Intramolecular Isopeptide Bonds Give Thermodynamic and Proteolytic Stability to the Major Pilin Protein of Streptococcus pyogenes

Gram-positive bacteria elaborate pili and do so without the participation of folding chaperones or disulfide bond catalysts. Sortases, enzymes that cut pilin precursors, form covalent bonds that link pilin subunits and assemble pili on the bacterial surface. We determined the x-ray structure of BcpA, the major pilin subunit of Bacillus cereus . The BcpA precursor encompasses 2 Ig folds (CNA 2 and CNA 3 ) and one jelly-roll domain (XNA) each of which synthesizes a single intramolecular amide bond. A fourth amide bond, derived from the Ig fold of CNA 1 , is formed only after pilin subunits have been incorporated into pili. We report that the domains of pilin precursors have evolved to synthesize a discrete sequence of intramolecular amide bonds, thereby conferring structural stability and protease resistance to pili.

Section: Discussionmentioning

confidence: 99%

Intramolecular amide bonds stabilize pili on the surface of bacilli

Budzik¹,

Poor

Faull

et al. 2009

“…We have shown that the intramolecular isopeptide bonds in Spy0128 strongly enhance thermodynamic stability and resistance to proteolysis (24). SpaA additionally contains a disulfide bond, conserved in the other C. diphtheriae major pilins, which might be expected to further enhance stability.…”

Section: Discussionmentioning

confidence: 99%

“…Mutagenesis has shown that a catalytic carboxyl group is essential for bond formation (4,5,24); this can be Asp or Glu, as shown by the use of Asp-241 in the M-domain and Glu-446 in the C-domain. The isopeptide moiety can have either cis or trans configuration, with corresponding bidentate or monodentate interaction with the essential carboxyl side chain.…”

Section: Discussionmentioning

confidence: 99%

The Corynebacterium diphtheriae shaft pilin SpaA is built of tandem Ig-like modules with stabilizing isopeptide and disulfide bonds

Kang

Paterson

Gaspar

et al. 2009

Self Cite

102

166

Cell-surface pili are important virulence factors that enable bacterial pathogens to adhere to specific host tissues and modulate host immune response. Relatively little is known about the structure of Gram-positive bacterial pili, which are built by the sortase-catalyzed covalent crosslinking of individual pilin proteins. Here we report the 1.6-Å resolution crystal structure of the shaft pilin component SpaA from Corynebacterium diphtheriae , revealing both common and unique features. The SpaA pilin comprises 3 tandem Ig-like domains, with characteristic folds related to those typically found in non-pilus adhesins. Whereas both the middle and the C-terminal domains contain an intramolecular Lys–Asn isopeptide bond, previously detected in the shaft pilins of Streptococcus pyogenes and Bacillus cereus , the middle Ig-like domain also harbors a calcium ion, and the C-terminal domain contains a disulfide bond. By mass spectrometry, we show that the SpaA monomers are cross-linked in the assembled pili by a Lys–Thr isopeptide bond, as predicted by previous genetic studies. Together, our results reveal that despite profound dissimilarities in primary sequences, the shaft pilins of Gram-positive pathogens have strikingly similar tertiary structures, suggesting a modular backbone construction, including stabilizing intermolecular and intramolecular isopeptide bonds.

“…In each case, the Thr residue is located on the first β-strand and the Gln residue is located on the last β-strand of the domain, reminiscent of the isopeptide bonds found in Gram-positive pili (5,14,21,22) (Fig. 1F).…”

Section: Resultsmentioning

confidence: 99%

Autocatalytically generated Thr-Gln ester bond cross-links stabilize the repetitive Ig-domain shaft of a bacterial cell surface adhesin

Kwon

Squire

Young

et al. 2013

Self Cite

Gram-positive bacteria are decorated by a variety of proteins that are anchored to the cell wall and project from it to mediate colonization, 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 cross-links, formed autocatalytically, in the pili from Streptococcus pyogenes has highlighted the role that such crosslinks can play in stabilizing such structures. We have investigated a putative cell-surface adhesin from Clostridium perfringens comprising an N-terminal adhesin domain followed by 11 repeat domains. The crystal structure of a two-domain fragment shows that each domain has an IgG-like fold and contains an unprecedented ester bond joining Thr and Gln side chains. MS confirms the presence of these bonds. We show that the bonds form through an autocatalytic intramolecular reaction catalyzed by an adjacent His residue in a serine protease-like mechanism. Two buried acidic residues assist in the reaction. By mutagenesis, we show that loss of the ester bond reduces the thermal stability drastically and increases susceptibility to proteolysis. As in pilin domains, the bonds are placed at a strategic position joining the first and last strands, even though the Ig fold type differs. Bioinformatic analysis suggests that similar domains and ester bond cross-links are widespread in Gram-positive bacterial adhesins. intramolecular ester bond | protein stability A striking feature of globular proteins is that despite the chemical diversity inherent in the side chains of their constituent amino acids, chemical reactions between these side chains are very rare. This may be explained by evolutionary selection, which minimizes reactions that could prejudice proper protein folding. Thus, the only common example of a covalent cross-link between protein side chains is the disulfide bond, which forms only in an appropriate redox environment when two Cys residues are brought together by protein folding. Nevertheless, some surprising examples of unexpected crosslinks have been brought to light by protein structure analysis or by the observation of unusual spectroscopic or biophysical properties. Examples include the Cys-Tyr bond in galactose oxidase (1), which provides a radical center; similar bonds in some catalases (2); the His-Tyr bond in cytochrome C oxidase (3); and the remarkable chromophore of GFP (4). These, and other examples, arise through intramolecular reactions facilitated by particular local environments.The recent discovery of isopeptide bonds joining Lys and Asn side chains in the proteins that make up pili on the Gram-positive bacterium Streptococcus pyogenes (5), as well as on other Gram-positive pathogens (6), has highlighted a class of proteins in which intramolecular cross-links seem to be remarkably prevalent. It includes not only Gram-positive pili but a number of other cell surface adhesins, known as mi...