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

Abstract

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.

Keywords: Enzyme mechanism; HDX-MS; IDH1; oncometabolite; structure-function relationships.

Fig. 1.. Pre-steady-state single-turnover kinetic features of IDH1 WT, R132H, and R132Q catalysis.

NADPH formation in the conventional reaction and consumption in the neomorphic reaction was monitored over the course of a single turnover (top plot) and compared with a control experiment lacking enzyme (bottom plot, in green). Residuals (middle plot) were obtained to assess goodness of a single exponential equation fit. A) IDH1 WT, conventional reaction. B) IDH1 R132H, neomorphic reaction. C) IDH1 R132Q, conventional reaction. D) IDH1 R132Q, neomorphic reaction.

Fig. 2.. IDH1 R132Q has lower deuterium uptake than R132H in both binary and quaternary complexes.

A) Plots of deuterium uptake encompassing residues 86–123, 168–209, and 260–291 (left), with the structural features of these residues shown in cartoon (right) for IDH1 R132Q, WT, and R132H. B) Plots of deuterium uptake for residues 144–191, 217–227, 257–267, and 295–354 (left), with the structural features of IDH1 R132Q, WT, and R132H encompassing these regions are shown in cartoon (right).

Fig. 3.. Crystal structure of NADP(H)-bound IDH1 R132Q.

A) The binary R132Q:NADP(H) complex is shown with each monomer highlighted using a slight color change. B) Dimer-based alignments of R132Q:NADP(H), WT:NADP(H), and R132H:NADP(H). C) Monomer-based alignments of R132Q:NADP(H), WT:NADP(H), and R132H:NADP(H). The inset features catalytic residues Y139 and K212 (though the latter residue drives catalysis in the monomer not shown), residue R132(H/Q), and the cofactor.

Figure 4.. Crystal structure of IDH1 R132Q bound to ICT, NADP(H) and Ca²⁺ that mimics the catalytic Mg²⁺.

A) The R132Q:NADP(H):ICT:Ca²⁺ complex is shown with each monomer highlighted using a slight color change. B) Dimer-based alignments of the R132Q:NADP(H):ICT:Ca²⁺/R132Q:NADP(H):Ca²⁺ dimer (cyan); R132H:NADP(H):ICT (wheat); R132H:NADP(H):αKG:Ca²⁺ (dark purple); and WT:NADP(H):ICT:Ca²⁺ (dark green). C) Monomer-based alignments of the R132Q:NADP(H):ICT:Ca²⁺/R132Q:NADP(H):Ca²⁺ dimer (dark and light cyan) with structures described in (B). For clarity, only the catalytic residues, residue R132X, cofactor, substrates, Ca²⁺ and hinge are shown in the inset. D) Dimer-based alignments of R132Q:NADP(H), R132Q:NADP(H):ICT:Ca²⁺, and R132Q:NADP(H):αKG:Ca²⁺. E) Monomer-based alignment of the IDH1 R132Q structures described in (D) with substrates, catalytic residues, and nearby secondary structure features highlighted.

Figure 5.. Crystal structure of IDH1 R132Q bound to αKG and NADP-adducts.

In (A-C) and (F), each R132Q monomer is highlighted using a slight change in color, with a description of the ligands listed below each monomer. A) R132Q:NADP(H):αKG:Ca²⁺/R132Q:NADP-αKG:Ca²⁺ dimer. B) R132Q:NADP-αKG:Ca²⁺/R132Q:NADP(H):αKG:Ca²⁺ dimer 1. C) R132Q:NADP-αKG:Ca²⁺/R132Q:NADP(H):Ca²⁺ dimer 2. D) Dimer-based alignments of R123Q αKG-bound structures with R132H:NADP(H):αKG:Ca²⁺ , G97D:NADP(H):αKG:Ca²⁺ , and WT:NADP(H):ICT:Ca²⁺ . E) Monomer-based alignment of αKG-containing R132Q monomers with structures described in (D). F) R132Q:NADP-TCEP:Ca²⁺/R132Q:NADP-TCEP:Ca²⁺ dimer. G) Alignment of adduct-containing R132Q monomers. H) Alignment of the non-substrate containing R132Q monomers.

Fig. 6.. Deuterium uptake by IDH1 WT, R132Q, and R132H in helices bounding the substrate binding pocket.

Deuterium uptake is shown as a gradient from red (high uptake) to blue (low uptake). A) Deuterium uptake by IDH1 WT, R132Q, and R132H upon no ligand treatment. These HDX-MS data were overlaid on NADP(H)-only bound forms of WT in all three cases, as the αKG helix was disordered in the NADP(H)-only bound forms of IDH1 R132Q and R132H. B) Deuterium uptake by WT and R132Q upon treatment with NADP⁺ and ICT, and by IDH1 R132Q and R132H upon treatment with NADPH and αKG. These HDX-MS data were overlaid on WT:NADP(H):ICT:Ca²⁺ , R132Q:NADP(H):ICT:Ca²⁺ and R132Q:NADP(H):αKG:Ca²⁺, or R132H:NADP(H): αKG:Ca²⁺ . C) Deuterium uptake by IDH1 WT and R132Q upon treatment with NADP⁺, ICT, and Ca²⁺, and by IDH1 R132Q and R132H upon treatment with NADPH, αKG, and Ca²⁺. These HDX-MS data were overlaid on the structures described in (B).

Fig. 7.. Hydrogen bond network facilitates a “seatbelt” that overlays NADP(H) in only some quaternary structures of IDH1.

A) Unlike the binary structure of IDH1 WT and quaternary structure of G97D:NADP(H):αKG:Ca²⁺ , the quaternary IDH1 WT complex forms a seatbelt over the NADP(H). B) Binary R132Q:NADP(H) and quaternary R132Q:NADP(H):αKG:Ca²⁺ structures do not form a seatbelt, while R132Q:NADP(H):ICT:Ca²⁺ and the most closed conformation of R132Q:NADP-αKG:Ca²⁺ for a seatbelt. C) No seatbelt is formed in the binary R132H:NADP(H), ternary R132H:NADP(H):ICT, or quaternary R132H:NADP(H):αKG:Ca²⁺ structures of IDH1 R132H^,.

Fig. 8.. Conformations and solvent accessibility of IDH1 WT, R132Q, and R132H upon substrate binding.

Helices displaying profound differences in alignment of the three forms of IDH1 are highlighted. The seatbelt feature is indicated on the α11 and α9 helices. A) Binary WT:NADP(H) collapses to a closed conformation upon ICT binding, though moderate levels of deuterium exchange are still permitted. B) Binary R132Q:NADP(H) collapses to a closed conformation upon ICT binding, showing improved catalytic efficiency for the conventional reaction and lower deuterium uptake compared to R132H. However, catalytic activity is much lower compared to WT. C) Binary R132H:NADP(H) collapses to a fully closed conformation only upon αKG binding, but a seatbelt is not formed and deuterium uptake remains high. D) Binary R132Q:NADP(H) forms semi-closed and closed conformations upon binding αKG and NADP-αKG, respectively, with a seatbelt successfully formed in the closed state. The αKG binding site was shifted away from the α9 helix, though catalytic activity was much higher than that seen in R132H.