Abstract:
Gram-positive bacteria are decorated by a variety of proteins that are anchored to the cell wall and project from it to mediate colonisation, attachment to host cells, and pathogenesis. These proteins, and protein assemblies such as pili, are typically long and thin yet must withstand high levels of mechanical stress and proteolytic attack. The recent discovery of intramolecular isopeptide bond crosslinks in the pili (Spy0128) from Streptococcus pyogenes, formed auto-catalytically between lysine and asparagine residues with the help of a nearby glutamic acid has highlighted the role that covalent crosslinks can play in stabilising proteins. This study aimed to identify the requirements for isopeptide bond formation and quantify their contribution to protein stability. The hypothesis that isopeptide bonds are a common feature in bacterial cell surface proteins with CnaA- and CnaB-type folds was also investigated. The role of the hydrophobic environment in isopeptide bond formation in the N-terminal domain of Spy0128 was studied by mutagenesis. Many of the mutations in the hydrophobic core around the bond-forming residues did not prevent isopeptide bond formation, but did result in decreases in thermal stability of up to 11 °C. The geometrical requirements for isopeptide bond formation were also explored by swapping the positions of the glutamic acid and asparagine residues involved in bond formation. A structure of this mutant was solved to 2.5 Å resolution and showed why the isopeptide bond did not form in this mutant; the resulting unpaired charged residues in the hydrophobic core significantly destabilised the Ndomain, and also disrupted isopeptide bond formation in the C-domain. The possibility that an isopeptide bond could be introduced to a protein that does not naturally have one was tested by protein engineering studies on the ancillary pilin FctB. This protein has an immunoglobulin (Ig)-like domain that is similar to N-terminal domain of Spy0128, but does not contain any of the residues required to form an isopeptide bond. Site directed mutagenesis was used to introduce lysine, asparagine and glutamic acid residues into the hydrophobic core of FctB, at positions that successfully led to the formation of an isopeptide bond. The presence of this bond was confirmed by both mass spectrometry and Xray crystallography; the crystal structure of FctB-iso, solved at 2.0 Å resolution, clearly showed electron density connecting the side chains of the lysine and asparagine residues. Introduction of the isopeptide bond to FctB increased its melting temperature by 10 °C, confirming the proposition that thermal stability could be enhanced in this manner. Bioinformatics studies indicated that isopeptide bonds are indeed a common feature of Grampositive cell-surface proteins. For experimental confirmation, two proteins (Bce0260 and Cpe0147) were chosen for further investigation. Bce0260 is a cell-surface protein from Bacillus cereus, comprising an N-terminal adhesin domain and 15 repeated domains, predicted to be Ig-like. Isopeptide bond formation in two of the repeat domains of Bce0260 was confirmed by mass spectrometry and mutagenesis. Cpe0147 is a putative cell-surface adhesin from Clostridium perfringens comprising an N-terminal adhesin domain followed by 11 repeat domains. Mass spectrometry of a two-domain construct (C2) showed a decrease of 33 Da from the predicted molecular mass, suggestive of two isopeptide bonds, but the sequence did not show Lys and Asn/Asp residues in the expected positions. Crystal structures of C2 and a single-domain construct (C1), solved at 1.9 Å and 1.1 Å resolution, respectively, showed that each domain has an IgG-like fold containing an unprecedented ester bond joining the side chains of a threonine and a glutamine residue. These were shown to form through an autocatalytic intramolecular reaction catalysed by an adjacent histidine residue in a serine protease-like mechanism, with two buried acidic residues assisting in the reaction. As with the isopeptide bonds in pili, the ester bonds are placed at a strategic position joining the first and last strands, and like isopeptide bonds were shown to be essential for stability. Removal of the ester bond from C1 by mutagenesis reduced the melting temperature from 68 °C to a level at which the domain appeared essentially unfolded. Further bioinformatic analyses suggested that these ester bond cross-links are present in the repeat domains of many other Gram-positive bacterial adhesins, raising the question of what other novel chemistry may be used for protein stabilisation.