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J Biol Chem, Vol. 275, Issue 18, 13654-13661, May 5, 2000

Solution Structure of the Chitin-binding Domain of Bacillus circulans WL-12 Chitinase A1^*

Takahisa Ikegami, Terumasa Okada, Masayuki Hashimoto^§, Shizuka Seino^§, Takeshi Watanabe^§, and Masahiro Shirakawa^¶

From the  Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan and the ^§ Department of Applied Biological Chemistry, Faculty of Agriculture, Niigata University, 8050 Ikarashi-2, Niigata 950-2181, Japan

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The three-dimensional structure of the chitin-binding domain (ChBD) of chitinase A1 (ChiA1) from a Gram-positive bacterium, Bacillus circulans WL-12, was determined by means of multidimensional heteronuclear NMR methods. ChiA1 is a glycosidase that hydrolyzes chitin and is composed of an N-terminal catalytic domain, two fibronectin type III-like domains, and C-terminal ChBD_ChiA1 (45 residues, Ala⁶⁵⁵-Gln⁶⁹⁹), which binds specifically to insoluble chitin. ChBD_ChiA1 has a compact and globular structure with the topology of a twisted -sandwich. This domain contains two antiparallel -sheets, one composed of three strands and the other of two strands. The core region formed by the hydrophobic and aromatic residues makes the overall structure rigid and compact. The overall topology of ChBD_ChiA1 is similar to that of the cellulose-binding domain (CBD) of Erwinia chrysanthemi endoglucanase Z (CBD_EGZ). However, ChBD_ChiA1 lacks the three aromatic residues aligned linearly and exposed to the solvent, which probably interact with cellulose in CBDs. Therefore, the binding mechanism of a group of ChBDs including ChBD_ChiA1 may be different from that proposed for CBDs.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Chitinase (EC 3.2.1.14) is a glycosyl hydrolase that catalyzes the hydrolytic degradation of chitin, a fibrous insoluble polysaccharide made of -1,4-N-acetyl-D-glucosamine residues. Chitinases are found in a wide variety of organisms that possess chitin as a constituent (fungi, insects, and crustaceans) and organisms that do not possess chitin as well (bacteria, plants, and vertebrates). The roles of chitinases in these organisms are diverse (1). Invertebrates require chitinases for partial degradation of old exoskeletons. Fungi produce chitinases to modify chitin, which is used as an important cell wall component. Bacteria produce chitinases to digest chitin and utilize it as carbon and energy sources. It is suggested that the production of chitinases by higher plants is a part of defense mechanisms against fungal pathogens (2).

Bacillus circulans WL-12 is a Gram-positive bacterium identified as being lytic for yeast and fungal cell walls (3). The bacterium has been reported to secrete multiple chitinases into culture medium containing chitin as an inducer (4, 5). Among these chitinases, A1 encoded by the chiA gene is thought to be the key enzyme in the chitinase system of this bacterium, because chitinase A1 (ChiA1)¹ is produced most abundantly and exhibits the highest activity as to the hydrolysis of colloidal chitin (4) and a high affinity to insoluble chitin. ChiA1 (M_r = ~74,000) contains three discrete functional domains: an N-terminal catalytic domain (CatD) (417 amino acid residues), a tandem repeat of fibronectin type III-like (FnIII) domains (duplicate 95 residues), and a C-terminal chitin-binding domain (45 residues) (6). Such a structural form of discrete catalytic and substrate-binding domains (7) as in ChiA1 has also been observed for some other polysaccharide-degrading enzymes, including cellulases (8, 9), xylanases (10), amylases, and a -1,3-glucanase (11).

The C-terminal chitin-binding domain (ChBD_ChiA1) is required for ChiA1 to bind specifically to insoluble chitin and to hydrolyze it efficiently (4, 6). We have found that ChBD_ChiA1 does not bind to chito-oligosaccharides or soluble derivatives of chitin but does bind to insoluble or crystalline chitin.² So far, two roles of the cellulose-binding domains (CBDs) of cellulases in their hydrolysis activities have been suggested: (i) CBDs may enhance the cellulase activities by concentrating the cellulases on a cellulose surface (13, 14), and (ii) CBDs may disrupt noncovalent interactions including hydrogen bonds between adjacent glucose units (15). The chitin-binding domains (ChBDs) are also considered to have similar roles to those of CBDs, but the roles of ChBDs remain unclear. Although a large number of studies have been performed on the structures of the CBDs of cellulases (8, 16-21), little is known about the tertiary structures of the ChBDs of chitinases.

