Molecular mechanisms of “off-on switch” of activities of human IDH1 by tumor-associated mutation R132H

Abstract

Human cytosolic NADP-IDH (IDH1) has recently been found to be involved in tumorigenesis. Notably, the tumorderived IDH1 mutations identified so far mainly occur at Arg132, and mutation R132H is the most prevalent one. This mutation impairs the oxidative IDH activity of the enzyme, but renders a new reduction function of converting α-ketoglutarate (αKG) to 2-hydroxyglutarate. Here, we report the structures of the R132H mutant IDH1 with and without isocitrate (ICT) bound. The structural data together with mutagenesis and biochemical data reveal a previously undefined initial ICT-binding state and demonstrate that IDH activity requires a conformational change to a closed pre-transition state. Arg132 plays multiple functional roles in the catalytic reaction; in particular, the R132H mutation hinders the conformational changes from the initial ICT-binding state to the pre-transition state, leading to the impairment of the IDH activity. Our results describe for the first time that there is an intermediate conformation that corresponds to an initial ICT-binding state and that the R132H mutation can trap the enzyme in this conformation, therefore shedding light on the molecular mechanism of the "off switch" of the potentially tumor-suppressive IDH activity. Furthermore, we proved the necessity of Tyr139 for the gained αKG reduction activity and propose that Tyr139 may play a vital role by compensating the increased negative charge on the C2 atom of αKG during the transfer of a hydride anion from NADPH to αKG, which provides new insights into the mechanism of the "on switch" of the hypothetically oncogenic reduction activity of IDH1 by this mutation.

AG-221, a First-in-Class Therapy Targeting Acute Myeloid Leukemia Harboring Oncogenic IDH2 Mutations

“…Comparing the quaternary complexes of IDH2 R140Q ·AG-221 and IDH2 R140Q ·αKG suggested that AG-221 allosterically stabilizes the open homodimer conformation, preventing the conformational change required for catalysis, consistent with the mode of inhibition documented for IDH1 R132H mutants (17,33).…”

Cancer-associated Isocitrate Dehydrogenase Mutations Inactivate NADPH-dependent Reductive Carboxylation

“…The IDH1(R132H) homodimers were insensitive to the presence of ICT. The fact that the maximal inhibition of NADPH consumption by NADP ϩ in the IDH1 WT/R132H heterodimers was greater than the maximal ICT inhibition was consistent with NADP ϩ binding converting IDH1 to an open inactive conformation that is overcome only by ICT (26,27). ICT was initially absent in the NADP ϩ titration in Fig.…”

“…This is due to the fact that IDH1 has a higher affinity for NADP ϩ and ICT than for NADPH and ␣KG. However, the ability of NADP ϩ to convert IDH1 into an inactive conformation that can be released only by ICT (26,27) also contributes to the potent regulation of IDH1 by NADP ϩ . These data suggest that reverse flow through IDH1 is promoted by an intracellular reducing environment (high NAD(P)H/NAD(P) ϩ ratio) and low production of citrate by the mitochondria.…”

Mutational patterns in oncogenes and tumour suppressors

“…Recent structural work suggests that the R132H IDH1 mutation hampers the conformational change from the initial isocitrate binding state to the pre-transition state, thus causing an impairment of enzyme function [43]. This alters the progression of this reaction causing the oncometabolite R(-)-2-hydroxyglutarate to be formed.…”