Related Experiment Video
Updated: May 28, 2025
Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale
Published on: March 14, 2019
Crystal structure of the plasmid-encoded R67 dihydrofolate reductase complexed with Congo red an amyloid binding dye
Akshay N Narendra1, Elizabeth E Howell2, Narendra Narayana3
1UCHealth Parkview Medical Center, 400 West 16th street, Pueblo, CO, 81003, USA.
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.
Insights
Bacterial dihydrofolate reductase (DHFR) R67 mutant shows high resistance to Trimethoprim (TMP). Structural analysis reveals Congo Red (CGR) binding interactions, suggesting potential for developing novel inhibitors.
Area of Science:
- Biochemistry
- Structural Biology
- Drug Discovery
Background:
- Plasmid-encoded R67 dihydrofolate reductase (DHFR) confers antibiotic resistance.
- R67 DHFR is highly resistant to Trimethoprim (TMP), unlike its chromosomal counterpart.
- Understanding R67 DHFR structure-inhibitor interactions is crucial for developing new antibiotics.
Purpose of the Study:
- Determine the high-resolution crystal structure of the Q67H mutant of R67 DHFR.
- Investigate the binding mode of the non-specific inhibitor Congo Red (CGR) within the R67 DHFR active site.
- Propose strategies for designing specific and potent R67 DHFR inhibitors.
Main Methods:
- X-ray crystallography at 1.15 Å resolution.
- Complex formation between Q67H mutant R67 DHFR and Congo Red (CGR).
- Analysis of electron density maps (Fo-Fc) to visualize inhibitor binding.
- Preliminary computational modeling studies.
Main Results:
- The structure reveals one naphthalene moiety of CGR bound in the active site pore, with flexibility in other parts of the molecule.
- CGR binds asymmetrically, interacting with residues from all four protomers of the tetrameric enzyme.
- Specific interactions include ionic, van der Waals, stacking, and hydrogen bonding.
- Modeling suggests targeting Lys32 residues could lead to potent inhibitors.
Conclusions:
- The determined structure provides insights into the binding of CGR to R67 DHFR.
- The findings support the potential of CGR fragments as scaffolds for developing novel DHFR inhibitors.
- A model for CGR binding to cross-β amyloid is proposed based on observed protein-inhibitor interactions.
