Yet more intramolecular cross-links in Gram-positive surface proteins.

The surface of Gram-positive bacteria comprises a single membrane and, typically, a thick layer of cross-linked peptidoglycan that imparts strength and rigidity. Anchored to this cell wall are protein assemblies, such as pili, and surface proteins, such as microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) that act as surface adhesins. These proteins are critical not only for bacterial binding to host surfaces in the early stages of infection but also for biofilm formation and immune evasion. In a series of studies published since 2007, these cell surface proteins and protein assemblies have been shown to contain isopeptide and thioester bonds, highly unusual intramolecular covalent linkages between amino acid side chains. These cross-links have either a structure-stabilizing role or may be directly involved in adhesion. Using an elegant combination of structural biology and mass spectrometry, Kwon et al. (1) reveal in PNAS an ester bond as yet another covalent linkage in a putative MSCRAMM from the Grampositive pathogen Clostridium perfringens. Remarkably, this ester bond, joining the side chains of a Thr and Gln residue, is equivalent to an unresolved acyl-enzyme intermediate, formed on an autocatalytic pathway that resembles the mechanism of serine proteases. Despite fundamental differences in the way they are encoded in bacterial genomes, following delivery/assembly at the cell surface, pili and many MSCRAMMs share important similarities. In such proteins, a series of repetitive domains (commonly referred to as stalk regions) are responsible for the length of the structure and allow an N-terminal region to be positioned away from the cell surface. At the C terminus, both pili and MSCRAMMs are anchored to the cell wall by sortases (2). Although many of the building blocks of Gram-positive surface proteins and pili bear some resemblance to their Gramnegative equivalents (they are variants of the Ig-like β-sandwich fold), their mechanisms of assembly are very different (3). As mentioned above, some of the more remarkable discoveries made since the molecular characterization of Gram-positive pili (4, 5) are the prevalence of intramolecular covalent linkages in the component proteins. The first of these bonds was discovered in the stalk protein of pili from the human pathogen Streptococcus pyogenes (6). These intramolecular isopeptide bonds have now been characterized experimentally in a wide-range of pilus subunits from many Gram-positive pathogens (reviewed in refs. 7–9) and also in the MSCRAMM FbaB (10). Intramolecular isopeptide bonds are most commonly formed between the side chains of Lys and Asn residues (Fig. 1) [although Lys-Asp bonds also exist (10, 11)]. The residues comprising these bonds are strategically positioned to bridge the first and either penultimate or last