Crystal structures of a quorum-quenching antibody

Abstract

A large number of Gram-negative bacteria employ N-acyl homoserine lactones (AHLs) as signaling molecules in quorum sensing, which is a population density-dependent mechanism to coordinate gene expression. Antibody RS2-1G9 was elicited against a lactam mimetic of the N-acyl homoserine lactone and represents the only reported monoclonal antibody that recognizes the naturally-occuring N-acyl homoserine lactone with high affinity. Due to its high cross-reactivity, RS2-1G9 showed remarkable inhibition of quorum sensing signaling in Pseudomonas aeruginosa, a common opportunistic pathogen in humans. The crystal structure of Fab RS2-1G9 in complex with a lactam analog revealed complete encapsulation of the polar lactam moiety in the antibody-combining site. This mode of recognition provides an elegant immunological solution for tight binding to an aliphatic, lipid-like ligand with a small head group lacking typical haptenic features, such as aromaticity or charge, which are often incorporated into hapten design to generate high-affinity antibodies. The ability of RS2-1G9 to discriminate between closely related AHLs is conferred by six hydrogen bonds to the ligand. Conversely, cross-reactivity of RS2-1G9 towards the lactone is likely to originate from conservation of these hydrogen bonds as well as an additional hydrogen bond to the oxygen of the lactone ring. A short, narrow tunnel exiting at the protein surface harbors a portion of the acyl chain and would not allow entry of the head group. The crystal structure of the antibody without its cognate lactam or lactone ligands revealed a considerably altered antibody-combining site with a closed binding pocket. Curiously, a completely buried ethylene glycol molecule mimics the lactam ring and, thus, serves as a surrogate ligand. The detailed structural delineation of this quorum-quenching antibody will aid further development of an antibody-based therapy against bacterial pathogens by interference with quorum sensing.

Figure 1

Structures of the two autoinducers (N-acyl homoserine lactones, AHLs) mediating quorum sensing in P. aeruginosa (1-2), of a lactam analog (3), and of haptens (RS1-3). The affinity constants of RS2-1G9 for 2 and 3 are listed.

Figure 2

Antibody combining site of RS2-1G9 bound to an AHL lactam mimetic (pink). The light and heavy chains are colored in yellow and blue, respectively. The σ_a-weighted 2F_o-F_c electron density map around the ligand is contoured at 1.4σ. The microenvironment at the very bottom of the binding pocket tolerates high-affinity binding of both the lactam and the lactone, since Asn^L34 does not form a hydrogen bond to the NH-group of the lactam. A potential hydrogen bond in the lactone complex model with the NH₂-group of Asn^L34 may account for its increased affinity with respect to the lactam. The displayed orientation of the terminal amide group of Asn^L34 is preferred due to formation of a hydrogen bond with Tyr^L32. CDR L3 is omitted for clarity.

Figure 3

Architecture of the antibody combining site of RS2-1G9. The CDRs of light and heavy chains are highlighted in yellow and blue, respectively. The lactam 3 is shown in pink. The tip of the CDR H3 loop bends over the ligand and largely seals it from bulk solvent.

Figure 4

High electrostatic and shape complementarity of the hapten analog in the antibody-combining site. A slice through the center of the binding site is shown. (a) and (b) correspond to the front and back view. The electrostatic potential was calculated in APBS and mapped onto the surface with the color code ranging from -30 kT/e (bright red) to +30 kT/e (dark blue).

Figure 5

Antibody combining site of RS2-1G9 (stereoview). Hydrogen bonds are shown as broken lines. Only Fab side chains that contact the lactam ligand (pink) are displayed. The hapten analog satisfies all its functional groups, except for the amide group of the lactam, which is crucial for the observed cross-reactivity of this antibody with an N-acyl homoserine lactone. A plethora of aromatic side chains surrounds the ligand. The residues between Leu^H97 and Asn^H100 of CDR H3 are omitted for clarity.

Figure 6

Ethylene glycol acts as a surrogate ligand in the “unliganded” antibody RS2-1G9 (lactam complex in grey, ethylene glycol complex in green, stereoview). This solvent molecule (yellow) substitutes for the lactam ring of the bound hapten derivative (pink) and fills the bottom of the ligand cavity. Strikingly, the bound ethylene glycol adopts an unfavorable, nearly eclipsed conformation that faithfully mimics part of the lactam ring. Ethylene glycol was added for cryoprotection of the crystals prior to data collection at cryogenic temperatures. This view also illustrates how CDRs H3 and L3 rearrange to close the N-acyl harboring tunnel of the binding site.

Figure 7

Comparison of the molecular surface representation of (a) the lactam complex and (b) the ethylene glycol complex of antibody RS2-1G9 reveals profound differences in the architecture of the antibody combining site. The lactam and the buried ethylene glycol (EG) are colored in pink and yellow, respectively.

Figure 8

Induced fit in RS2-1G9 (lactam complex in grey, ethylene glycol complex in green, stereoview). Upon ligand binding, the main-chain and side-chain atoms of CDR H3 undergo the largest rearrangements, while L3, L1, and H2 move to a minor extent. L2 and H1 are essentially not in contact to the hapten derivative and, hence, are not displayed. In particular, the tip of H3 completely reorganizes upon ligand binding to accommodate the lactam in the binding pocket (induced fit). For clarity, Tyr^H98, Tyr^H99, Asn^H100, and Asn^H100A are labeled in both structures.