The structures of at least three chitinases have already been determined. They are (i) endochitinase from barley seeds, Hordeum vulgare (22), (ii) hevamine with combined chitinase and lysozyme activities from a plant, Hevea brasiliensis (23), and (iii) chitinase A from a Gram-negative soil bacterium, Serratia marcescens (24). However, the two plant chitinases do not contain ChBDs that are separated from their catalytic domains. It is suggested that the remaining chitinase, S. marcescens chitinase A, contains a putative ChBD in its N-terminal region (ChiN) (residues Ala²⁴-His¹³⁷) (1), but no sequence similarity exists between ChiN and ChBD_ChiA1 (see "FnIII Domains"). Therefore, the three-dimensional structure of a ChBD whose function has clearly been identified is necessary for elucidation of its binding mechanism with chitin and its role in the subsequent catalytic activity. Further, some CBDs studied so far have the ability to bind to soluble cello-oligosaccharides (17) and chitin (25) as well as crystalline cellulose. On the other hand, ChBD_ChiA1 only binds to insoluble chitin; specifically, it does not bind to soluble chito-oligosaccharides, soluble chitin derivatives, or cellulose.² Thus, it is important to approach the mechanism by which this rather small domain consisting of 45 amino acids exhibits this binding specificity to insoluble chitin from the viewpoint of its tertiary structure.

Here we present the three-dimensional structure of the chitin-binding domain (Ala⁶⁵⁵-Gln⁶⁹⁹) of B. circulans WL-12 chitinase A1 (ChBD_ChiA1) determined by NMR using a uniformly ¹⁵N-labeled domain. To the best of our knowledge, this is the first reported structure of a ChBD that binds specifically to chitin. Analysis involving structures and sequential alignments allowed us to suggest that the chitin-binding mechanism of ChBD_ChiA1 is different from the currently proposed cellulose-binding mechanism of the CBDs of cellulases, which are supposed to interact with cellulose via three aromatic rings arranged linearly on the surfaces of CBDs.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Sample Preparation-- Cells of Escherichia coli BL21(DE3) harboring the expression pET3a plasmid (Novagen, Madison, WI) with the ChBD_ChiA1 gene were grown in M9 minimal medium containing 100 µg/ml ampicillin, 0.1% glycerol, 4.0 g/liter D-glucose, and 0.5 g/liter ¹⁵NH₄Cl as a sole nitrogen source. The cells were incubated at 30 °C with shaking, and expression of the protein was induced by the addition of 0.5 mM isopropyl -D-thiogalactopyranoside for 24 h. The cells were collected by centrifugation and disrupted by sonication in 100 mM Tris-HCl buffer (pH 8.0) containing 10 mM phenylmethylsulfonyl fluoride and 1 mM EDTA. Proteins in the soluble fraction were collected by ammonium sulfate precipitation (60% saturation) and then dialyzed against 5 mM sodium phosphate buffer (pH 6.0). The solution was applied to a chitin affinity column (Chitin EX, Funakoshi, Japan) equilibrated previously with the same buffer. After washing the column with 20 mM sodium phosphate buffer (pH 6.0) containing 1 M sodium chloride and with 20 mM sodium acetate buffer (pH 5.5), the protein was eluted with 20 mM acetic acid (pH 3.0). About 15 mg of purified and uniformly ¹⁵N-labeled ChBD_ChiA1 was obtained from 1 liter of an M9 minimal culture. The obtained protein has a T7 tag consisting of 14 residues (H₂N-Met-Ala-Ser-Met-Thr-Gly-Gly-Gly-Gly-Met-Gly-Arg-Gly-Ser-) derived from the expression vector at its N terminus. The protein was stable and did not form a precipitate, at least upon changes of the solution in the pH range of 3-9, a NaCl concentration range of 20-1000 mM, and a temperature range of 4-40 °C. The samples for most of the NMR measurements comprised 2.0 mM ¹⁵N-labeled ChBD_ChiA1 in 10 mM KH₂PO₄-K₂HPO₄ (pH 6.0) and 10 mM deuterated dithiothreitol in 90% H₂O, 10% ²H₂O or 1.2 mM ¹⁵N-labeled ChBD_ChiA1 in 100 mM KH₂PO₄-K₂HPO₄ (pH 6.0) and 10 mM deuterated dithiothreitol in 99.8% ²H₂O. Protein concentrations were estimated using the calculated molar absorption coefficient at 280 nm (₂₈₀ = 20,970). The calculated weight of the molecule, without the T7 tag region, is 5,018.

