Functional analysis of conserved aromatic amino acids in the discoidin domain of Paenibacillus β-1,3-glucanase
© Cheng et al; licensee BioMed Central Ltd. 2009
Received: 13 September 2009
Accepted: 25 November 2009
Published: 25 November 2009
The 190-kDa Paenibacillus β-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 β-sandwich fold.
The discoidin domain (DS domain) is a structural and functional motif that is appended, singly or in tandem, to various eukaryotic and prokaryotic proteins . The first DS domain was identified in the amoeba Dictyostelium discoideum and described as a lectin with high affinity for galactose and galactose derivatives . It should be noted that the domain is also referred to as F5/8C due to its presence at the carboxyl-terminus of blood coagulation factors V and VIII. The DS domain binds a wide variety of ligand molecules, including phospholipids, carbohydrates, and partner proteins, thus enabling its cognate protein to participate in various physiological functions such as cellular adhesion [3, 4], migration , neural development [6, 7], and nutrition assimilation [8, 9]. A subgroup of the domain possessing carbohydrate-binding ability is also classified as the carbohydrate-binding module family 32 (CBM32) . Due to the recent progress of genome projects, the number of CBM32 members has increased significantly over a short period time. However, most of these members have not been functionally characterized.
The structure of several DS domains has been determined and deposited in the PDB . The DS domain comprises approximately 150 amino acid residues, arranged into a β-sandwich fold with several flexible loops. Presumably, the β-sandwich fold is stabilized predominantly by hydrophobic interactions. The variability within the loops has been suggested to account for the diverse binding spectrum of the DS domain . Co-crystallizations of CBM32 members and their ligands, such as the module of Clostridium perfringens N-acetylglucosaminidase with β-galactosyl-1,4-β-N-acetylglucosamine or the module of Micromonospora viridifaciens sialidase with lactose, demonstrate that the protruding loops form the ligand binding site [13, 14].
Recently, a β-1,3-glucanase consisting of 1793 amino acid residues [GenBank: DQ987544] was isolated from Paenibacillus sp. BCRC 17245 and was characterized . This β-1,3-glucanase (referred to as LamA hereafter) is highly modular, containing a signal sequence, three repeats of the S-layer homologous module, a segment with unknown function, a catalytic module of glycoside hydrolase family 16 (GH16), four repeats of CBM4 family, and a F5/8C module from amino- to carboxyl-terminus. Differential properties between two truncated proteins (GH16 and GH16 tagged with the F5/8C module) suggested that the carboxyl-terminal F5/8C has an ability to complex with polysaccharides containing β-1,3-, β-1,3-1,4-, and β-1,4-glucosidic linkages and such ability conferred greater antifungal activities to GH16 on the growth of Candida albicans and Rhizoctonia solani. In addition, the presence of the F5/8C module enhances the hydrolyzing activity of the catalytic module to various polysaccharides. To better understand the F5/8C module in terms of its structure and function, the module alone was expressed in E. coli and characterized biochemically in this study. In addition, functions of several conserved aromatic amino acid residues in the module were investigated by mutagenesis.
Materials and methods
Laminarin, chitin (from crab shells), and lichenan were purchased from Sigma, while Avicel PH101 was purchased from Fluka. The chitin was treated with phosphoric acid prior to use .
pET-C and pET-CF were used for expression of the truncated proteins GH16 and the GH16 fused with F5/8C, respectively . To express the F5/8C module, the pET-F plasmid was generated by PCR-based deletion mutagenesis (QuickChange Site-Directed Mutagenesis Kit, Stratagene) using pET-CF as the template. The PCR was conducted for 35 cycles (95°C, 30 s; 60°C, 30 s; 72°C, 6 min) followed by a 10 min extension at 72°C in a 50 μl solution that contained 10 ng pET-CF, 0.32 mM each of the 5'-phosphorylated primers (5'-TATGCAGGGAATACGGTCTCC and 5'-CGAATTCGGATCCTGGCTGTG), 0.2 mM of each of the dNTPs, and 2.5 U of Pfu polymerase. The PCR product was recovered and self-ligated to become pET-F. A similar PCR protocol, with specific divergent primers, was used to create the specific mutation of pET-F for the production of the mutant F5/8C module. The desired mutations were confirmed with ABI Prism 3773 auto sequencer.
