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

Abstract

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 ␤-sheet and the latter a two-stranded ␤-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.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 (1-5). The antimicrobial activity is initially identified for chitin-binding proteins extracted from plants (6 -7), 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). It has been well demonstrated that this domain is indispensable for the antimicrobial activity and exhibits a significant conservation in primary sequence (Ͼ40%) and in three-dimensional (3D) 1 structure (9 -12). 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,(13)(14)(15)(16). Kawabata et al. (1) 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) 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) 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. EXPERIMENTAL PROCEDURESAn invertebrate...

Carbohydrate-binding modules: fine-tuning polysaccharide recognition

“…The fold comprises predominantly coil, but does have two small β-sheets and a small region of helix ( Figure 1). This fold can accommodate a surprisingly extended binding site as exemplified by WGA, [52]); (e) family 2 CBM, Cf CBM2, from Cellulomonas fimi (PDB code 1EXG [81]); (f) family 9 CBM, TmCBM9-2, from T. maritima in complex with cellobiose (PDB code 1I82 [33]); (g) family 32 CBM, MvCBM32, from Micromonospora viridifaciens in complex with galactose (PDB code 1EUU [65]); (h) family 5 CBM, EcCBM5, from Erwinia chrysanthemi (PDB code 1AIW [82]); (i) family 13 CBM, SlCBM13, from S. lividans in complex with xylopentaose (PDB code 1MC9 [48]); (j) family 1 CBM, TrCBM1, from Trichoderma reesi (PDB code 1CBH [83]); (k) family 10 CBM, CjCBM10, from Cellvibrio japonicus (PDB code 1E8R [84]); (l) family 18 CBM from Urtica dioca in complex with chitotriose (PDB code 1EN2 [85]); (m) family 14 CBM, tachychitin, from Tachypleus tridentatus (PDB code 1DQC [86]). Bound ligands are shown as 'liquorice' representations, while bound metal ions are shown as a blue spheres.…”

Binding of the AVR4 Elicitor of Cladosporium fulvum to Chitotriose Units Is Facilitated by Positive Allosteric Protein-Protein Interactions

“…This blue shift indicates that one of the Trp residues becomes more solvent-buried upon complexation, which is most likely Trp 71 because the corresponding residue in Tachycitin is solvent-exposed (9). In contrast, Trp 63 would remain solvent-buried because Trp 63 is involved in a hydrophobic interaction in the core of the protein with Tyr 38 (again based on the structure of Tachycitin) (9). This hydrophobic interaction is strictly conserved based on the high degree of conservation of both aromatic residues in the CBM14 family.…”

“…However, CBM18 is only fused to the plant-specific family 19 catalytic domain, whereas chitinases of mammals and invertebrates utilize CBM14 in combination with the family 18 catalytic domain. Sequence homology is missing between the two motifs, but the three-dimensional structure of Tachycitin revealed that CBM14 and CBM18 partially share their tertiary structure (9).…”

Retroactive, a membrane‐anchored extracellular protein related to vertebrate snake neurotoxin‐like proteins, is required for cuticle organization in the larva of Drosophila melanogaster

“…4A). It is important to note that Rtv does not contain any known chitin binding do-main or any other motif found in cuticle proteins (Andersen et al, 1995;Shen and Jacobs-Lorena, 1999;Suetake et al, 2000Suetake et al, , 2002 and, therefore, defines a new class of cuticle proteins.…”

“…However, an instructive role for fiber orientation is assigned to the so-called plaques at the apical plasma membrane that are thought to harbor enzymes required for chitin synthesis (Locke, 1976). On the molecular level, it has been proposed that ␤-sheet conformation in the conserved domains of cuticular proteins such as the R&R chitin binding motif and the tachycitin-like chitin binding domain may be crucial for chitin binding (Suetake et al, 2000;Rebers and Willis, 2001;Hamodrakas et al, 2002;Togawa et al, 2004) and that this association may be responsible for the characteristic organization of the laminae (Fraenkel and Rudall, 1947;Iconomidou et al, 1999). Commonly, aromatic amino acids have been demonstrated to be implicated in the interaction between chitin binding proteins and their substrate (Asensio et al, 1998;Rebers and Willis, 2001;Hardt and Laine, 2004;Katouno et al, 2004).…”