NMR Spectroscopy-- All NMR experiments were performed with a Bruker DRX500 or DRX800 spectrometer equipped with a triple resonance (¹H, ¹⁵N, and ¹³C) probe with a self-shielded triple axis gradient coil. Most spectra were recorded at 310 K. For ¹H and ¹⁵N resonance assignments (26), two-dimensional ¹⁵N-¹H HSQC, two-dimensional TOCSY (with a mixing time of 50 ms), three-dimensional ¹⁵N-edited TOCSY (with a mixing time of 70 ms), and two-dimensional H(NN)H TOCSY and three-dimensional (H)NNH TOCSY (with mixing times of 308 ms) (27), spectra were acquired. The numbers of complex points and spectral widths in the three-dimensional (H)NNH TOCSY experiment with the DRX500 were 20, 620.2 Hz (¹⁵N, F₁, and F₂) and 681, 8012.8 Hz (¹H, F₃). For dihedral angle constraints, a two-dimensional HMQC-J spectrum (26) was acquired with the DRX800. The ¹⁵N dimension was recorded with an acquisition time of 403 ms (400 complex data sets). The digital resolution was 0.5 Hz after zero-filling and subsequent Fourier transformation. For interproton distance constraints (26), two-dimensional NOESY and three-dimensional ¹⁵N-edited NOESY spectra were acquired with the DRX800. The mixing time in both experiments was 150 ms. The two-dimensional TOCSY and two-dimensional NOESY experiments were performed with samples dissolved in 99.8% ²H₂O, and the other experiments were performed with samples dissolved in 90% H₂O/10% ²H₂O. Amide proton exchange with the solvent was monitored with a series of ¹⁵N-¹H-HSQC spectra obtained every 30-60 min at 298 K after dissolving lyophilized and protonated ¹⁵N-labeled ChBD_ChiA1 (pH 6.0) in ²H₂O. The signals remaining at eight times within 22 h were identified. In all experiments, the ¹H carrier was set to the frequency of water resonance (4.692 ppm), and the ¹⁵N carrier was set to 118.32 ppm. The two-dimensional ¹⁵N-¹H HSQC, three-dimensional ¹⁵N-edited NOESY, two-dimensional H(NN)H TOCSY, and three-dimensional (H)NNH TOCSY experiments included the WATERGATE and Water-flip-back techniques (26). The three-dimensional ¹⁵N-edited TOCSY experiment included the sensitivity enhancement and gradient echo methods for the indirect ¹⁵N dimension (26). All other indirect dimensions were recorded in the States-TPPI manner (26). The NMR data were processed and analyzed using the nmrPipe (28) and Pipp (29) software packages, respectively. Stereospecific assignments of the methyl groups of the leucine and valine residues were achieved with 15% fractionally ¹³C-labeled ChBD_ChiA1 dissolved in 99.8% ²H₂O as described (30).

Structure Calculation-- The NOE connectivities derived from strong, medium, and weak cross-peaks were categorized and assumed to correspond to the upper limits for interproton distances of 3.0, 4.0, and 5.0 Å, respectively. Pseudoatom corrections were applied to upper bound constraints involving methyl, methylene, and aromatic ring protons as described (31). The NOE list was not filtered to remove restraints of no structural value. The distance constraints for the hydrogen bonds were applied for slowly exchanging amides, i.e. 2.8-3.3 Å for N-O pairs and 1.8-2.3 Å for H-O pairs. A pair of these constraints was used for each hydrogen bond to restrict the length and angle of the bond (32, 33). The -strands were aligned on the basis of the interstrand NOE connectivities among H and H^N atoms. Hydrogen bond pairs between the amide hydrogens with slow chemical exchange rates and carbonyl oxygens were determined in accordance with the alignment of the  strands. The backbone torsion angles, , were estimated from the scalar ³J_HNH coupling constants derived from the HMQC-J spectrum. The angle constraints used were 65 ± 25° for ³J_HNH <4.7 Hz, 120 ± 40° for ³J_HNH >8.5 and <9.9 Hz, and 120 ± 20° for ³J_HNH >10.0 Hz. At the stage of structural constraint collection, the program DYANA version 1.5 (molecular dynamics in a torsion angle space with 25,000 steps) was used (34). Finally, an ensemble of 100 structures of ChBD_ChiA1 was calculated with the program X-PLOR version 3.851 (35), the standard simulated annealing protocol (sa.inp file) with 18,000 steps at a high temperature and 9,000 steps for the cooling being used. The T7 tag region at the N terminus was excluded from the structure calculations. The best 30 structures were analyzed with MOLMOL (36) and with AQUA and PROCHECK-NMR (37) software. The secondary structures were determined on the basis of the main chain hydrogen bond patterns, the interstrand NOE connectivities, and the results of AQUA and PROCHECK-NMR analyses (37).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Resonance Assignments-- The amide ¹H and ¹⁵N resonances were assigned at first using two-dimensional H(NN)H TOCSY and three-dimensional (H)NNH TOCSY spectra (27), magnetization being transferred from an amide ¹⁵N spin to those of both preceding and following residues through a ¹⁵N homonuclear Hartmann-Hahn mixing (TOCSY) scheme. Thus, the two-dimensional and three-dimensional spectra provided observable resonances at the frequencies of (^Hi, ^Hj) and (^Ni, ^Nj, ^Hj), respectively, where i equals j  1, j, or j + 1. Figs. 1, A and B, show examples of the sequential connectivities of the amide groups of residues (Tyr⁶⁶²-Tyr⁶⁷⁰) in the two-dimensional H(NN)H TOCSY and three-dimensional (H)NNH TOCSY spectra, respectively. The combination of these two-dimensional and three-dimensional spectra provided secure amide sequential connectivities, which amounted to 63% of the expected connectivities. Next, additional information regarding ¹H^N-¹H^N distances and amino acid types was obtained from the three-dimensional ¹⁵N-edited NOESY and three-dimensional ¹⁵N-edited TOCSY spectra, respectively. This information compensated for the ambiguity of the amide sequential connectivities that were not resolved in the above ³J_NN-based experiments. Finally, we accomplished the assignment of all the amide resonances except for those of Asn⁶⁷¹, Gly⁶⁷², the T7 tag region, and three proline residues. The region containing Asn⁶⁷¹ and Gly⁶⁷² may undergo a conformational exchange, because these residues form the turn connecting the ₂- and ₃-strands (see "Structure Description"). Fig. 1C shows a two-dimensional ¹⁵N-¹H HSQC spectrum with the amide resonance assignments. The well dispersed resonance peaks in both the ¹H and ¹⁵N dimensions reflect the stable structure of ChBD_ChiA1. The proton resonances of the  sites and side chains were assigned using the two-dimensional TOCSY and three-dimensional ¹⁵N-edited TOCSY spectra. The methyl groups of the four leucine and two valine residues were assigned stereospecifically using 15% fractionally ¹³C-labeled ChBD_ChiA1. The methyl groups of the other two residues, Leu⁶⁷⁸ and Val⁶⁵⁸, could not be assigned stereospecifically owing to their overlapping ¹H chemical shifts. Overall, we assigned 95% of the expected ¹H and ¹⁵N resonances of the main chain and side chains.

