Significance We describe an unprecedented type of intramolecular cross-link in a protein molecule, which we have found in the repetitive domains of a cell surface adhesin from the Gram-positive organism Clostridium perfringens . From high-resolution crystal structures of the protein, coupled with MS, we show that these domains contain intramolecular ester bonds joining Thr and Gln side chains. These bonds are generated autocatalytically by a serine protease-like mechanism and provide the long, thin protein with greatly enhanced mechanical strength and protection from proteolytic attack. The bonds provide an intriguing parallel with the internal isopeptide bonds that stabilize Gram-positive pili. Bioinformatics analysis suggests that these intramolecular ester bonds are widespread and common in cell surface adhesion proteins from Gram-positive bacteria.
The pili expressed on the surface of the human pathogen Streptococcus pyogenes play an important role in host cell attachment, colonisation and pathogenesis. These pili are built from two or three components, an adhesin subunit at the tip, a major pilin that forms a polymeric shaft, and a basal pilin that is attached to the cell wall. Assembly is carried out by specific sortase (cysteine transpeptidase) enzyme. These components are encoded in a small gene cluster within the S. pyogenes genome, often together with another protein, SipA, whose function is unknown. We show through functional assays, carried out by expressing the S. pyogenes pilus components in Lactococcus lactis, SipA from the clinically important M1T1 strain is essential for pilus assembly, and that SipA function is likely to be conserved in all S. pyogenes. From the crystal structure of SipA we confirm that SipA belongs to the family of bacterial signal peptidases (SPases), which process the signal-peptides of secreted proteins. In contrast to a previous arm-swapped SipA dimer, this present structure shows that its principal domain closely resembles the catalytic domain of SPases and has a very similar peptide-binding cleft, but it lacks the catalytic Ser and Lys residues characteristic of SPases. In SipA these are replaced by Asp and Gly residues, which play no part in activity. We propose that SipA functions by binding a key component at the bacterial cell surface, in a conformation that facilitates pilus assembly.
In the fission yeast, Schizosaccharomyces pombe, tolerance to high sodium and lithium concentrations requires the functioning of the sod2, Na+/H+ antiporter. We have directly measured the activity of this antiporter and demonstrated reconstitution of the activity in gene deletion strains. In addition, we have shown that it can be transferred to, and its antiporter activity detected in, the budding yeast, Saccharomyces cerevisiae, where it also confers sodium and lithium tolerance. Proton flux through the S. pombe Na+/H+ antiporter was directly measured using microphysiometry. The direction of transmembrane proton flux mediated by this antiporter was reversible, with protons being imported or exported in response to the external concentration of sodium. This bidirectional activity was also detected in S. cerevisiae strains expressing sod2 and expression of this gene complemented the sodium and lithium sensitivity resulting from inactivation of the ENA1/PMR2 encoded Na+-exporting ATPases. This suggests that antiporters or sodium pumps can be utilized interchangeably by S. cerevisiae to regulate internal sodium concentration. Potent inhibitors of mammalian Na+/H+ exchangers were found to have no effect on sod2 activity. The proton flux mediated by sod2 was also found to be unaffected by perturbation of membrane potential or the plasma membrane proton gradient.
Abstract Poly-γ-glutamate tails are a distinctive feature of archaeal, bacterial, and eukaryotic cofactors, including the folates and F 420 . Despite decades of research, key mechanistic questions remain as to how enzymes successively add glutamates to poly-γ-glutamate chains while maintaining cofactor specificity. Here, we show how poly-γ-glutamylation of folate and F 420 by folylpolyglutamate synthases and γ-glutamyl ligases, non-homologous enzymes, occurs via processive addition of L -glutamate onto growing γ-glutamyl chain termini. We further reveal structural snapshots of the archaeal γ-glutamyl ligase (CofE) in action, crucially including a bulged-chain product that shows how the cofactor is retained while successive glutamates are added to the chain terminus. This bulging substrate model of processive poly-γ-glutamylation by terminal extension is arguably ubiquitous in such biopolymerisation reactions, including addition to folates, and demonstrates convergent evolution in diverse species from archaea to humans.
Abstract The overall stability of globular protein structures is marginal, a balance between large numbers of stabilizing non-covalent interactions and a destabilizing entropic term. Higher stability can be engineered by introduction of disulfide bonds, provided the redox environment is controlled. The discovery of stabilizing isopeptide bond crosslinks, formed spontaneously between lysine and asparagine (or aspartic acid) side chains in certain bacterial cell-surface proteins suggests that such bonds could be introduced by protein engineering as an alternative protein stabilization strategy. We report the first example of an isopeptide bond engineered de novo into an immunoglobulin-like protein, the minor pilin FctB from Streptococcus pyogenes . Four mutations were sufficient; lysine, asparagine and glutamic acid residues were introduced for the bond-forming reaction, with a fourth Val/Phe mutation to help steer the lysine side chain into position. The spontaneously-formed isopeptide bond was confirmed by mass spectrometry and X-ray crystallography, and was shown to increase the thermal stability by 10 °C compared with the wild type protein. This novel method for increasing the stability of IgG-like proteins has potential to be adopted by the field of antibody engineering, which share similar β-clasp Ig-type domains.
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