Reprogramming the specificity of sortase enzymes.
Ontology highlight
ABSTRACT:
SUBMITTER: Dorr BM
PROVIDER: S-EPMC4169943 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Pili are elongated structures that protrude from bacteria and increase their virulence. The Streptococcus pnuenomae pilus island 1 pili are composed of three subunits, RrgA, RrgB, and RrgC, and are assembled by three class C sortase C (SrtC) enzymes: SrtC-1, SrtC-2, and SrtC-3. Pilin subunits are recognized by SrtC proteins through a pentapeptide sorting signal, and while previous studies have sought to characterize the selectivities of SrtC isoforms for these subunits, the molecular mechanisms underlying these interactions remain unclear. Here, we report a series of molecular dynamics simulations of each SrtC enzyme with the sorting signals of RrgA, RrgB, and RrgC to determine the structural and thermodynamic basis of pilin recognition. Results show that, in accordance with previous studies, both SrtC-1 and SrtC-3 are selective for RrgB, while SrtC-2 is selective for RrgA. This specificity is tuned by the sorting signal binding conformation in which the first two residue sidechains complement hydrophobic residues around the active site, while the third residue projects away from the catalytic triad and makes specific interactions based on its charge and reach. Together, these results provided atomic-scale descriptions of the SrtC substrate selectivity mechanisms and extend the emerging model of pilin construction in S. pnuenomae.
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Project description:Streptococcus agalactiae, also referred to as Group B Streptococcus (GBS), is one of the most common causes of life-threatening bacterial infections in infants. In recent years cell surface pili have been identified in several Gram-positive bacteria, including GBS, as important virulence factors and promising vaccine candidates. In GBS, three structurally distinct types of pili have been discovered (pilus 1, 2a and 2b), whose structural subunits are assembled in high-molecular weight polymers by specific class C sortases. In addition, the highly conserved housekeeping sortase A (SrtA), whose main role is to link surface proteins to bacterial cell wall peptidoglycan by a transpeptidation reaction, is also involved in pili cell wall anchoring in many bacteria. Through in vivo mutagenesis, we demonstrate that the LPXTG sorting signal of the minor ancillary protein (AP2) is essential for pilus 2a anchoring. We successfully produced a highly purified recombinant SrtA (SrtA(?N40)) able to specifically hydrolyze the sorting signal of pilus 2a minor ancillary protein (AP2-2a) and catalyze in vitro the transpeptidation reaction between peptidoglycan analogues and the LPXTG motif, using both synthetic fluorescent peptides and recombinant proteins. By contrast, SrtA(?N40) does not catalyze the transpeptidation reaction with substrate-peptides mimicking sorting signals of the other pilus 2a subunits (the backbone protein and the major ancillary protein). Thus, our results add further insight into the proposed model of GBS pilus 2a assembly, in which SrtA is required for pili cell wall covalent attachment, acting exclusively on the minor accessory pilin, representing the terminal subunit located at the base of the pilus.
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Project description:Sortases are a group of enzymes displayed on the cell-wall of Gram-positive bacteria. They are responsible for the attachment of virulence factors onto the peptidoglycan in a transpeptidation reaction through recognition of a pentapeptide substrate. Most housekeeping sortases recognize one specific pentapeptide motif; however, Streptococcus pyogenes sortase A (SpSrtA WT) recognizes LPETG, LPETA and LPKLG motifs. Here, we examined SpSrtA's flexible substrate specificity by investigating the role of the β7/β8 loop in determining substrate specificity. We exchanged the β7/β8 loop in SpSrtA with corresponding β7/β8 loops from Staphylococcus aureus (SaSrtA WT) and Bacillus anthracis (BaSrtA WT). While the BaSrtA-derived variant showed no enzymatic activity toward either LPETG or LPETA substrates, the activity of the SaSrtA-derived mutant toward the LPETA substrate was completely abolished. Instead, the mutant had an improved activity toward LPETG, the preferred substrate of SaSrtA WT.
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