Capturing the Dynamic Conformational Changes of Human Isocitrate Dehydrogenase 1 (IDH1) upon Ligand and Metal Binding Using Hydrogen-Deuterium Exchange Mass Spectrometry.

Human isocitrate dehydrogenase 1 (IDH1) is a highly conserved metabolic enzyme that catalyzes the interconversion of isocitrate and α-ketoglutarate. Kinetic and structural studies with IDH1 have revealed evidence of striking conformational changes that occur upon binding of its substrates, isocitrate and NADP+, and its catalytic metal cation. Here, we used hydrogen-deuterium exchange mass spectrometry (HDX-MS) to build a comprehensive map of the dynamic conformational changes experienced by IDH1 upon ligand binding. IDH1 proved well-suited for HDX-MS analysis, allowing us to capture profound changes in solvent accessibility at substrate binding sites and at a known regulatory region, as well as at more distant local subdomains that appear to support closure of this protein into its active conformation. HDX-MS analysis suggested that IDH1 is primarily purified with NADP(H) bound in the absence of its metal cation. Subsequent metal cation binding, even in the absence of isocitrate, was critical for driving large conformational changes. WT IDH1 folded into its fully closed conformation only when the full complement of substrates and metal was present. Finally, we show evidence supporting a previously hypothesized partially open conformation that forms prior to the catalytically active state, and we propose this conformation is driven by isocitrate binding in the absence of metal.

Active site remodeling in tumor-relevant IDH1 mutants drives distinct kinetic features and potential resistance mechanisms.

Mutations in human isocitrate dehydrogenase 1 (IDH1) drive tumor formation in a variety of cancers by replacing its conventional activity with a neomorphic activity that generates an oncometabolite. Little is understood of the mechanistic differences among tumor-driving IDH1 mutants. We previously reported that the R132Q mutant unusually preserves conventional activity while catalyzing robust oncometabolite production, allowing an opportunity to compare these reaction mechanisms within a single active site. Here, we employ static and dynamic structural methods and observe that, compared to R132H, the R132Q active site adopts a conformation primed for catalysis with optimized substrate binding and hydride transfer to drive improved conventional and neomorphic activity over R132H. This active site remodeling reveals a possible mechanism of resistance to selective mutant IDH1 therapeutic inhibitors. This work enhances our understanding of fundamental IDH1 mechanisms while pinpointing regions for improving inhibitor selectivity.

Active site remodeling in tumor-relevant IDH1 mutants drives distinct kinetic features and potential resistance mechanisms.

Mutations in human isocitrate dehydrogenase 1 (IDH1) drive tumor formation in a variety of cancers by replacing its conventional activity with a neomorphic activity that generates an oncometabolite. Little is understood of the mechanistic differences among tumor-driving IDH1 mutants. We previously reported that the R132Q mutant uniquely preserves conventional activity while catalyzing robust oncometabolite production, allowing an opportunity to compare these reaction mechanisms within a single active site. Here, we employed static and dynamic structural methods and found that, compared to R132H, the R132Q active site adopted a conformation primed for catalysis with optimized substrate binding and hydride transfer to drive improved conventional and neomorphic activity over R132H. This active site remodeling revealed a possible mechanism of resistance to selective mutant IDH1 therapeutic inhibitors. This work enhances our understanding of fundamental IDH1 mechanisms while pinpointing regions for improving inhibitor selectivity.

Active site remodeling in tumor-relevant IDH1 mutants drives distinct kinetic features and potential resistance mechanisms.

Mutations in human isocitrate dehydrogenase 1 (IDH1) drive tumor formation in a variety of cancers by replacing its conventional activity with a neomorphic activity that generates an oncometabolite. Little is understood of the mechanistic differences among tumor-driving IDH1 mutants. We previously reported that the R132Q mutant uniquely preserves conventional activity while catalyzing robust oncometabolite production, allowing an opportunity to compare these reaction mechanisms within a single active site. Here, we employed static and dynamic structural methods and found that, compared to R132H, the R132Q active site adopted a conformation primed for catalysis with optimized substrate binding and hydride transfer to drive improved conventional and neomorphic activity over R132H. This active site remodeling revealed a possible mechanism of resistance to selective mutant IDH1 therapeutic inhibitors. This work enhances our understanding of fundamental IDH1 mechanisms while pinpointing regions for improving inhibitor selectivity.