Molecular Characterization of Monoclonal Antibodies that Inhibit Acetylcholinesterase by Targeting the Peripheral Site and Backdoor Region

Yves Bourne,Pascale Marchot,Patricia Lamourette,Didier Boquet,Sosthène Essono,Ludovic Renault,Jacques Grassi,Grégoire Mondielli,Ashley M Buckle

doi:10.1371/journal.pone.0077226

Abstract

The inhibition properties and target sites of monoclonal antibodies (mAbs) Elec403, Elec408 and Elec410, generated against Electrophorus electricus acetylcholinesterase (AChE), have been defined previously using biochemical and mutagenesis approaches. Elec403 and Elec410, which bind competitively with each other and with the peptidic toxin inhibitor fasciculin, are directed toward distinctive albeit overlapping epitopes located at the AChE peripheral anionic site, which surrounds the entrance of the active site gorge. Elec408, which is not competitive with the other two mAbs nor fasciculin, targets a second epitope located in the backdoor region, distant from the gorge entrance. To characterize the molecular determinants dictating their binding site specificity, we cloned and sequenced the mAbs; generated antigen-binding fragments (Fab) retaining the parental inhibition properties; and explored their structure-function relationships using complementary x-ray crystallography, homology modeling and flexible docking approaches. Hypermutation of one Elec403 complementarity-determining region suggests occurrence of antigen-driven selection towards recognition of the AChE peripheral site. Comparative analysis of the 1.9Å-resolution structure of Fab408 and of theoretical models of its Fab403 and Fab410 congeners evidences distinctive surface topographies and anisotropic repartitions of charges, consistent with their respective target sites and inhibition properties. Finally, a validated, data-driven docking model of the Fab403-AChE complex suggests a mode of binding at the PAS that fully correlates with the functional data. This comprehensive study documents the molecular peculiarities of Fab403 and Fab410, as the largest peptidic inhibitors directed towards the peripheral site, and those of Fab408, as the first inhibitor directed toward the backdoor region of an AChE and a unique template for the design of new, specific modulators of AChE catalysis.

Highlights

Acetylcholinesterase (AChE, EC 3.1.1.7) terminates cholinergic neurotransmission by rapidly catalyzing hydrolysis of the neurotransmitter, acetylcholine, at neuronal and neuromuscular synapses [1,2,3]
Inhibitor binding at the peripheral anionic site (PAS) appears to limit the catalytic rate by a combination of steric and electrostatic blockade of ligand trafficking through the gorge and by altering the active center conformation [9,10,11,12]
Crystal structures of fasciculin 2 (Fas2)-AChE complexes revealed the large surface area and multiple electrostatic and hydrophobic anchoring points solicited by the bound toxin at the PAS, along with apparent occlusion of the AChE gorge by the Fas2 central finger, loop II, all features being consistent with the nano- to picomolar affinity of the complex [13,26]

Summary

Introduction

Acetylcholinesterase (AChE, EC 3.1.1.7) terminates cholinergic neurotransmission by rapidly catalyzing hydrolysis of the neurotransmitter, acetylcholine, at neuronal and neuromuscular synapses [1,2,3]. This electronegative patch confers on the Fab408 molecule a clear anisotropic distribution of surface charges with a moderate dipole moment of 484 Debyes and a vector oriented toward the H-chain N-terminus, opposite to the CDRs. This is consistent with the Fab408 neutral pI value (Figure 1; Table 1) and its interaction with the BDR of EeAChE, which is remote by more than 100° from the axis linking PAS residue Trp281 (homologous to mAChE Trp286) to active site residue Ser202 (mAChE Ser203) and roughly aligns with the dipole vector of the subunit (cf below).

Results

Conclusion