View larger version (34K): [in this window] [in a new window]   Fig. 1.   NMR spectra of ChBDChiA1. The spectra were obtained with 2.0 mM 15N-labeled ChBDChiA1 at pH 6.0 and 310 K. A, a portion of the two-dimensional H(NN)H TOCSY spectrum acquired with a DRX500 in 43 h. Each cross-peak correlates the amide 1H chemical shifts of the neighboring two residues. The sequential connectivities from Ala661 to Tyr670 are indicated by quadrangles. B, strips taken from the three-dimensional (H)NNH TOCSY spectrum (27) acquired with the DRX500 in 70 h. The strips are taken from slices at the backbone amide 15N (F2) frequency of each residue ranging from Tyr662 to Tyr670. The 15N (F2) frequency values in parentheses represent the original values of the folded resonances. The experiment correlates amide 1H (F3) and 15N (F2) chemical shifts of each residue to 15N (F1) chemical shifts of the preceding and following residues. The sequential connectivities of the amide groups are indicated by vertical lines, at the middle of which autocorrelated peaks are located. The peaks with asterisks have negative intensities due to folding in the 15N dimensions. C, two-dimensional 15N-1H-HSQC spectrum acquired with a DRX800. The assignments of the backbone amide groups are indicated. The assignments in parentheses are those of side chain amide groups of asparagine, glutamine, and tryptophan residues.

Constraints for the Structure Calculation-- The distance constraints based on NOE were extracted from two-dimensional NOESY and three-dimensional ¹⁵N-edited NOESY spectra with mixing times of 150 ms. By comparing the intensities of well resolved cross-peaks in a series of preliminary two-dimensional NOESY spectra obtained with different mixing times, i.e. 50, 100, 150, 200, and 250 ms (data not shown), we judged that the mixing time of 150 ms did not cause severe spin diffusion during the NOE transfer of magnetization. Fig. 2A summarizes the sequential and medium-range NOE connectivities along with the secondary structures, ¹H chemical shift indices (38), amide hydrogen exchange rates, and ³J_HNH coupling constants observed for ChBD_ChiA1. The -strand regions are characterized by the strong intensities of the NOE cross-peaks between  protons and the amide protons of the subsequent residues (d_{N(i, i + 1)}). The locations of the -strands are also indicated by positive deviations of the ¹H chemical shift values from those observed in random coils, which are represented by the ¹H chemical shift index of +1 (38), slow rates of exchange of the amide protons with the solvent, and ³J_HNH coupling constants larger than 8.5 Hz. Interstrand NOE connectivities were observed among H and H^N atoms (¹H^N-¹H^N, ¹H-¹H^N, and ¹H-¹H), as shown by the arrows in Fig. 2B. Overall, 493 NOE-based distance, 20 hydrogen bond, and 33 dihedral angle constraints were collected and used for the structure calculations (Table I).

