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. 2011 May 13;286(19):17351-8.
doi: 10.1074/jbc.M111.231258. Epub 2011 Mar 23.

Structural basis for ligand recognition and discrimination of a quorum-quenching antibody

Robert N Kirchdoerfer  1 , Amanda L Garner, Caralyn E Flack, Jenny M Mee, Alexander R Horswill, Kim D Janda, Gunnar F Kaufmann, Ian A Wilson
Affiliations

Structural basis for ligand recognition and discrimination of a quorum-quenching antibody

Robert N Kirchdoerfer et al. J Biol Chem. .

Abstract

In the postantibiotic era, available treatment options for severe bacterial infections caused by methicillin-resistant Staphylococcus aureus have become limited. Therefore, new and innovative approaches are needed to combat such life-threatening infections. Virulence factor expression in S. aureus is regulated in a cell density-dependent manner using "quorum sensing," which involves generation and secretion of autoinducing peptides (AIPs) into the surrounding environment to activate a bacterial sensor kinase at a particular threshold concentration. Mouse monoclonal antibody AP4-24H11 was shown previously to blunt quorum sensing-mediated changes in gene expression in vitro and protect mice from a lethal dose of S. aureus by sequestering the AIP signal. We have elucidated the crystal structure of the AP4-24H11 Fab in complex with AIP-4 at 2.5 Å resolution to determine its mechanism of ligand recognition. A key Glu(H95) provides much of the binding specificity through formation of hydrogen bonds with each of the four amide nitrogens in the AIP-4 macrocyclic ring. Importantly, these structural data give clues as to the interactions between the cognate staphylococcal AIP receptors AgrC and the AIPs, as AP4-24H11·AIP-4 binding recapitulates features that have been proposed for AgrC-AIP recognition. Additionally, these structural insights may enable the engineering of AIP cross-reactive antibodies or quorum quenching vaccines for use in active or passive immunotherapy for prevention or treatment of S. aureus infections.

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Figures

FIGURE 1.
FIGURE 1.
The agr operon. A, the agrBDCA genes encode the AgrB, AgrD, AgrC, and AgrA proteins, which are all involved in the biosynthesis of AIPs. The propeptide AgrD is processed by AgrB and SpsB into AIP-4, which is sensed by the two-component regulatory system AgrC and AgrA. Phosphorylated AgrA activates transcription at the P2 and P3 promoters. Autoinduction of the agr operon leads to induction the response regulator, RNA III, and changes in gene expression. AP4–24H11 sequesters AIP-4 and prevents the activation of AgrC. B, schematic representation of the four AIPs encoded by agr groups I–IV. The residues of AIP-4 are numbered to illustrate and clarify the nomenclature.
FIGURE 2.
FIGURE 2.
High agr specificity of quorum-sensing signaling inhibition mediated by AP4-24H11 in S. aureus. S. aureus strains containing a plasmid with an agr P3-YFP promoter fusion (pDB59) were used to examine the quorum-quenching activity of AP4-24H11. Reporter strains representing each of the four agr groups were constructed and incubated with AP4-24H11 in concentrations ranging from 0.2 to 9.8 × 10−5 mg/ml. As controls (Ctrl), assays containing no antibody or an isotype control antibody (0.2 mg/ml) were included. For agr inhibition controls, supernatants (sup) from MW2 containing AIP-3 or LAC containing AIP-1 were included as indicated. Using AP4-24H11, strong inhibition of agr group IV and modest inhibition of agr group I was observed, whereas minimal inhibition of agr group II and III was evident only at high AP4-24H11 doses. Error bars represent S.D. of quadruplicate samples, and each experiment was repeated.
FIGURE 3.
FIGURE 3.
Crystal structure of AIP-4·AP4-24H11. A, structure of AP4-24H11 Fab with AIP-4 inserted between the heavy chain (blue) and light chain (red) in the combining site. B, electrostatic surface of the 24H11 binding site (+3.0 kV (blue) to −3.0 kV (red)). The hydrophobic residues AIP-4 Phe6, Ile7, and Met8 are buried in a hydrophobic pocket. The negative potential beneath the ring arises from GluH95. A sodium ion is modeled as a purple sphere.
FIGURE 4.
FIGURE 4.
Antibody recognition of the AIP-4 main chain conformation. GluH95 in CDRH3 extends toward the center of the AIP-4 macrocyclic ring. The GluH95 side chain makes hydrogen bonds to each of the amide nitrogens of AIP-4 residues 5–8.
FIGURE 5.
FIGURE 5.
Molecular interactions between AP4-24H11 and AIP-4 side chains. Close-up illustration of the interactions of AIP-4 macrocycle side chains with the antibody combining site of AP4-24H11. A, the aromatic ring of AIP-4 Tyr5 is positioned 3.5–4.0Å from TyrH33, creating a CH-π bond. B, Phe6 of AIP-4 makes a CH-π bond with TyrL49 with the ζ-carbon of AIP-4 Phe6 positioned 4.1–4.5Å from the TyrL49 aromatic ring. Ile7 does not engage in any specific interactions, but the leucine side chain found in other AIPs might create steric clashes within the binding site. C, Met8 of AIP-4 packs along the GluH95 side chain and part of the main chain, which might prevent interactions with the branched side chains found in other AIPs at this position.
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