E. coli BL21(DE3) strain, harboring the desired derivatives of pET-F, was grown at 30°C in 500 mL of 2× TY medium (16 g/L tryptone, 10 g/L yeast extract, and 5 g/L NaCl) supplemented with ampicillin 100 μg/mL until the OD600 reached around 1. Isopropyl β-D-1-thiogalactopyranoside (final concentration of 1 mM) was added to the medium and the culture was continued for 7-8 h. The cells were harvested by centrifugation, suspended in 10 mL lysis buffer (20 mM Tris-HCl [pH8.0], 500 mM NaCl, 10% glycerol, 5 mM phenylmethylsulfonyl fluoride, and 5 mM β-mercaptoethanol), and disrupted by ultrasonic treatment. The clarified protein solution was mixed with Ni-NTA resin (Promega) in the presence of 10 mM imidazole. The mixed resin was washed extensively with wash buffer (10 mM phosphate [pH 7.0], 500 mM NaCl, 10% glycerol, and 40 mM imidazole), and the bound protein was eluted with wash buffer containing 500 mM imidazole. The eluted protein was then passed through a Sephacryl S-300 gel filtration column (Hiprep 16/60, Pharmacia) equilibrated with 10 mM sodium acetate [pH 5.8] and 10% glycerol for further analysis and molecular weight estimation. Protein concentration was determined by the Coomassie blue method using bovine serum albumin (BSA) as the standard.
The sedimentation velocity study was carried out with an analytical ultracentrifuge (Beckman Coulter XL-A) at 20°C and 42000 rpm. The concentration of the protein sample was adjusted to an OD280 of 0.5 (0.17 mg/mL) in 10 mM phosphate buffer [pH 7.0] and 10% glycerol. The UV absorption at 280 nm was scanned 200 times in continuous mode and the data were analyzed with Sedfit94 software to obtain the differential concentration distribution c(s).
The circular dichroism (CD) spectrum within the range of 190 to 360 nm was recorded at room temperature using a Jasco J-815 CD spectrometer fitted with a quartz cell of 5 mm path length. The concentration of the protein sample was adjusted to 0.2 mg/mL in a 10 mM phosphate buffer [pH 7.0]. The data were collected using JWSTDA32 software and analyzed with a CONTINLL program in CDPro software. The protein set SP37 was used as the reference during analysis.
Differential scanning calorimetry (DSC) was performed using a calorimeter (N-DSCIII, TA). Measurements were made with a scan rate of 2°C/min over a 20-80°C range, using a sample concentration of 1 mg/mL in the 10 mM phosphate buffer [pH 7.0]. The data were analyzed with CpCalc OLE 2.0 software.
Substrate binding assays
The affinity of the protein to soluble polysaccharides was assayed by affinity electrophoresis  in which 0.3% (w/v) laminarin was included in a native 12% polyacrylamide gel (PAGE). Electrophoresis was performed at 80 V at 4°C.
The affinity of the protein for insoluble polysaccharides was determined by a pull-down assay; the protein (20 μg/mL) and substrates at indicated concentrations were mixed in a final volume of 300 μL in a 50 mM sodium acetate buffer [pH 5.8]. After 1 h of shaking at 37°C, the supernatant was recovered by centrifugation at 16,000 × g for 5 min. The pellet was washed once and suspended in 300 μL of the same buffer. The fraction of protein in the supernatant and the pellet was estimated by 12% sodium dodecyl sulfate (SDS)-PAGE and Coomassie blue staining. The apparent binding affinity (K d) of the protein for chitin was calculated using GraFit5 software according to the dependence of the protein fraction bound to chitin on the amounts of chitin used. K d was defined as the amount of chitin required for reaching 1/2 of the maximal binding of the protein.
