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

Abstract

Inosine monophosphate dehydrogenase (IMPDH) catalyzes the rate-limiting step of GTP biosynthesis. The multilevel regulation of the enzyme complicates its inhibition to treat diseases such as retinitis pigmentosa (RP) where modulation of the isozyme IMPDH1 is disrupted by mutations in a GTP-binding site on its regulatory CBS domain. Here, we hypothesize that upsetting the ATP-binding site 1 on the CBS domain, which contributes to all levels of the enzyme regulation, can endow it with the conformational features characteristic of the inhibited form of IMPDH1. This concept was validated by introducing a rationally selected mutation, I157V, which was appraised in silico followed by experimental structural characterizations using circular dichroism, intrinsic and extrinsic fluorescence, and thermal denaturation of the purified mutant, comparatively with a wild type enzyme in its active extended conformation. Molecular dynamics yielded a mutant conformation matching the experimental models of inactive IMPDH in terms of various global measures, local features and energy landscapes. Consistently, CD profiles, fluorescence spectra and thermal data confirmed a rigid, compressed and stable conformation with appropriate secondary structural compositions, surface properties and overall fold, respectively. The mutation could thus mimic the allosteric inhibition of the catalytic domain by the GTP-bound CBS domain. Disrupted ATP Site 1 also suppresses the enzyme assembly to octamers and filaments. Therefore, designing molecules to target Site 1 is suggested as a potent strategy to cope with the enzyme regulatory defects in RP using the proposed dual suppression approach.