Development, Structure, and Mechanism of Synthetic Antibodies that Target Claudin and Clostridium perfringens Enterotoxin Complexes

Abstract

Strains of the Gram-positive bacterium Clostridium perfringens produce a two-domain enterotoxin (CpE) that afflict millions of humans and domesticated animals annually by causing prevalent gastrointestinal illnesses. CpEs C-terminal domain (cCpE) binds cell surface receptors then its N-terminal domain restructures to form a membrane-penetrating 𝛽-barrel pore, which is toxic to epithelial cells of the gut. The claudin family of membrane proteins are the receptors for CpE, and also control the architecture and function of cell/cell contacts called tight junctions that create barriers to intercellular transport of solutes. CpE binding disables claudin and tight junction assembly and induces cytotoxicity via 𝛽-pore formation, disrupting gut homeostasis. Here, we aimed to develop probes of claudin/CpE assembly using a phage display library encoding synthetic antigen-binding fragments (sFabs) and discovered two that bound complexes between human claudin-4 and cCpE. We established each sFabs unique modes of molecular recognition, their binding affinities and kinetics, and determined structures for each sFab bound to ~35 kDa claudin-4/cCpE in three-protein comprised complexes using cryogenic electron microscopy (cryoEM). The structures reveal a recognition epitope common to both sFabs but also that each sFab distinctly conforms to bind their antigen, which explain their unique binding equilibria. Mutagenesis of antigen/sFab interfaces observed therein result in further binding changes. Together, these findings validate the structures and uncover the mechanism of targeting claudin-4/cCpE complexes by these sFabs. Based on these structural insights we generate a model for CpEs cytotoxic claudin-bound 𝛽-pore that predicted that these two sFabs would not prevent CpE cytotoxicity, which we verify in vivo with a cell-based assay. This work demonstrates the development and targeting mechanisms of sFabs against claudin/cCpE that enable rapid structural elucidation of these small membrane protein complexes using a cryoEM workflow. It further provides a structure-based framework and therapeutic strategies for utilizing these sFabs as molecular templates to target claudin/CpE assemblies, obstruct CpE cytotoxicity, and treat CpE-linked gastrointestinal diseases that cause substantial economic and quality of life losses throughout the world.

Cryo-EM Structures of Clostridium perfringens Enterotoxin Bound to its Human Receptor, Claudin-4

“…43 This sFab, called CpE Obstructing Protein-1 (COP-1), binds hsCLDN-4 and was used recently by us to enable a high resolution structure of the hsCLDN-4/cCpE complex by cryo-EM. 44,45 After exchanging the solubilizing detergent for hsCLDN-4 from UDM to 2,2didecylpropane-1,3-bis-β-D-maltopyranoside (LMNG), we made 1 mg of hsCLDN-4/CpETryp complexes-henceforth known as small complex(es). CpETryp was chosen based on its improved crystallizability and biological role of in activating CpE cytotoxicity.…”

“…Details of negative stain EM imaging and data processing of small complexes conducted at the RTSF Cryo-EM Microscopy Facility at Michigan State University have been reported previously. 44 For cryo-EM of the small complex, fractions containing both proteins from SEC in Cryo-EM Buffer (20 mM Hepes pH 7.4, 100 mM NaCl, and 0.003% LMNG) were pooled and concentrated to 4.4 and 8.0 mg/mL using a 50 kDa MWCO concentrator (Millipore). 3.5 μL of sample at each concentration were applied to UltrAuFoil 1.2/1.3 300 mesh (Quantifoil) grids that were glow-discharged for 60 seconds at 15 mA using a Pelco easiGlow (Ted Pella Inc) instrument.…”