding and hydrophobic interactions, there are fewer interactions with the two N-acetylallosamine sugars in the -2 and -3 subsites (see Table IV). The sugar in the -2 subsite makes two hydrogen bonds to Asn-100, via the O4 and O6 atoms (Fig. 3). Further hydrogen bonds are formed from O3 to Glu-297 and from Trp-358 to O7. The methyl on the N-acetyl group binds in a hydrophobic pocket formed by Tyr-267, Met-300, and Leu-362 (Fig. 3). The -3 sugar stacks with Trp-31, whereas a hydrogen bond is formed with the side chain of Glu-297 (Fig. 3). Two ordered water molecules mediate several inhibitor-protein hydrogen bonds (Fig. 3). Residues 266–337 form the α/β domain in HCHT, which is absent in the smaller family 18 chitinases such as hevamine and the fungal chitinases (Figs. 2 and 4). Therefore, these smaller enzymes have a more solvent exposed -2 subsite and almost no interactions with the N-acetylallosamine at -3 (18Terwisscha van Scheltinga A.C. Armand S. Kalk K.H. Isogai A. Henrissat B. Dijkstra B.W. Biochemistry. 1995; 34: 15619-15623Crossref PubMed Scopus (313) Google Scholar). Enzymology—A large number of allosamidin derivatives have been synthesized and characterized for their biological activity (reviewed in Refs. 13Sakuda S. Chitin Enzymology. 2. Atec Edizioni, Grottammare, Italy1996: 203-212Google Scholar and 14Berecibar A. Grandjean C. Siriwardena A. Chem. Rev. 1999; 99: 779-844Crossref PubMed Scopus (315) Google Scholar). Here, we have focused on three derivatives (DEME, METH, and GLCB; Fig. 1) for which enzymological data with several chitinases is already available (compiled in Ref. 13Sakuda S. Chitin Enzymology. 2. Atec Edizioni, Grottammare, Italy1996: 203-212Google Scholar) (Table III). We have determined the apparent IC50 values of these derivatives against human chitinase using a standard assay with the fluorescent substrate 4-methylumbelliferyl-chitotriose (4MU-NAG3) (Table III). The IC50 for ALLO (40 nm) has been reported previously (38Renkema, G. H. (1997) Chitotriosidase, Studies on the Human Chitinase. Ph.D. thesis, University of Amsterdam, Amsterdam, The NetherlandsGoogle Scholar). Removal of one of the methyl groups on the allosamizoline moiety leads to an ∼20-fold increase in affinity (DEME; Fig. 1), compared with ALLO. If an extra methyl group is added to the O6 hydroxyl on the -3 allosamine, a similar increase in inhibition is observed (METH; Fig. 1 and Table III). If both these modifications are combined together with epimerization at carbon C3, only a 5-fold stronger inhibition is measured (GLCB; Fig. 1 and Table III), compared with ALLO. These data (together with other demethylallosamidin derivatives not discussed here (13Sakuda S. Chitin Enzymology. 2. Atec Edizioni, Grottammare, Italy1996: 203-212Google Scholar)) suggest that the major effect on inhibition is the large increase in binding upon removal of one of the methyl groups on the allosamizoline moiety. The inhibition data of these derivatives on other chitinases (Table III) shows that there are two different classes: one that, similar to HCHT, shows a 10–100-fold drop for DEME compared with ALLO (the chitinases from S. cerevisiae and C. albicans) and another that does not show this effect (the chitinases from Trichoderma harzianum and Bombyx mori). In addition, HCHT and the chitinases from T. harzianum and B. mori bind ALLO 10–1000-fold better than the fungal chitinases from S. cerevisiae and C. albicans. Inspection of the HCHT·ALLO structure (Fig. 3) and a sequence alignment (Fig. 2) reveals two potential reasons for this difference in inhibition. First, the S. cerevisiae and C. albicans chitinases are similar to the relatively small plant chitinase hevamine, which lacks the extra α/β domain that gives the active site a groove character and provides several contacts with the inhibitor (Tyr-267, Glu-297, and Met-300 in HCHT; Figs. 3 and 4). In addition, Met-210 and Met-356, two hydrophobic residues that form part of the pocket for the allosamizoline methyl groups, are conserved in HCHT, T. harzianum, and B. mori chitinases but replaced by more hydrophilic residues in the small fungal chitinases.Table IIIApparent IC50 values of the allosamidin derivatives against chitinases from different speciesS. cerevisiaeC. albicansT. harzianumB. moriHumanALLO550001000013004840DEME48011001300811.9METH60000140001900652.6GLCB81013002600658.0 Open table in a new tab Comparison of the Complexes—Despite the wealth of synthetic and natural allosamidins described in the literature, currently only complexes of family 18 chitinases with native ALLO have been determined (19van Aalten D.M.F. Komander D. Synstad B. Gåseidnes S. Peter M.G. Eijsink V.G.