Biology subjects

Huxford, T.

Publications and source records attributed to Huxford, T..

3 recordsLinked to original sources

bioRxiv2026

Cryo-EM reveals alternative modes of dimerization driving activation of IKK

The inhibitor of {kappa}B kinase (IKK) complex integrates diverse cellular inflammatory responses, and induces transcription factor NF-{kappa}B. The molecular mechanism by which IKK becomes catalytically active in response to signaling remains unclear despite structural knowledge of the individual IKK1/, IKK2/{beta}, and NEMO/IKK{gamma} protein components within its hetero-oligomeric assembly. Cryo-EM of the IKK2/{beta} homodimer bound to an associating NEMO/IKK{gamma} protein fragment, reveals multiple conformers. Mutual exclusivity of dimeric conformers, canonical versus alternate, is reflected in and dependent upon order-to-disorder transition of the canonical 6-helical bundle dimerization interface. Correlation of this unusual structural plasticity of IKK2/{beta} with its biochemical and cellular activities suggests mechanistic possibilities for how association with its partner scaffold protein NEMO/IKK{gamma} and polyubiquitin chains might dictate catalytic activation of IKK through distinct IKK2/{beta} conformers. SignificanceThe inhibitor of {kappa}B kinase (IKK) complex is central to inflammatory signaling via the NF-{kappa}B family transcription factors. Its activation mechanism has remained unclear. Cryo-EM analysis reveals that the constituent kinase IKK2/{beta} adopts structurally distinct, mutually exclusive dimeric conformations controlled by an ordered-to-disordered transition at its canonical dimerization interface. Stabilization of select IKK2/{beta} conformers by the scaffold protein NEMO in association with poly-ubiquitin chains is regulated through modular architecture and structural plasticity of distinctive kinase-associated domains, present only in kinases of this family. This unique regulatory mechanism governing catalytic activation of IKK2/{beta} provides a conceptual framework for targeting dysregulated NF-{kappa}B signaling in human diseases.

immunology↗

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↗