Targeting a Cryptic Pocket in a Protein-Protein Contact by Disulfide-Induced Rupture of a Homodimeric Interface

Area of Science:

• Biochemistry
• Structural Biology
• Drug Discovery

Background:

• Protein-protein interfaces are key therapeutic targets, but studying monomeric states of homodimers is challenging.
• The enzyme tRNA-guanine transglycosylase (TGT) is essential for Shigellosis pathogenesis and functions as a homodimer.
• Previous attempts to crystallize monomeric TGT failed due to dimer formation during crystallization.

Purpose of the Study:

• To develop a method for accessing the monomeric state of TGT to enable drug design.
• To identify novel binding sites at the TGT dimer interface.
• To demonstrate the druggability of the TGT interface for therapeutic intervention.

Main Methods:

• Site-directed mutagenesis to introduce cysteine residues for disulfide bond formation.
• Crystallography to capture a quasi-monomeric state under oxidizing conditions.
• Fragment-based drug discovery (FBDD) via soaking and NMR spectroscopy to identify and validate binders.

Main Results:

• A disulfide linkage was formed in a TGT variant, stabilizing a quasi-monomeric state with a novel packing geometry.
• A previously buried loop-helix motif was exposed to the surface in the quasi-monomeric state.
• Fragment screening identified hits binding to a cryptic site near the exposed motif, with validated binding confirmed by NMR.

Conclusions:

• Introducing a disulfide cross-link is a viable strategy to capture quasi-monomeric states of homodimers.
• This approach provides access to previously inaccessible structural information and binding sites.
• The TGT interface is druggable, and this method facilitates the development of modulators to interfere with dimer formation for treating Shigellosis.