Antifungal activity assays
Macroconidia of Fusarium oxysporum f. sp. lycopersici and conidia of Glomerella cingulata were washed out from the respective hypha with a 10 mM sodium acetate buffer [pH 5.8] containing 10% glycerol and adjusted to a concentration of 2 × 105/mL. To determine the protein's effect on conidial germination, equal volumes of the conidia solution and protein sample (12.5 μM) were mixed and incubated at 22°C under moisture condition for 16 h. Growth of hypha from the conidia was observed under a light microscope and the hyphal length was measured using Leica Image-ProPlus 4.5 software.
Protein expression and purification
Mutational effects on protein structure
Mutational effects on polysaccharide-binding activity
According to the sequence information, a variety of bacterial glycoside hydrolases have been suggested to have one or more DS modules. These hydrolases include, but are not limited to, α-1,3-glucanase , β-1,3-glucanase [15, 20], β-galactosidase , cellulase , chitosanase , α-1,2-mannanase , hyaluronidase [25–27], sialidase [28, 29], alginate lyase . Structural and biochemical studies on the modules of M. viridifaciens sialidase , Y. enterocolitica polygalacturonic acid-binding protein , and C. perfringens N-acetylglucosaminidase  have indicated that galactose or its derivatives are the basic units for recognition by family 32 CBMs. In this study, the F5/8C module of LamA was expressed alone in E. coli and was characterized in vitro. The affinity electrophoresis results and the pull-down assays indicated that the F5/8C module can bind to laminarin as well as many other insoluble polysaccharides, including chitin, lichenan, and cellulose, suggesting a wide substrate-binding spectrum of family 32 CBMs.
With the broad binding specificity, the carboxyl-terminal appended F5/8C module should increase the attachment of LamA to its natural substrates, for instance plant and fungal cell walls that often comprise cellulose, pectin, β-1,3-glucan, β-1,3-1,4-glucan, chitin and others. As a consequence, the F5/8C module increases the encountering frequency between the catalytic GH16 module and the β-1,3-glucosidic linkages within the complex substrates. In other words, the F5/8C module should be able to increase the "effective concentration" of the catalytic module on the surface of the substrates. The enhanced antifungal activity of the GH16 module by F5/8C on the growths of C. albican and R. solani, and F. oxysporum and G. cingulata (data presented here) support this notion.
A small fraction of the F5/8C module of LamA form dimers. The recombinant lactadherin C2 domain exists as a monomer in solution, but forms a dimer using a part of a β-strand (S7) as the contact area when it was packed in the crystal . A similar result was observed in the crystal structure of the C2 domain of human factor V . These observations suggest that dimerization under certain circumstances, such as high protein concentration, may be a general property of the DS domain. Dimerization of LamA, presumably anchored on cell wall by its S-layer homologous modules, may increase the binding valence of the protein and further increase the binding avidity of the Paenibacillus strain to its assimilated polysaccharides.
The mutations of W1679, W1688, and Y1768 did not cause dramatic changes in the binding affinity of the F5/8C module to chitin. These data suggest that substrate binding is not the primary function of these residues. Otherwise, a much greater impacts would have been expected. For example, mutations of the conserved tryptophan residues of the CBM on Cellulomonas fimi CenA caused 30- to 50-fold decreases in cellulose-binding affinity . Furthermore, mutations of the CBM on Thermotoga maritima Lam16A caused 30- to 150-fold decreases in celluolose-binding .
There is increasing documentation that bacterial glycoside hydrolases contain DS domains. However, the properties and functions of this domain are mostly speculative rather than characterized. Therefore, in this study, we characterized the DS domain in LamA of Paenibacillus sp. BCRC 17245. The DS domain can bind a variety of polysaccharides and maximize the inherent catalytic function of LamA. Furthermore, the importance of the conserved aromatic residues in the protein's conformation and stability can be interpreted based on the structure of mammalian DS domains, despite no more than 16% similarity in amino acid sequences between the domains of LamA and mammalian proteins. This suggests that the core of the β-sandwich fold has been conserved while the DS domain has evolved to have various ligand-binding functions. The W-R-W triad is particularly critical for the formation and maintenance of the β-sandwich fold based on the previous study on retinoschisin, crystal structure of mammalian DS domains, and data presented here.
This work was supported by National Science Council, ROC: NSC 94-2320-B-127-002.
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