View larger version (32K): [in this window] [in a new window]   Fig. 2.   Summary of the structure information obtained in the NMR experiments. A, summary of the sequential and medium range NOE connectivities, secondary structures, chemical shift indices, amide hydrogen exchange rates, 3JHNH coupling constants, and solvent accessibility values for ChBDChiA1. The NOE connectivities are represented by bars, the size of which indicates the NOE intensity (strong, medium, or weak). The notation dN(i, i + 1), for example, represents the connectivity between the  proton resonance of a residue (i) and the amide proton resonance of the subsequent residue (i + 1) in the sequence. Amide protons that were exchanged slowly at pH 6.0 and 298 K are indicated. The residues with life times of >0.5 h and <4 h are indicated by open circles, >4 h and <18 h by half closed circles, and >18 h by closed circles. The three-bond scalar coupling constants between spins 1HN and 1H (3JHNH) of <4.9 Hz are indicated by open boxes, >4.9 Hz and <8.5 Hz by one-third closed boxes, >8.5 Hz and <10.0 Hz by two-thirds closed boxes, and >10.0 Hz by closed boxes. The chemical shift indices (CSI) (38) are plotted for 1H resonances. Upper bars, +1; lower bars, 1; horizontal lines, 0. The solvent accessibility was calculated with the program MOLMOL (36) for the side chain of each residue and is shown by the bar height ranging from 0 to 60%. The figure was produced with the program VINCE (Rowland Institute for Science). B, the distance information defining the -sheets of ChBDChiA1. The intra- and interstrand NOEs are indicated by arrows. The hydrogen bonds used for the structure calculations are indicated by dotted lines. The residues constituting the -strands are labeled with black boxes. C, schematic diagram of the -strands of ChBDChiA1. The diagram is drawn in the same direction as in B.

Structure Determination-- Solution structures were calculated through the standard simulated annealing protocol in the program X-PLOR 3.851 (35). Out of 100 calculated structures, 30 final structures, which showed the lowest energy values, no distance constraint violation of >0.3 Å, and no dihedral angle constraint violation of >5°, were selected for further analyses. Superpositioning of these 30 structures is shown in Fig. 3A, and a summary of the constraints and structural statistics is given in Table I. The backbones converged well, as indicated by the r.m.s. deviation of 0.326 Å from the mean structure for the backbone C', C, N atoms of all the residues except for the N-terminal Ala⁶⁵⁵ and C-terminal Gln⁶⁹⁹ residues. The r.m.s. deviation for all heavy atoms in the same regions is 0.700 Å. The Asn⁶⁷¹ and Gly⁶⁷² residues, whose amide signals could not be observed in ¹H-¹⁵N HSQC spectra, are in the loop region connecting the ₂- and ₃-strands, and the calculated coordinates of the loop diverged due to the few constraints in the loop.

View larger version (42K): [in this window] [in a new window]   Fig. 3.   Tertiary structures of ChBDChiA1. A, a stereoview of the best-fit superpositioning of the final 30 structures, which were calculated by means of the simulated annealing procedure of X-PLOR 3.851 (35). The backbone atoms (N, C, and C') in the regions colored purple (Trp656-Tyr670 and Lys673-Leu698) are superimposed. The side chains of the residues forming the hydrophobic core are also shown in magenta. B, schematic ribbon drawing of the representative structure. The secondary structure elements and both end residues of each -strand are shown. The figure was drawn with the programs MOLSCRIPT (50) and RASTER3D (51). The direction of the molecule is the same as in A. C, mapping of the electrostatic potential on the solvent-accessible surface of ChBDChiA1. Blue, a positive potential; red, a negative potential. The right-hand image was generated from the left one by 180° rotation about the vertical axis. The molecular orientation in the left-hand image is almost the same as in A and B. The figures were generated with the program GRASP (52).

Almost the same structures were obtained upon calculation with the program DYANA 1.5 (34) with the same constraints as those used in the X-PLOR calculation. The average DYANA target function value of the best 30 structures selected out of the 100 calculated ones was 0.32 ± 0.10 (one S.D.), and the r.m.s. deviations of all 60 structures that were calculated with X-PLOR and DYANA were 0.354 Å for the backbone atoms and 0.713 Å for all of the heavy atoms in the same regions as described above.

Structure Description-- ChBD_ChiA1 has a compact and globular structure, as shown in Fig. 3B. It contains two antiparallel -sheets (Fig. 2C). One sheet is composed of three strands designated as ₂ (Gln⁶⁶⁶-Tyr⁶⁷⁰), ₃ (Lys⁶⁷³-Cys⁶⁷⁷), and ₅ (Trp⁶⁹⁶-Leu⁶⁹⁸), while the other is composed of two strands designated as ₁ (Thr⁶⁶⁰-Tyr⁶⁶²) and ₄ (His⁶⁸¹-Ser⁶⁸³). No region characteristic of an -helix exists. ₅ consists of three residues and forms an antiparallel -sheet with the three C-terminal residues of ₃. Fig. 2B shows the hydrogen bond networks and NOE connectivities among these five -strands that were used for the structure calculation. The two antiparallel -sheets formed by these hydrogen bond networks fold into the topology of a twisted -sandwich with an angle of about 45° between the sheets. The  sheet formed by ₂, ₃, and ₅ makes a flat surface on the molecule, and the loop connecting ₄ and ₅ runs on the opposite side to the surface. The core region is formed by the hydrophobic and aromatic residues shown in Fig. 3A, i.e. Trp⁶⁵⁶ (10.6), Tyr⁶⁶² (10.1), Val⁶⁶⁸ (1.7), Tyr⁶⁷⁰ (9.5), Tyr⁶⁷⁵ (5.4), Cys⁶⁷⁷ (1.3), Leu⁶⁹⁵ (6.8), and Trp⁶⁹⁶ (3.1%), the values in parentheses indicating the solvent accessibility averaged over the 30 calculated structures for the side chains. This hydrophobic core and the hydrogen bond networks observed among the -strands make the overall structure rigid and compact. Such a character was also expected from the presence of the amide hydrogen atoms that were not exchanged with the solvent for as long as 18 h at pH 6.0 and 298 K (the residues with closed circles in Fig. 2A). ChBD_ChiA1 contains only three charged residues (+Lys⁶⁷³, +Lys⁶⁷⁶, and Glu⁶⁸⁸), and the surface of the molecule is dominated by noncharged residues, as shown in Fig. 3C.

