Multiple Length Peptide-Pheromone Variants Produced by Streptococcus pyogenes Directly Bind Rgg Proteins to Confer Transcriptional Regulation

Aggarwal, Chaitanya; Jimenez, Juan Cristobal; Nanavati, Dhaval; Federle, Michael J.

doi:10.1074/jbc.m114.583989

Cited by 46 publications

(68 citation statements)

References 32 publications

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“…As shown in Fig 9B, S . suis has a high affinity for its cognate XIP-C7 with a K d similar to that observed for SHPs and Rggs (Table 2) [39]. Additionally, the binding mechanism is driven by hydrogen bonding and likely electrostatic interactions as indicated by the large enthalpy, which again emphasizes the importance of the electropositive pocket (Fig 5C).…”

Section: Resultssupporting

confidence: 62%

Pheromone Recognition and Selectivity by ComR Proteins among Streptococcus Species

Shanker¹,

Morrison²,

Talagas³

et al. 2016

PLoS Pathog

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107

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Natural transformation, or competence, is an ability inherent to bacteria for the uptake of extracellular DNA. This process is central to bacterial evolution and allows for the rapid acquirement of new traits, such as antibiotic resistance in pathogenic microorganisms. For the Gram-positive bacteria genus Streptococcus, genes required for competence are under the regulation of quorum sensing (QS) mediated by peptide pheromones. One such system, ComRS, consists of a peptide (ComS) that is processed (XIP), secreted, and later imported into the cytoplasm, where it binds and activates the transcription factor ComR. ComR then engages in a positive feedback loop for the expression of ComS and the alternative sigma-factor SigX. Although ComRS are present in the majority of Streptococcus species, the sequence of both ComS/XIP and ComR diverge significantly, suggesting a mechanism for species-specific communication. To study possible cross-talk between streptococcal species in the regulation of competence, and to explore in detail the molecular interaction between ComR and XIP we undertook an interdisciplinary approach. We developed a ‘test-bed’ assay to measure the activity of different ComR proteins in response to cognate and heterologous XIP peptides in vivo, revealing distinct ComR classes of strict, intermediate, and promiscuous specificity among species. We then solved an X-ray crystal structure of ComR from S. suis to further understand the interaction with XIP and to search for structural features in ComR proteins that may explain XIP recognition. Using the structure as a guide, we probed the apo conformation of the XIP-binding pocket by site-directed mutagenesis, both in test-bed cultures and biochemically in vitro. In alignments with ComR proteins from other species, we find that the pocket is lined by a variable and a conserved face, where residues of the conserved face contribute to ligand binding and the variable face discriminate among XIP peptides. Together, our results not only provide a model for XIP recognition and specificity, but also allow for the prediction of novel XIP peptides that induce ComR activity.

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Section: Resultssupporting

confidence: 62%

Pheromone Recognition and Selectivity by ComR Proteins among Streptococcus Species

Shanker¹,

Morrison²,

Talagas³

et al. 2016

PLoS Pathog

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107

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“…To identify inhibitors of Rgg function, we developed a fluorescence polarization (FP) assay whereby drug and druglike compounds (Prestwick Chemical) were screened for their ability to disrupt the binding of fluorescent-labeled synthetic SHP peptides (FITC-SHP) to Rgg Sp (26) or Rgg2 Sd (Fig. 3 C-D).…”

Section: Resultsmentioning

confidence: 99%

“…1B) with similar affinities, and Rgg2 Sp and Rgg3 Sp bind to identical DNA-regulatory elements upstream of their target promoters (21,26). The net response of S. pyogenes to SHP pheromone is the robust expression of Rgg2/3-controlled promoters; however, Rgg2 Sp and Rgg3 Sp affect transcription by different mechanisms.…”

mentioning

confidence: 99%

Rgg protein structure–function and inhibition by cyclic peptide compounds

Parashar

Aggarwal

Federle

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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100

