# Crystal structure of the plasmid-encoded R67 dihydrofolate reductase complexed with Congo red an amyloid binding dye

## TL;DR

This study reveals the crystal structure of a drug-resistant bacterial enzyme bound to Congo red, offering insights into potential new inhibitor designs.

## Contribution

The paper presents a high-resolution crystal structure of R67 DHFR with Congo red, revealing unique binding interactions and proposing a model for amyloid binding.

## Key findings

- Congo red binds asymmetrically in the active site pore of R67 DHFR, unlike its crystallographic symmetry.
- The naphthalene moiety of Congo red interacts with residues 66–68 through multiple non-covalent interactions.
- Modeling suggests Congo red fragments could target Lys32 to create potent inhibitors for the enzyme.

## Abstract

Plasmid-encoded bacterial R67 dihydrofolate reductase (DHFR) catalyzes the same reaction as the chromosomal counterpart but is highly resistant to the widely used antibiotic Trimethoprim (TMP) unlike the chromosomal enzyme. The structure of Q67H mutant of R67 DHFR complexed with a non-specific inhibitor Congo red (CGR) has been determined at 1.15 Å resolution. In the Fo-Fc map, one of the two naphthalene moieties in CGR is clearly observed, however, the biphenyl linker and the other naphthalene moiety are not seen owing to flexibility. CGR does not utilize its twofold axis to align with any of the three crystallographic twofold axes of the tetrameric protein instead, it binds like the asymmetrical folate and NADP+ at any one of the four symmetry-related positions in the active site pore. The naphthalene moiety with exocyclic sulphonate ion and amino group, interacts with residues 66–68 from all four protomers via metal-based ionic, van der Waals, stacking, and hydrogen bonding interactions. Preliminary modeling studies suggest variant fragments of CGR targeting one or both Lys32 residues at the site of enlarging pore may yield specific and potent inhibitors. Based on the CGR – protein interactions in the present work, we propose a putative model for the binding of CGR to cross-β amyloid.