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. 2009 Nov 25:8:62.
doi: 10.1186/1475-2859-8-62.

Functional analysis of conserved aromatic amino acids in the discoidin domain of Paenibacillus beta-1,3-glucanase

Yueh-Mei Cheng  1 , Feng-Chia Hsieh, Menghsiao Meng
Affiliations

Functional analysis of conserved aromatic amino acids in the discoidin domain of Paenibacillus beta-1,3-glucanase

Yueh-Mei Cheng et al. Microb Cell Fact. .

Abstract

The 190-kDa Paenibacillus beta-1,3-glucanase (LamA) contains a catalytic module of the glycoside hydrolase family 16 (GH16) and several auxiliary domains. Of these, a discoidin domain (DS domain), present in both eukaryotic and prokaryotic proteins with a wide variety of functions, exists at the carboxyl-terminus. To better understand the bacterial DS domain in terms of its structure and function, this domain alone was expressed in Escherichia coli and characterized. The results indicate that the DS domain binds various polysaccharides and enhances the biological activity of the GH16 module on composite substrates. We also investigated the importance of several conserved aromatic residues in the domain's stability and substrate-binding affinity. Both were affected by mutations of these residues; however, the effect on protein stability was more notable. In particular, the forces contributed by a sandwiched triad (W1688, R1756, and W1729) were critical for the presumable beta-sandwich fold.

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Figures

Figure 1
Figure 1
Monomer to dimer ratio of the F5/8C module of LamA. (A) Elution profile of the purified protein using a Sephacryl S-300 column. The Vo indicates the void volume. The estimated molecular masses of the eluted peaks are indicated. (B) Sedimentation velocity studies. The condition for analytical ultracentrifugation and data analysis are described in the Materials and Methods. (C) Glutaraldehyde cross-linking of the purified F5/8C module. The protein (0.65 mg/mL) was treated with 0.05% glutaraldehyde at room temperature for 1 h and resolved on a 12% SDS-PAGE gel. Lanes 1 and 2 contain the protein sample without and with the treatment of glutaraldehyde, respectively. (D) Glutaraldehyde cross-linking of two larger truncated proteins of LamA (CBF and CB3). The proteins (0.3 mg/mL) were treated with 0.05% glutaraldehyde at room temperature for 40 min and resolved on an 8% SDS-PAGE gel. The arrows point to the putative migration zones of monomeric, dimeric, and multimeric CBF after glutaraldehyde treatment.
Figure 2
Figure 2
Binding of the F5/8C module of LamA to insoluble polysaccharides. The purified protein (20 μg/mL) and the indicate substrate (25 mg/mL) were thoroughly mixed at 37°C for 1 h. The amount of protein remaining in the supernatant (S) and co-precipitating with the substrate (P) were examined by SDS-PAGE.
Figure 3
Figure 3
Effects of the truncated proteins derived from LamA on conidial germination. Germination of F. oxysporum macroconidia (A) and G. cingulata conidia (B) in a buffer containing 6.25 μM of either BSA, the GH16 module, the F5/8C module, or the GH16-F5/8C fusion protein. The culture conditions are described in the Materials and Methods. The germination rate (%) and hyphal length (μm) are indicated in panels A and B, respectively.
Figure 4
Figure 4
Amino acid sequence alignment of several DS domains. (A) Schematic representation of the domain organization of LamA. (B) Sequence alignment, based on the ClustalV method, of the F5/8C module of LamA (Psp; GenBank ABJ15796) with that of Paenibacillus fukuinensis chitosanase-glucanase (Pfu; GenBank BAB64835), Cellvibrio japonicus cbp32B (Cja; GenBank ACE83872), Saccharophagus degradans β-1,3-glucanase (Sde; GenBank ABD82184), retinoschisin (Ret; NP_000321), the C2 domain of human coagulation factor V (Fa5; GenBank AAB59401), the C2 domain of human coagulation factor VIII (Fa8; GenBank AAA52484), and the C2 domain of lactadherin of Bos taurus (Lac; NP_788783). Sequence similarities between the DS domain of Psp and Pfu, Cja, Sde, Ret, Fa5, Fa8, and Lac are 39, 36.9, 44.2, 13.0, 13.0, 14.5, and 16.8%, respectively. Conserved residues are shown in bold, while residues mutated in this study are marked with black dots. Sequences that constitute protruding loops in Fa5, Fa8, and Lac are underlined according to PDB code 1CZS, 1D7P, and 3BN6, respectively. Please note these Proteins can be searched and accessed via http://www.ncbi.nlm.nih.gov/sites/entrez?db=Protein&itool=toolbar
Figure 5
Figure 5
The circular dichroism spectra of wild-type and mutant F5/8C modules of LamA. The experimental conditions are described in the Materials and Methods.
Figure 6
Figure 6
Differential scanning microcalorimetric plots of wild-type and mutant F5/8C modules of LamA. The experimental conditions are described in the Materials and Methods.
Figure 7
Figure 7
Affinity electrophoresis of wild-type and mutant F5/8C modules. The indicated proteins were separated by native 12% PAGE (A) and native PAGE including 0.3% (w/v) laminarin in the separation gel (B). Open arrows point to the migration positions of the wild-type F5/8C module. The relative mobility (RM) of each protein compared with BSA under the given conditions is indicated.
Figure 8
Figure 8
Dependence of the protein fraction bound to chitin on the chitin concentrations. Each of the indicated proteins (20 μg/mL) was thoroughly mixed with chitin at the indicated concentrations at 37°C for 1 h. The fractions of the protein that remaining in the supernatant (S) and co-precipitating with chitin (P) were determined by SDS-PAGE. The apparent binding affinity (Kd) was calculated using GraFit5 software. The data were averages from two independent experiments, each with triplicate samples.
Figure 9
Figure 9
Structure of the C2 domain of human factor V. A ribbon plot is shown highlighting the β-sandwich fold. Side chains labeled W47, W57, Y88, W99, R137, L149 correspond to W2119, W2129, Y2160, W2171, R2209, and L2221 of mature factor V, respectively.
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