Structural Characterization of Native Autoinducing Peptides and Abiotic Analogues Reveals Key Features Essential for Activation and Inhibition of an AgrC Quorum Sensing Receptor in Staphylococcus aureus

Abstract

Staphylococcus aureus is a major human pathogen that uses quorum sensing (QS) to control virulence. Its QS system is regulated by macrocyclic peptide signals (or autoinducing peptides (AIPs)) and their cognate transmembrane receptors (AgrCs). Four different specificity groups of S. aureus have been identified to date (groups I–IV), each of which uses a different AIP:AgrC pair. Non-native ligands capable of intercepting AIP:AgrC binding, and thereby QS, in S. aureus have attracted considerable interest as chemical tools to study QS pathways and as possible anti-virulence strategies for the treatment of infection. We recently reported a set of analogs of the group-III AIP that are capable of strongly modulating the activity of all four AgrC receptors. Critical to the further development of such ligands is a detailed understanding of the structural features of both native AIPs and non-native analogs that are essential for activity. Herein, we report the first three-dimensional structural analysis of the known native AIP signals (AIPs-I–IV) and several AIP-III analogs with varied biological activities using NMR spectroscopy. Integration of these NMR studies with the known agonism and antagonism profiles of these peptides in AgrC-III revealed two key structural elements that control AIP-III (and non-native peptide) activity: (1) a tri-residue hydrophobic “knob” essential for both activation and inhibition, and (2) a fourth anchor point on the exocyclic tail needed for receptor activation. These results provide strong structural support for a mechanism of AIP-mediated AgrC activation and inhibition in S. aureus, and should facilitate the design of new AgrC ligands with enhanced activities (as agonists or antagonists) and simplified chemical structures.

Characterization of structural elements in native autoinducing peptides and non-native analogues that permit the differential modulation of AgrC-type quorum sensing receptors in Staphylococcus aureus

“…Second, we sought to characterize structural motifs involved in potential inter-staphylococcal AIP:AgrC receptor interactions; thus, we also determined the structures of native AIP signals from S. epidermidis and S. lugdunensis and compared them to a native AIP signal in S. aureus . Third, we evaluated the structure of a new AIP-III analogue, AIP-III D4N, which we previously hypothesized should act as an AgrC-III agonist, 10 and found that it indeed acts as an AgrC-III agonist and adopts the expected 3-D conformation, strengthening our hypothesis regarding the structural requirements needed for AgrC-III receptor activation. Overall, we were able to identify and refine a series of different structural motifs that are required for inhibition or activation of different S. aureus AgrC receptors (I–III) by AIP-type ligands.…”

“…In our previous study, we determined the solution-structure of AIP-II by NMR and found that this peptide was relatively unstructured (at least under our experimental conditions). 10 However, a closer evaluation of the two hydrophobic residues within the macrocycle (Leu8 and Phe9) revealed that their side chains projected in the same direction from the ring in all of the calculated structures (total 20 structures). 10 In view of this structural trend, we reasoned that the macrocyclic portion of AIP-II may be capable of adopting a defined structure in the absence of the exocyclic tail.…”

“…The AIP-I D5N tail projects more toward the upper right side of the macrocycle, while the tail of AIP-III projects toward the upper left side of the macrocycle (Figure 2H). 10 The closer similarity of the structure of AIP-I D5N to that of AIP-III, compared to AIP-I and AIP-I D5A, correlates with the ability of AIP-I D5N to activate the AgrC-III receptor, while both AIP-I and AIP-I D5A are potent AgrC-III inhibitors (Table 3). Indeed, the presence of the knob motif in all three AIP-I analogues supports our previous hypothesis regarding the requirement for the triangular knob motif for AgrC-III modulation (both inhibition and activation).…”

“…10 AIP-I D5A was found to be less structured relative to AIP-I (RMSD values over 3-fold higher, Table S-11; see Figure S-1A for 10-structure ensemble of AIP-I D5A), but within the macrocycle a similar triangular hydrophobic knob motif was apparent, composed of the Phe6, Ile7, and Met8 side chains projecting from one face (Figures 2A–C). The exocyclic tail projects back from the macrocycle in both AIP-I D5A and AIP-I; however, the orientation of the tail differs between the two structures.…”

“…Towards this goal, we recently reported the 3-D solution-phase structures of the four native AIP signals (I–IV) in S. aureus and several synthetic AIP-III analogues as determined using NMR spectroscopy. 10 This past study allowed us to identify two critical structural motifs within AIP-type ligands that confer inhibition and activation of the AgrC-III receptor – (i) a hydrophobic patch (or “knob”) on the macrocycle essential for receptor binding and (ii) an additional hydrophobic contact or “anchor” on the N-terminal tail critical for receptor activation. In the absence of the anchor, peptides containing a hydrophobic knob were found to inhibit the AgrCIII receptor, presumably by outcompeting the native ligand.…”