Abstract
Streptococcus pyogenes and other Gram-positive bacterial pathogens present long macromolecular filaments known as pili on their surface that mediate adhesion and colonization. These pili are covalent polymers, assembled by sortases. Typically, they comprise a putative adhesin at their tip, a backbone subunit present in multiple copies and a basal subunit that is covalently anchored to the peptidoglycan layer of the cell surface. The crystal structures of pilin subunits revealed the presence of unusual covalent linkages in these proteins, including intramolecular isopeptide and internal thioester bonds. The intramolecular isopeptide bonds in backbone pilins are important for protein stability. Here, using both the wild-type protein and a set of mutants, we assessed the proteolytic and thermal stability of the S. pyogenes pilus tip adhesin Spy0125, in the presence and absence of its intramolecular isopeptide and internal thioester bonds. We also determined a crystal structure of the internal thioester bond variant Spy0125Cys426Ala. We find that mutations in the intramolecular isopeptide bonds compromise the stability of Spy0125. Using limited proteolysis and thermal denaturation assays, we could separate the contribution of each intramolecular isopeptide bond to Spy0125 stability. In contrast, mutation in the internal thioester bond had a lesser effect on protein stability and the crystal structure is essentially identical to wild type. This work suggests that the internal thioester in Spy0125, although having a minor contributory role, is not required for protein stability and must have a different primary function, most likely mediating a covalent interaction with host cell ligands. Proteins 2014; 82:517–527. © 2013 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.
Highlights
The composition and assembly of long, flexible pili presented on the surface of Gram-positive bacterial pathogens have been extensively studied over the last 10 years
We present the results of proteolytic and thermal stability assays showing that the intramolecular isopeptide bonds are the most important for the overall stability of the Spy0125-CTR protein
We have not been able to determine the subtle differences between the fragments in these bands. These results show that susceptibility of Spy0125-CTR to limited proteolysis is conferred by the mutation of the intramolecular isopeptide bonds in the protein but not mutation of the internal thioester bond
Summary
The composition and assembly of long, flexible pili presented on the surface of Gram-positive bacterial pathogens have been extensively studied over the last 10 years These pili are found on the surface of clinically relevant pathogens including Bacillus cereus, Bacillus anthracis, Corynebacterium diphtheriae,[1] Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, and others Gram-positive bacterial pili typically comprise repeats of a major pilin subunit, in a beads-on-a-string-type arrangement They are assembled by the action of sortases, enzymes that catalyze the formation of intermolecular isopeptide bonds between the C-terminal carboxyl group of one subunit and a specific lysine side chain of the subunit in the chain.[6] In addition to the major subunit, pili contain a distinct minor pilin at the tip of the structure and most, not all, contain a second minor pilin at the base.[7] Typically, the genes encoding pilus subunits and pilus-specific sortases are clustered in bacterial genomes.[8] In three-component pili, the basal pilin is used to anchor
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