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8979-8984Crossref PubMed Scopus (407) Google Scholar, 20Bortone K. Monzingo A.F. Ernst S. Robertus J.D. J. Mol. Biol. 2002; 320: 293-302Crossref PubMed Scopus (70) Google Scholar, 22Terwisscha van Scheltinga A.C. Kalk K.H. Beintema J.J. Dijkstra B.W. Structure. 1994; 2: 1181-1189Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). The complexes of HCHT with the DEME, METH, and GLCB allosamidin derivatives (Fig. 1) show no significant backbone conformational changes and superimpose with root mean square deviations of 0.32, 0.31, and 0.32 Å on HCHT Cα atoms, respectively. Analysis of the binding pocket shows that although several key hydrogen bonds are conserved (Table IV and Fig. 3), there are differences in hydrogen bonding and side chain conformation. Demethylallosamidin—In the HCHT·DEME structures, where the allosamidin lacks one of the methyl groups on the allosamizoline (Fig. 1), the remaining methyl group points toward the oxygen side of the oxazoline ring, creating a small void that is filled by Glu-140 and Asp-138 rotating up to 30 degrees around χ1/2. This brings the Asp-138 Oδ2 atom closer to the allosamizoline nitrogen that carries the remaining methyl group, almost allowing formation of a hydrogen bond (distance 3.7 Å) (Fig. 3). This could explain the observed increase in affinity that is observed for DEME (Table III). There are no further noticeable conformational changes in the -1 binding site. The residues surrounding the allosamizoline moiety are the only ones that are highly conserved in family 18 chitinases (Figs. 2, 3, 4). Analysis of sequence differences does not reveal an amino acid change that is consistent with the different changes in inhibition when comparing ALLO and DEME binding to a range of chitinases (Table III). However, residues 95, 208, 210, and 356 do form part of the hydrophobic pocket for the allosamizoline methyl groups and are not conserved. It is possible that several concerted changes at these positions are responsible for the two types of effects on inhibition (i.e. no change or 10–100-fold stronger inhibition; Table III) for DEME. Methylallosamidin—The structure of the HCHT·METH complex reveals that introduction of a methyl group on O6 of the -3 allosamine displaces an ordered water molecule from the binding pocket (Figs. 1 and 3). This ordered water molecule hydrogen bonds with DEME but not with ALLO (and is therefore not shown in Fig. 3-ALLO). In HCHT, addition of the methyl group to ALLO appears to increase the inhibition (Table III). A possible explanation could be the entropic gain through displacement of the ordered water molecule, yet a similar effect is not observed in the other chitinases (Table III). In the absence of structural data for these chitinases it is difficult to explain this, in particular because the entire α/β domain is missing in the smaller fungal chitinases (Fig. 2). Glucoallosamidin B—In the GLCB derivative three modifications are combined: removal of one of the allosamizoline methyls (as in DEME), addition of a methyl on O6 of the -3 allosamine (as in METH), and epimerization of the -2 allosamine to a glucosamine (Fig. 1). The HCHT·GLCB complex (Fig. 3) shows several changes compared with the HCHT·ALLO complex. In general, the GLCB molecule appears to be shifted about 0.5 Å toward the reducing end of the binding cleft (Fig. 3). This leads to weakening of the key hydrogen bonds in the -1 subsite (Table IV), which may be partially responsible for the weaker inhibition of HCHT, compared with the DEME and METH derivatives. Also, changes similar to those in the HCHT·DEME (rotation of Asp-138 and Glu-140) and the HCHT·METH complex (displacement of an ordered water molecule) are observed (Fig. 3). In addition, the equatorial O3 oxygen is no longer able to hydrogen bond another ordered water molecule observed in the HCHT·ALLO complex. The displacement of this water molecule leads to the loss of the water-mediated hydrogen bond with Arg-269 (Fig. 3). At the same time, however, the equatorial configuration of the O3 hydroxyl allows formation of the hydrogen bond with the pyranose oxygen of the -3 sugar, which is generally found in glucopolymers. The trends observed in the GLCB inhibition data (Table III) are similar to those for DEME, suggesting that removal