The similarity of the tertiary structure of ChBD_ChiA1 to known structures was examined with the DALI server version 2.0 (39), pairs with Z scores of more than 2.0 being assumed to be similar. The results showed that the structure of ChBD_ChiA1 is similar to that of the CBD of endoglucanase Z (CBD_EGZ) (Protein Data Bank code 1aiw) secreted by a Gram-negative and plant pathogenic bacterium, Erwinia chrysanthemi (8), as indicated by the Z score of 2.8 and r.m.s. deviation (C atoms) of 2.4 Å (Fig. 4). No other similar structure (characterized by a Z score > 2.0) was found in the DALI data base. The sequence of CBD_EGZ is also similar to that of ChBD_ChiA1 (Fig. 5) (CLUSTAL W (40) alignment score, 19%) (see "Discussion").

View larger version (27K): [in this window] [in a new window]   Fig. 4.   Structural comparison of CBDEGZ and ChBDChiA1. A, schematic ribbon drawing of the structure of the 62-amino acid C-terminal cellulose-binding domain of endoglucanase Z (Cel5) (CBDEGZ) secreted by E. chrysanthemi determined by NMR by Brun et al. (8); B, drawing of ChBDChiA1. The aromatic and hydrophobic residues conserved well among various chitinases, which are indicated by the gray backgrounds in Fig. 5, are also shown. The corresponding residues between CBDEGZ and ChBDChiA1 are drawn in the same colors. The two cysteine residues of CBDEGZ forming the disulfide bridge (Cys4-Cys61) are also shown in orange in A. The three residues of CBDEGZ, which are involved in the cellulose-binding (Trp18, Trp43, and Tyr44), are colored green in A. Both images were drawn with the program MOLMOL (36).

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Amino acid sequence alignment of ChBDChiA1 with the domains of other chitinases and CBDEGZ. After alignment with the program CLUSTAL W (40), all of the sequences were further aligned on the basis of the three-dimensional structures of ChBDChiA1 and CBDEGZ. Amino acid residues well conserved are indicated by gray backgrounds. The amino acid sequences shown are for B. circulans WL-12 chitinase D (53) (ii), S. marcescens 2170 chitinase C (54) (iii), Aeromonas sp. strain 10S-24 chitinase II (55) (iv, vii), Janthinobacterium lividum (v, viii) chitinase (56), Aeromonas sp. strain 10S-24 chitinase (57) (vi), Aeromonas caviae extracellular chitinase A (58) (ix), Alteromonas sp. strain O-7 chitinase 85 (12) (x), and the C-terminal CBD of E. chrysanthemi endoglucanase Z (EGZ or Cel5) (8) (xi). The sequences are classified into two groups, i.e., the ChBDChiA1 group displayed in the upper part (lines i-v) and the CBDEGZ group displayed in the lower part (lines vi-xi). The numbers at the left and right of each sequence represent the first and last residue positions in the sequence, respectively. The numbers at the top and bottom represent the sequence numbers of ChBDChiA1 and CBDEGZ, respectively. The boxes at the top and bottom represent the  strand regions of ChBDChiA1 and CBDEGZ, respectively. The three residues of CBDEGZ (Trp18, Trp43, and Tyr44), which are involved in the cellulose-binding, are indicated by closed circles at the bottom. The residues of ChBDChiA1 proposed to be candidates for the interaction with chitin are indicated by diamonds at the top. The numbers in brackets indicates the aligned scores estimated with the program CLUSTAL W (40) between ChBDChiA1 and the respective sequences. The score corresponds to 100 for identical sequences.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Comparison of ChBD_ChiA1 and CBDs-- Chitin differs chemically from cellulose only in that each C2 hydroxyl (-OH) group in cellulose is replaced by an acetamide (-NHCOCH₃) group in chitin. Thus, the mechanism by which ChBD_ChiA1 binds to chitin was expected to be similar to the mechanism by which CBDs bind to cellulose. The most accepted model for the binding of CBDs to cellulose is that aromatic rings arranged in the flat face of a CBD are stacked on every other pyranose ring of polysaccharides through hydrophobic interactions (20). The involvement of aromatic residues in the interactions has been observed using NMR (41), site-directed mutagenesis (16, 25), and chemical modification (42). In CBD_EGZ, for example, three aromatic residues, Trp¹⁸, Trp⁴³, and Tyr⁴⁴, the latter two of which are conserved widely in CBDs, are localized on one face of CBD_EGZ, and are exposed to the solvent, as shown in Fig. 4A (8). Recently, Simpson et al. confirmed the involvement of these three aromatic residues in the interaction with cellulose on site-directed mutagenesis of the intact endoglucanase Z (also referred to as Cel5) (25).

