Neutralizing Antibodies Against Allosteric Proteins: Insights From a Bacterial Adhesin

Abstract

Allosteric proteins transition between 'inactive' and 'active' states. In general, such proteins assume distinct conformational states at the level of secondary, tertiary and/or quaternary structure. Different conformers of an allosteric protein can be antigenically dissimilar and induce antibodies with a highly distinctive specificities and neutralizing functional effects. Here we summarize studies on various functional types of monoclonal antibodies obtained against different allosteric conformers of the mannose-specific bacterial adhesin FimH - the most common cell attachment protein of Escherichia coli and other enterobacterial pathogens. Included are types of antibodies that activate the FimH function via interaction with ligand-induced binding sites or by wedging between domains as well as antibodies that inhibit FimH through orthosteric, parasteric, or novel dynasteric mechanisms. Understanding the molecular mechanism of antibody action against allosteric proteins provides insights on how to design antibodies with a desired functional effect, including those with neutralizing activity against bacterial and viral cell attachment proteins.

Keywords: FimH; adhesin; attachment; conformation; inhibition.

Figure 1.. Structure of FimH and type 1 pili.

Panel (a) – electron micrograph of E. coli, with type 1 pili, or fimbria, seen protruding from the surface on all sides. Arrow indicates one fimbrial tip. Panel (b) – cartoon structure of the fimbrial tip (PDB ID 3JWN [27]). Panel (c) – cartoon structure of the FimH lectin domain in the inactive or low affinity state (LAS), anchored to the pilin domain in the fimbrial tip (PDB ID 3JWN [27]). Panel (d) – cartoon structure of the FimH lectin domain in the active or high affinity state (HAS), when not anchored to the pilin domain, with mannose from the co-crystal (PDB ID 1UWF [46]). Color coding for (c) and (d): red spheres – side chain atoms of residues of the lectin domain that bind mannose (in HAS); green spheres – side chain atoms of residues of the lectin domain that interact with the pilin domain (in LAS); in orange – residues in other lectin domain regions that change most dramatically between LAS and HAS [27]; purple spheres – atoms of mannose ligand attached to HAS[46].

Figure 2.. Effects of toggle mutations in FimH.

Panel (a) – Orientation of L34, V35, and Y64 in the cross-sectional view of the inactive (PDB ID 3JWN [27]) and active (PDB ID 1UWF [46]) conformations of the FimH lectin domain. Panel (b) – core repacking model of the allosteric transition induced by domain separation. LD – lectin domain; PD – pilin domain. The circles indicate the location of residues discussed in the text; red = V35, blue = L34, black = V64, green = V28, yellow = A115; grey – interdomain-interacting residues in the pilin domain (do not change the core vs. surface orientation).

Figure 3.. Allosteric activating anti-LIBS antibodies.

Panel (a) – anti-LIBS antibody (mAb21) enhances bacterial binding to uroepithelial cells, where *** indicates p < 0.0001. (Adapted with permission from [90].) Panels (b) and (c) – the mannose-induced mAb34 epitope in the FimH lectin domain in the LAS (PDB ID: 3JWN [27]) and HAS (PDB ID 1UWF [46]), respectively. Panels (d) and (e) – the interdomain-wedged mAb21 epitope in the LAS and HAS, respectively. Color coding for (b) though (e): red and green spheres as in the Figure 1cd above; in blue and yellow - side chain atoms of the mAb-binding critical epitope residues that do (yellow) or do not (blue) overlap with residues that would otherwise be labelled green or red; in pink – pilin domain.

Figure 4.. Comparison of the different types of conformational regulation that have been shown by antibodies to FimH.

The clusters of circles show the typical location of epitopes for antibodies in each category, with the darker color identifying the preferred epitope conformation that is stabilized by antibody, and the lighter color identifying an epitope conformation that is not recognized by antibody. The gray arrows indicate the direction of conformational regulation.

Figure 5.. Orthosteric and parasteric inhibitory antibodies.

Panels (a) and (b) – orthosteric mAb475 epitope in the FimH lectin domain in the LAS (PDB ID: 3JWN [27]) and HAS (PDB ID 1UWF [46]), respectively. Panel (c) - effect of mAb475 on bacterial binding to a bladder epithelial cell line, in comparison with the effects of soluble α-methyl-mannose (αmm) and mAb21 [89]. Panel (d) - bacteria recovered from mice bladders one day after inoculation with bacteria that were pretreated with mAb475 versus mAb21 [89]. Panels (e) and (f) - parasteric mAb923 epitope in the LAS (PDB ID: 3JWN [27]) and HAS (PDB ID 1UWF [46]), respectively. Panel (g) - effect of mAb926 versus other antibodies on detachment of bacteria already forming a biofilm on a mannosylated surface [94]. Panel (h) - bacteria recovered from mice bladders one day after inoculation with bacteria that were pretreated with mAb926 versus mAb475 [94]. Color coding as in Figure 3, so that the yellow and blue combined indicate the mAb epitope. Panels c and d adapted with permission from [89], and panel g and h adapted with permission from [94].

Figure 6.. Dynasteric kinetic trapping antibody mAb824.

Panel (a) - FimH lectin domain in the LAS (PDB ID: 3JWN [27]); Panel (b) - FimH lectin domain in the HAS (PDB ID 1UWF [46]). As in prior figures, the residues that interact with the mannose ligand and pilin domain are in red and green, respectively. Both panels show the mAb824 functional epitope (blue) and, partially, structural epitope (grey spheres) [77] that also include the toggle residues L34 (crimson) and V35 (turquoise). Note that while the functional epitope does not change conformations between LAS and HAS, the structural epitope does, with the toggle residues switching between core and solvent orientation.