Dihydrofolate reductase: multiple conformations and alternative modes of substrate binding

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Lactobacillus casei dihydrofolate reductase (DHFR) forms a complex with folate and NADP+.
• This complex exists in solution as a mixture of three slowly interconverting conformations.
• The relative proportions of these conformations are pH-dependent.

Purpose of the Study:

• To assign all aromatic proton resonances for folate and NADP+ in all three conformational states of the DHFR complex.
• To investigate the structural differences within the enzyme's active site across these conformations.

Main Methods:

• Nuclear Magnetic Resonance (NMR) spectroscopy, including two-dimensional exchange experiments.
• Comparison of COSY spectra of the enzyme-ligand complex at different pH values.
• Analysis of Nuclear Overhauser Effects (NOEs) to determine proximity of protons.

Main Results:

• Resonances for nicotinamide protons and the pteridine 7-proton of folate show distinct chemical shifts across the three conformations.
• Protein conformation shows only minor changes between low and high pH states, suggesting localized active site differences.
• NOEs indicate no major movement of the nicotinamide ring, but specific NOEs involving the pteridine 7-proton differ between conformations.

Conclusions:

• The active site of Lactobacillus casei DHFR undergoes localized structural rearrangements in response to pH changes.
• These conformational changes involve specific interactions with the bound folate and NADP+ cofactors.
• NMR provides detailed insights into the dynamic structural landscape of enzyme-ligand complexes.