Huxford, T. | Search bioRxiv

bioRxiv2024

Active site remodeling in IDH1 mutants drives distinct kinetic 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.

biochemistry↗

bioRxiv2023

Dual-specific autophosphorylation of kinase IKK2 enables phosphorylation of substrate IkappaBalpha without requiring ATP

Rapid and high-fidelity phosphorylation of serine residues at positions 32 and 36 of I{kappa}B by IKK2, a prototypical Ser/Thr kinase, is critical for canonical NF-{kappa}B activation. Here, we report that IKK2 not only phosphorylates substrate serine residues and autophosphorylates its own activation loop, but also autophosphorylates at a tyrosine residue proximal to the active site and is, therefore, a dual-specificity kinase. We observed that mutation of Y169, an autophosphorylatable tyrosine located at the DFG+1 (DLG in IKK1) position, to phenylalanine renders IKK2 incapable of catalyzing phosphorylation at S32 within its I{kappa}B substrate. We also observed that mutation of the phylogenetically conserved ATP-contacting residue K44 in IKK2 to methionine converts IKK2 to an enzyme that no longer catalyzes specific phosphorylation of I{kappa}B at S32 or S36, but rather directs phosphorylation of I{kappa}B at other residues. Lastly, we report evidence of a phospho-relay from autophosphorylated IKK2 to I{kappa}B in the presence of ADP. These observations suggest an unusual evolution of IKK2, in which autophosphorylation of tyrosine(s) in the activation loop, and the conserved ATP-contacting K44 residue provide its signal-responsive substrate specificity and ensure fidelity during NF-{kappa}B activation.

biochemistry↗