Despite the similarity between the overall topologies of CBD_EGZ and ChBD_ChiA1, ChBD_ChiA1 lacks a region that corresponds to the loop ranging from Val¹⁴ to Gln²² of CBD_EGZ (colored purple in Fig. 4A). This loop region of CBD_EGZ contains Trp¹⁸, which is one of the three aromatic residues that are involved in the interaction with cellulose. Moreover, the other two aromatic residues, which correspond to Trp⁴³ and Tyr⁴⁴ in CBD_EGZ, are also missing or replaced by another aromatic residue, His⁶⁸¹, in ChBD_ChiA1 (Fig. 4B).

We recently showed that ChBD_ChiA1 does not interact with soluble substrates such as hexa-N-acetylchitohexaose, 40% deacetylated chitin, carboxymethyl chitin, or ethylene glycol chitin by means of both NMR chemical shift mapping experiments and isothermal titration calorimetry.² Moreover, ChBD_ChiA1 binds specifically to chitin but not to cellulose. Thus, ChBD_ChiA1 probably recognizes the solid surface conformation of crystallin chitin but not that of cellulose or the flexible conformations of soluble substrates. On the other hand, the binding specificities of the CBDs of some cellulases are rather broad. CBD_CBHI, CBD_CBHII (41), CBD_EGI (17), and CBD_XYLA (10) bind to both crystalline cellulose and soluble cello-oligosaccharides, and CBD_EGZ (CBD_Cel5) (25), CBD_CbpA (43), CBD_CBHII (44), CBD_Cex (18), CBD_CenA (45), and Cip-CBD (20) bind to both cellulose and chitin. Thus, we suggest that the binding mechanism of ChBD_ChiA1 is different from that of CBDs.

Two Groups of ChBDs-- Brun et al. (8) pointed out that the stWWst motif, which corresponds to Ala⁴¹, Asn⁴², Trp⁴³, Tyr⁴⁴, Thr⁴⁵, and Ala⁴⁶ in CBD_EGZ, is not conserved in ChBD_ChiA1. Fig. 5 shows the sequence alignment of ChBD_ChiA1 with the domains of other chitinases and CBD_EGZ. In CBD_EGZ and the five chitinases listed on the lower lines (Fig. 5, vi-xi), the Trp-Trp, Tyr-Trp, or Trp-Tyr (Arom-Arom) sequence is conserved at the site where CBD_EGZ holds the Trp⁴³-Tyr⁴⁴ sequence. On the contrary, ChBD_ChiA1 and the four chitinases listed on the upper lines (i-v) have no such sequence at the corresponding site. The lack of the Arom-Arom sequence in ChBD_ChiA1 is more apparent in the tertiary structure shown in Fig. 4B. Therefore, the chitinases in the group including ChBD_ChiA1 (the ChBD_ChiA1 group) probably have different binding surfaces for substrates from those of the chitinases and cellulases in the other group including CBD_EGZ (the CBD_EGZ group).

We searched for the residues of ChBD_ChiA1 that may be involved in the binding to chitin. The criteria for the search were that (i) such residues are well conserved at least in the ChBD_ChiA1 group (lines i-v in Fig. 5); (ii) they exist on the surface of the molecule; and (iii) they are hydrophobic or aromatic residues contributing to the hydrophobic interaction with chitin. The last criterion is based on the observation that more ChBD_ChiA1 bound to chitin at a pH nearer to its isoelectric point (about 9.0) or in the presence of more NaCl in the solution.² The residues that meet these criteria are His⁶⁸¹ (27.1), Thr⁶⁸² (34.4), Trp⁶⁸⁷ (22.5), Pro⁶⁸⁹ (38.6), and Pro⁶⁹³ (45.5), the values in parentheses indicating the solvent accessibility. These residues are localized on one face of the molecule, which is different from the interaction surface of CBD_EGZ, as shown in Fig. 6. Further, Trp⁶⁸⁷ and Pro⁶⁹³ are specific residues to the ChBD_ChiA1 group, i.e. they are not conserved in the CBD_EGZ group (lines vi-xi in Fig. 5). In CBD_EGZ, the three aromatic residues involved in the interaction with cellulose, Trp¹⁸ (49.2), Trp⁴³ (36.0), and Tyr⁴⁴ (45.9%), are exposed much on the surface of the molecule, but in ChBD_ChiA1 no aromatic residue with such high solvent accessibility was found. His⁶⁸¹ (27.1) and Trp⁶⁸⁷ (22.5%) are the two aromatic residues that are most exposed to the solvent. Instead, Thr⁶⁸² (34.4) and Pro⁶⁸⁹ (38.6%) in ChBD_ChiA1 have much higher solvent accessibility values than the corresponding residues of CBD_EGZ, Thr⁴⁵ (5.0) and Pro⁴⁹ (0.9%), respectively. Thus, in the ChBD_ChiA1 group, residues such as threonine and proline as well as aromatic residues may also be involved in the interaction with chitin. Since ChBD_ChiA1 binds only to the crystalline form of chitin, the domain is expected to recognize the characteristic conformation of crystallin chitin, such as extended polysaccharide chains aligned in parallel to each other. Thus, the binding specificity of ChBD_ChiA1 to solid chitin is probably due to the different binding mechanism from that of CBD_EGZ despite their similar tertiary structures.