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Peptide pheromone cell-cell signaling (quorum sensing) regulates the expression of diverse developmental phenotypes (including virulence) in Firmicutes, which includes common human pathogens, e.g., Streptococcus pyogenes and Streptococcus pneumoniae. Cytoplasmic transcription factors known as "Rgg proteins" are peptide pheromone receptors ubiquitous in Firmicutes. Here we present X-ray crystal structures of a Streptococcus Rgg protein alone and in complex with a tight-binding signaling antagonist, the cyclic undecapeptide cyclosporin A. To our knowledge, these represent the first Rgg protein X-ray crystal structures. Based on the results of extensive structure-function analysis, we reveal the peptide pheromone-binding site and the mechanism by which cyclosporin A inhibits activation of the peptide pheromone receptor. Guided by the Rgg-cyclosporin A complex structure, we predicted that the nonimmunosuppressive cyclosporin A analog valspodar would inhibit Rgg activation. Indeed, we found that, like cyclosporin A, valspodar inhibits peptide pheromone activation of conserved Rgg proteins in medically relevant Streptococcus species. Finally, the crystal structures presented here revealed that the Rgg protein DNA-binding domains are covalently linked across their dimerization interface by a disulfide bond formed by a highly conserved cysteine. The DNA-binding domain dimerization interface observed in our structures is essentially identical to the interfaces previously described for other members of the XRE DNA-binding domain family, but the presence of an intermolecular disulfide bond buried in this interface appears to be unique. We hypothesize that this disulfide bond may, under the right conditions, affect Rgg monomer-dimer equilibrium, stabilize Rgg conformation, or serve as a redox-sensitive switch.

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“…Our lab has previously shown that shp3 has greater potential to activate Rgg-SHP signaling than shp2 ; using a series of gene replacements and by constructing chimeric shp s, it was determined that the difference in activation potential lies in the non-effector N-terminal portion of the peptides [23]. Furthermore, mature pheromones (C-terminal eight amino acids; Table 1) bind the Rgg proteins and induce P shp signaling with similar EC 50 values [13,23]. However, we have also shown that an aminopeptidase, PepO, degrades SHPs and limits signaling, with SHP2-C8 inactivated more efficiently than SHP3-C8 [19].…”

Section: Resultsmentioning

confidence: 99%

“…pyogenes [5], but is not required to produce XIP in S . mutans [12], and additional processing steps may occur in the final maturation of either peptide [13]. However, the mechanism by which XIP and SHP peptides are exported from the cell remains poorly understood for the model systems of S .…”

Section: Introductionmentioning

confidence: 99%

PptAB Exports Rgg Quorum-Sensing Peptides in Streptococcus

Chang

Federle

2016

PLoS ONE

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A transposon mutagenesis screen designed to identify mutants that were defective in peptide-pheromone signaling of the Rgg2/Rgg3 pathway in Streptococcus pyogenes generated insertions in sixteen loci displaying diminished reporter activity. Fourteen unique transposon insertions were mapped to pptAB, an ABC-type transporter recently described to export sex pheromones of Enterococcus faecalis. Consistent with an idea that PptAB exports signaling peptides, the pheromones known as SHPs (short hydrophobic peptides) were no longer detected in cell-free culture supernatants in a generated deletion mutant of pptAB. PptAB exporters are conserved among the Firmicutes, but their function and substrates remain unclear. Therefore, we tested a pptAB mutant generated in Streptococcus mutans and found that while secretion of heterologously expressed SHP peptides required PptAB, secretion of the S. mutans endogenous pheromone XIP (sigX inducing peptide) was only partially disrupted, indicating that a secondary secretion pathway for XIP exists.

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Multiple Length Peptide-Pheromone Variants Produced by Streptococcus pyogenes Directly Bind Rgg Proteins to Confer Transcriptional Regulation

Abstract: Significance: Bioactivity and receptor-ligand affinity assays developed herein provide a platform to discover quorum-sensing inhibitors.

Cited by 46 publications

References 32 publications

Pheromone Recognition and Selectivity by ComR Proteins among Streptococcus Species

Pheromone Recognition and Selectivity by ComR Proteins among Streptococcus Species

Rgg protein structure–function and inhibition by cyclic peptide compounds

PptAB Exports Rgg Quorum-Sensing Peptides in Streptococcus

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