Table 1.
Crystallographic data and refinement statistics for TCLL and its complex with GlcNAc.
Figure 1.
Characterization of native and deglycosylated TCLL.
A. SDS-PAGE showing purity and difference in masses of glycosylated and deglycosylated TCLL. B. Intact mass spectrum of glycosylated TCLL with major species of molecular mass of 33,440 Da. Several other associated peaks indicate species with different carbohydrate content. An isoform of TCLL was also observed at 32, 206 Da. C. Intact mass spectrum of deglycosylated TCLL. It shows a single species indicating a single subunit and absence of sugar moiety. The molecular mass is also lower than its glycosylated native form as expected.
Figure 2.
Effect of GlcNAc on the intrinsic fluorescence of TCLL and ITC analysis.
A. Addition of GlcNAc to 0.5 µM solution of protein in 25 mM phosphate buffer saline, pH 7.2 at increasing concentration resulted in quenching of spectra. The emission spectra were recorded between 291–500 nm upon excitation at 290 nm. No shift in wavelength maxima from 316 nm was observed. B. Typical ITC thermograms and theoretical fits of the integrated peak to the experimental data for binding of TCLL with GlcNAc. The data (filled squares) were fitted nicely to a single binding sites model, and the solid lines represent the best fit.
Figure 3.
Alignment of TCLL with GH18 family members such as hevamine, PPL2, concanavalin B, XIP-I, XAIP and SCCTS1. The alignment was done using the program CLUSTALW [38] and figure was prepared using ESPRIPT [36]. The conserved residues are represented in black background. The key active site residues for chitinase activity are shown by arrows.
Figure 4.
The crystal structure of TCLL.
A Cartoon diagram representation of TCLL in (A) top view orientation and (B) side view orientation. The α-helices (α1 to α8) are shown in cyan and extra helices (α1 and α8) in orange. The β-strands (β1 to β8) are shown in TV blue and extra strands (β2′, β2′′ and β3′) in violet. Connecting loops are in wheat color. Two units of GlcNAc which is N-glycosylated at Asn146, two molecules of MPD and sodium acetate are shown in green color. Three disulphide bonds are indicated by red arrows. Unusual loop that protrudes out from domain are shown by black arrow in (B). The hydrogen bond network formed by extra strands is highlighted.
Figure 5.
The crystal structure of the TCLL-GlcNAc complex and interactions of TCLL residues with GlcNAc.
The TCLL-GlcNAc complex shows two GlcNAc sites denoted as S1 and S2. The S1 is formed by two loops, α3β4 and α4β5 and one helix α2 and S2 is formed by two loops β4α4 and β5α5 and one helix α5. GlcNAc binding sites are focused and interactions are shown. At S1, GlcNAc is stabilized through hydrogen bonds with residues Gln74, Tyr121, Asp123, Arg152 and three water molecules. At S2, GlcNAc is stabilized through hydrogen bonds with residues Glu132, Tyr167, Tyr168, Lys171 and eight water molecules in the pocket. Hydrogen bonds are shown in black dotted lines, water molecules in orange spheres, GlcNAc is shown as stick with purple blue carbons at S1 and cyan carbons at S2. Residues interacting with GlcNAc are shown as stick with brown carbons at S1 and wheat color carbons at S2. The 2Fo−Fc electron density maps are shown around 5 Å area of GlcNAc surroundings at S1 and S2 sites in TKI-PPT complex structure contoured at 1.0 σ.
Figure 6.
Substrate binding subsites and active site residues of TCLL.
Hevamine (2HVM and 1KQY) was superimposed onto TCLL and the interacting residues with GlcNAc moiety are shown. Hevamine residues are in cyan sticks with purple label and the corresponding TCLL residues are in green sticks with black label. The active site residues of hevamine are shown in pink sticks with red label and corresponding residues of TCLL are Ala128, Val130 and Phe186 show mutations of key catalytic residues.