View larger version (32K): [in this window] [in a new window]   Fig. 6.   The residues that may interact with chitin. The side chain atoms of these residues are shown in a space-filling model on a ribbon representation of ChBDChiA1 in stereo.

We anticipate that the hydrophobic effect is the major contribution to the interaction between ChBD_ChiA1 and chitin, as mentioned above. However, since ChBD_ChiA1 is eluted from a chitin affinity column at pH 3.0, the negatively charged unique residue, Glu⁶⁸⁸, may also be involved in the interaction (Fig. 3C). The involvement of polar residues (Gln, Asn) in the interaction with cellulose through hydrogen bonding to oxygen atoms or hydroxyl groups of glucose moieties has been observed in the CBDs (20, 46).

FnIII Domains-- The N-terminal domain of chitinase A from a soil bacterium, S. marcescens, is likely to interact with chitin because the intact S. marcescens chitinase A exhibits significant chitin-binding activity (47) and comprises this N-terminal domain (ChiN) (residues Ala²⁴-His¹³⁷) and a C-terminal catalytic domain (residues Val¹⁵⁹-Ala⁴⁴² and Asp⁵¹⁷-Val⁵⁶³), which is significantly homologous to the catalytic domain of B. circulans chitinase A1 (ChiA1). However, no similarity was found in the sequence (CLUSTAL W (32) alignment score, 10%), overall topology, or arrangements of the aromatic rings between ChiN and ChBD_ChiA1. ChiN (114 residues, 11 -strands) is more than twice the size of ChBD_ChiA1 (45 residues, five -strands), and the tertiary structure of ChiN rather resembles those of the FnIII modules found in various animal proteins despite no sequence similarity between ChiN and FnIII domains (48). Although ChiA1 also contains two consecutive FnIII domains between the catalytic (CatD) and binding (ChBD) domains (49), these FnIII domains, interestingly, do not bind to chitin (6). Since ChiN has nine aromatic residues, some of them may be involved in the interaction with chitin in the similar way to how the three consecutive aromatic rings of CBDs interact with cellulose.

A Disulfide S-S Bond-- CBD_EGZ has a disulfide bridge (Cys⁴-Cys⁶¹) between the two extremities of the domain, whereas ChBD_ChiA1 has no disulfide bridge at the corresponding site (Fig. 4). To be precise, Cys⁶¹ of CBD_EGZ corresponds to the outside of the C-terminal end of ChiA1, and no cysteine residue exists around the region in ChiA1 corresponding to the region containing Cys⁴ in CBD_EGZ. As a protein engineering approach, the introduction of a disulfide bridge into ChiA1 would increase the stability of the protein against changes in heat, pH, ionic strength, etc. more and would make a mutated protein more appropriate for various industrial uses.

	FOOTNOTES

^* This work was supported by grants from the Ministry of Education, Science and Culture of Japan (to M. S. and T. I.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The atomic coordinates and the structure factors (code 1ed7) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^¶ To whom correspondence should be addressed. Fax: 81-743-72-5579; Tel.: 81-743-72-5571; E-mail: shira@bs.aist-nara.ac.jp.

² Hashimoto, M., Ikegami, T., Seino, S., Ohuchi, N., Fukada, H., Sugiyama, J., Shirakawa, M., and Watanabe, T. (2000) J. Bacteriol. 182, in press.

	ABBREVIATIONS

The abbreviations used are: ChiA1, B. circulans WL-12 chitinase A1; CBD, cellulose-binding domain; ChBD, chitin-binding domain; HMQC, heteronuclear multiple quantum correlation; HSQC, heteronuclear single quantum correlation; NOE, nuclear Overhauser effect; NOESY, NOE spectroscopy; TOCSY, total correlation spectroscopy; FnIII, fibronectin type III-like domain; r.m.s., root mean square.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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