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 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.

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). Traces represent an average of four technical replicates. Residuals (middle plot) were obtained to assess the goodness of a single exponential equation fit in the top plots. Kinetic parameters were calculated and reported as +/−SEM resulting from deviation of the mathematical 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 86–120, 168–182, and 269–291 (left) are shown with the structural features of these residues shown in cartoon (right) for IDH1 R132Q, WT, and R132H. B Plots of deuterium uptake for residues 168–191, 217–227, 257–267, and 305–354 (left) are shown, with the structural features of IDH1 R132Q, WT, and R132H encompassing these regions indicated in cartoon (right). Each point represents the mean of three technical replicates.

Fig. 3. Crystal structure of NADP(H)-bound IDH1 R132Q shows a typical open conformation.

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) (red), WT:NADP(H) (black), and R132H:NADP(H) (light green). C Monomer-based alignments of the structures in (B). D The view show in (C) was simplified to highlight catalytic residues Y139 and K212 (though the latter residue drives catalysis in the monomer not shown as this is a monomer-based alignment), residue R132(H/Q), and the cofactor.

Fig. 4. IDH1 R132Q bound to ICT, NADP(H) and Ca²⁺ is in a closed, catalytically competent conformation.

A The R132Q:NADP(H):ICT:Ca²⁺ complex is shown with each monomer highlighted using a slight color change. B Monomer-based alignments of R132Q:NADP(H):ICT:Ca²⁺/R132Q:NADP(H):Ca²⁺ monomers (dark and light cyan) with WT:NADP(H):ICT:Ca²⁺ (dark green); R132H:NADP(H):ICT (wheat); and R132H:NADP(H):αKG:Ca²⁺ (dark purple). C For clarity, only the catalytic residues, residue R132X, cofactor, substrates, Ca²⁺ and hinge are shown in the same orientation for the structures shown in (B).

Fig. 5. Crystal structure of IDH1 R132Q bound to αKG and NADP-adducts are in closed and semi-closed conformations.

In (A–C) and (E), a description of the ligands present is listed below each monomer. A R132Q:NADP(H):αKG:Ca²⁺/R132Q:NADP-αKG:Ca²⁺ dimer. Each R132Q monomer is highlighted using a slight change in color. B R132Q:NADP-αKG:Ca²⁺/ R132Q:NADP(H):αKG:Ca²⁺ dimer 1 (yellow) aligned with the dimer shown in (A) (magenta). C R132Q:NADP-αKG:Ca²⁺/R132Q:NADP(H):Ca²⁺ dimer 2 (orange) aligned with the dimer shown in (A) (magenta). D Monomer-based alignment of ICT- and αKG-containing R132Q monomers. E R132Q:NADP-TCEP:Ca²⁺/R132Q:NADP-TCEP:Ca²⁺ dimer. F Monomer-based alignment of adduct-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⁺ form 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 in some of our crystallographic snapshots. The αKG binding site was shifted away from the α9 helix, though catalytic activity was much higher than that seen in R132H.

Fig. 9. Possible mechanisms of IDH1 R132Q selective mutant IDH1 inhibitor resistance.

We have reported previously that IDH1 R132Q binds selective mutant IDH1 inhibitors poorly. A A previously solved structure of a selective IDH1 R132H inhibitor (6O2Y) was aligned to WT, R132H, and R132Q binary complexes. In (B–E), residues A111-V121 are shown as a surface. B The structure of the inhibitor bound to a R132H:NADP(H) complex. Residues A111-V121 in R132Q:NADP(H) (C) and in WT:NADP(H) (D) obstruct the inhibitor binding pocket. E The inhibitor could be accommodated in the structure of R132H:NADP(H).