Rationally-Stabilized Inhibited State of IMPDH Proposes a Novel Targeting Approach Involving Its Cross-Domain and...

Area of Science:

• Biochemistry
• Enzyme kinetics
• Molecular biology

Background:

• Inosine monophosphate dehydrogenase (IMPDH) is crucial for GTP biosynthesis, but its complex regulation hinders effective inhibition for diseases like retinitis pigmentosa (RP).
• RP involves mutations in IMPDH1's GTP-binding site, disrupting enzyme regulation.
• The ATP-binding site 1 on the CBS domain influences allosteric regulation of IMPDH.

Purpose of the Study:

• To investigate if targeting ATP Site 1 on the CBS domain can induce an inhibited IMPDH1 conformation.
• To validate the hypothesis that mutating ATP Site 1 can mimic allosteric inhibition.

Main Methods:

• In silico analysis of a rationally selected mutation (I157V).
• Experimental characterization using circular dichroism, fluorescence spectroscopy, and thermal denaturation.
• Comparison of mutant and wild-type IMPDH1 conformations.
• Molecular dynamics simulations.

Main Results:

• Molecular dynamics simulations showed the I157V mutation induced a conformation matching experimentally observed inactive IMPDH.
• Circular dichroism, fluorescence, and thermal denaturation data confirmed a rigid, stable, and compressed mutant conformation.
• The mutation disrupted IMPDH assembly into octamers and filaments.

Conclusions:

• Targeting ATP Site 1 is a viable strategy to mimic allosteric inhibition of IMPDH.
• This approach offers a dual suppression strategy for enzyme regulatory defects in RP.
• Designing molecules to target ATP Site 1 could be a potent therapeutic approach for RP.