of one of the allosamizoline methyls is the dominating effect. Design of Specific Allosamidin Derivatives—Inhibition data of allosamidin and its derivatives show that there are significant differences in inhibition against the different chitinases (Table III). This suggests that if allosamidin is used as a template in novel synthetic studies aimed at designing derivatives against a specific chitinase, it would be possible to engineer a certain degree of specificity. This is important if such derivatives are used as antibiotics against human pathogens, as these molecules should not inhibit human chitinase, which has been suggested to be part of an innate defense against chitinous pathogens (29Boot R.G. Blommaart E.F.C. Swart E. van der Vlugt K.G. Bijl N. Moe C. Place A. Aerts J.M.F.G. J. Biol. Chem. 2001; 276: 6770-6778Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar, 30Boot R.G. Renkema G.H. Verhoek M. Strijland A. Bliek J. de Meulemeester T.M.A.M.O. Mannens M.M.A.M. Aerts J.M.F.G. J. Biol. Chem. 1998; 273: 25680-25685Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar, 31Choi E.H. Zimmerman P.A. Foster C.B. Zhu S. Kumaraswami V. Nutman T.B. Chanock S.J. Genes Immun. 2001; 2: 248-253Crossref PubMed Scopus (129) Google Scholar). The structures described here allow for an evaluation of the potential for structure-based design of specific allosamidins. When sequence conservation is interpreted in the context of the HCHT·ALLO complex (Fig. 4) it appears that the only residues that are conserved and form part of the binding site are those interacting with the allosamizoline (Figs. 2, 3, 4). This would suggest it is difficult to make allosamizoline derivatives that are specific for certain chitinases. Yet not all residues contacting the allosamizoline are conserved (Fig. 2), and the differential inhibition for the DEME derivative (Table III) demonstrates it is possible to exploit these differences. For instance, Asn-100 makes hydrogen bonding interactions with the -2/-3 sugars (Fig. 3 and Table IV) yet is only present in the human chitinase. In general, there is no sequence conservation in the residues surrounding the -2 and -3 subsites (Figs. 2 and 4). This is especially true for the smaller S. cerevisiae and C. albicans chitinases, which lack the extra α/β domain that harbors several residues that are seen to interact with the allosamidins in the complexes described here (Figs. 2, 3, 4 and Table IV). Thus, the binding site in HCHT has a deep groove character, whereas in hevamine (and the closely related small fungal chitinases) it is a shallow pocket (22Terwisscha van Scheltinga A.C. Kalk K.H. Beintema J.J. Dijkstra B.W. Structure. 1994; 2: 1181-1189Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar) (Fig. 4). Hence, it should be possible to design derivatives that have larger groups on the -2 and -3 sugars, fitting only the smaller, more open, chitinases. Alternatively, moieties could be introduced that interact specifically with side chains lining the deeper grooves of the larger chitinases. Recently, an additional mammalian chitinase has been described that is mainly expressed in the stomach (29Boot R.G. Blommaart E.F.C. Swart E. van der Vlugt K.G. Bijl N. Moe C. Place A. Aerts J.M.F.G. J. Biol. Chem. 2001; 276: 6770-6778Abstract Full Text Full Text PDF PubMed Scopus (431) Google Scholar). This protein has 52% sequence identity with the human macrophage chitinase and also contains the additional α/β fold. Given the different expression patterns and the fact that this additional mammalian chitinase has a pH optimum of around 2, it is likely that it plays a different role than the human macrophage chitinase and has enough sequence differences to allow development of chitinase-specific inhibitors. The structures of the human chitinase in complex with allosamidin and its derivatives have given new insights into the molecular mechanisms and specificity of these potent family 18 chitinase inhibitors. The dimethyl derivative, 10- to 100-fold more potent than allosamidin against most chitinases, appears to bind more strongly because of possible extra interactions with conserved residues that are part of the family 18 chitinase sequence signature. Modifications of the -2 and -3 N-acetylallosamines lead to displacement of ordered water molecules and altered hydrogen bonding with the protein. The structures could be used for further structure-based optimization of allosamidin.

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