Abstract
The Gel/Gas/Phr family of fungal beta(1,3)-glucanosyltransferases plays an important role in cell wall biogenesis by processing the main component beta(1,3)-glucan. Two subfamilies are distinguished depending on the presence or absence of a C-terminal cysteine-rich domain, denoted "Cys-box." The N-terminal domain (NtD) contains the catalytic residues for transglycosidase activity and is separated from the Cys-box by a linker region. To obtain a better understanding of the structure and function of the Cys-box-containing subfamily, we identified the disulfide bonds in Gas2p from Saccharomyces cerevisiae by an improved mass spectrometric methodology. We mapped two separate intra-domain clusters of three and four disulfide bridges. One of the bonds in the first cluster connects a central Cys residue of the NtD with a single conserved Cys residue in the linker. Site-directed mutagenesis of the Cys residue in the linker resulted in an endoplasmic reticulum precursor that was not matured and underwent a gradual degradation. The relevant disulfide bond has a crucial role in folding as it may stabilize the NtD and facilitate its interaction with the C-terminal portion of a Gas protein. The four disulfide bonds in the Cys-box are arranged in a manner consistent with a partial structural resemblance with the plant X8 domain, an independent carbohydrate-binding module that possesses only three disulfide bonds. Deletion of the Cys-box in Gas2 or Gas1 proteins led to the formation of an NtD devoid of any enzymatic activity. The results suggest that the Cys-box is required for proper folding of the NtD and/or substrate binding.
Highlights
JULY 4, 2008 VOLUME 283 NUMBER 27 required for cell adhesion and virulence
The organization of domains of GH72ϩ proteins resembles that of many polysaccharide-hydrolyzing enzymes such as mannanases, chitinases, cellulases, and glucanases that are composed of the following distinct domains: a putative catalytic module and a noncatalytic module connected by a flexible region [20, 21]
In this work we have focused on the characterization of disulfide bonds in Gas1 and Gas2, two homologous GH72ϩ proteins of S. cerevisiae that share 53% of amino acid identity in the NtDϩL region, and 41% in the remaining part of the protein
Summary
General Strategy Used to Identify Disulfide-linked Peptides— Our analytical strategy of the protein under study consists of cleavage between adjacent Cys residues and mass spectrometric analysis of the peptide mixture (see supplemental Fig. S1 for a flow chart). Expression and Purification of Recombinant Gas Proteins— pHIL-S1-derived plasmids, linearized with BglII, were transformed into Pichia pastoris strain GS115 (his4), and the selection of HisϩMuts mutants and protein induction were performed as described previously [27]. To test for (1,3)-glucanosyltransferase activity, 1–1.5 g of the purified protein were incubated with 3 mM reduced laminarioligosaccharide containing 13 glucose residues (rG13), and the assay was performed as described previously [27]. For the binding assays to the yeast cell wall (1,3)-glucan, 2 g of purified sGas1p or sGas1-⌬Ser were incubated for 30 min at 4 °C in 5 mM acetate buffer, pH 5.5, with 10 or 50 mg (wet weight) of alkali-insoluble fraction of cell walls obtained, as described previously [33], and washed once with 100 mM Tris-HCl, pH 7.5, twice with 10 mM Tris-HCl, pH 7.5, and twice with 5 mM sodium acetate, pH 5.5. Secondary structure was predicted using the structure prediction methods PSIPRED [37] and Jnet [38]
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