Ogbu, C. P. | Search bioRxiv

bioRxiv2024

Biophysical Basis of Paracellular Barrier Modulation by a Pan-Claudin-Binding Molecule

Claudins are a 27-member protein family that form and fortify specialized cell contacts in endothelium and epithelium called tight junctions. Tight junctions restrict paracellular transport across tissues by forming molecular barriers between cells. Claudin-binding molecules thus hold promise for modulating tight junction permeability to deliver drugs or as therapeutics to treat tight junction-linked disease. The development of claudin-binding molecules, however, is hindered by their intractability and small targetable surfaces. Here, we determine that a synthetic antibody fragment (sFab) we developed binds directly to 10 claudin subtypes with nanomolar affinity by targeting claudins paracellular-exposed surface. Application of this sFab to cells that model intestinal epithelium show that it opens the paracellular barrier comparable to a known, but application limited, tight junction modulator. This novel pan-claudin-binding molecule can probe claudin or tight junction structure and holds potential as a broad modulator of tight junction permeability for basic or translational applications.

biophysics↗

bioRxiv2023

Cryo-EM structures of a synthetic antibody against 22 kDa claudin-4 reveal its complex with Clostridium perfringens enterotoxin

Claudins are a family of [~]25 kDa membrane proteins that integrate into tight junctions to form molecular barriers at the paracellular spaces between endothelial and epithelial cells. Humans have 27 subtypes, which homo- and hetero-oligomerize to impart distinct properties and physiological functions to tissues and organs. As the structural and functional backbone of tight junctions, claudins are attractive targets for therapeutics capable of modulating tissue permeability to deliver drugs or treat disease. However, structures of claudins are limited due to their small sizes and physicochemical properties--these traits also make therapy development a challenge. We have developed a synthetic antibody fragment (sFab) that binds human claudin-4 and used it to resolve structures of its complex with Clostridium perfringens enterotoxin (CpE) using cryogenic electron microscopy (cryo-EM). The resolution of the structures reveals the architectures of 22 kDa claudin-4, the 14 kDa C-terminal domain of CpE, and the mechanism by which this sFab binds claudins. Further, we elucidate the biochemical and biophysical bases of sFab binding and demonstrate that this molecule exhibits subtype-selectivity by assaying homologous claudins. Our results provide a framework for developing sFabs against hard-to-target claudins and establishes the utility of sFabs as fiducial markers for determining cryo-EM structures of this small membrane protein family at resolutions that surpass X-ray crystallography. Taken together, this work highlights the ability of sFabs to elucidate claudin structure and function and posits their potential as therapeutics for modulating tight junctions by targeting specific claudin subtypes.

biophysics↗