Imamoto, A. | Search bioRxiv

bioRxiv2026

Trans-Allosteric Activation Releases Distinct Conformational Traps in Kinase Heterodimers

Protein kinases function as dynamic, mechanically coupled nodes, yet the conformational drivers of multimeric activation remain unclear. Here, we present AlloQuant, a computational suite that translates AlphaFold3 structural ensembles into quantitative metrics of kinase regulation, including internal network rigidity, metastable-state populations, and sub-angstrom conformational drivers. Applying AlloQuant to CDK1, we demonstrate that binding of the Cyclin B1 (CCNB1) cofactor mechanically decouples a hyper-rigid inactive kinase core, allowing activating phosphorylation (pT161) to subsequently re-impose localized tension on the catalytic machinery. Conversely, the C-terminal Src kinase (CSK) faces a distinct conformational trap. While nucleotide-free monomeric CSK spontaneously samples a pre-active geometry, ATP binding excludes the active C-In conformation in all but 1 of 225 models. We show that docking partner engagement overcomes this blockade. Autophosphorylation of SRC at the activation loop (Y419) redistributes SRC conformational states without altering bulk rigidity. This redistribution is structurally coupled to the conformational state of CSK via the regulatory spine, not the catalytic machinery. Rather than mechanically deforming CSK, SRC engagement acts by conformational selection, committing roughly a quarter of CSK molecules to a fully active state. Thus, trans-allosteric kinase activation operates by defining the accessible conformational landscape of the receiver kinase. That control is exerted through mechanical remodeling in cofactor-dependent complexes and through conformational selection in transient kinase-kinase heterodimers. These findings establish AlloQuant as a general framework for quantifying how a binding partner reshapes a kinase's conformational landscape, applicable across the kinome because it assigns landmarks by profile-HMM alignment.

biophysics↗

bioRxiv2025

Minimal Perturbation of Activation Loop Dynamics Rewires KinaseSignaling

Enzymes are central to life, with their catalytic activity often shaped by the dynamic conformations of regulatory loops. In hub enzymes such as tyrosine kinases, the activation loop critically controls substrate specificity, catalytic efficiency, and downstream signaling, shaping cellular fate. Yet, the molecular mechanisms by which loop dynamics encode these functions remain incompletely understood. Here, we used SRC kinase as a model to dissect how minimal perturbations of the activation loop reprogram kinase behavior. By generating and characterizing multiple variants, we identified a triple-deletion mutant with altered loop dynamics. Structural and biochemical analyses revealed that this variant explores distinct loop conformations and exhibits a subtle shift in substrate preference toward more acidic motifs. These fine-tuned conformational changes translated into specific cellular signaling outcomes, as demonstrated by phosphoproteomic profiling. Comparative analysis across species further showed that nature exploits similar loop remodeling strategies to modulate kinase function. Together, our findings provide a blueprint for rationally tuning kinase activity and offer a generalizable framework for rewiring signaling pathways in diverse cellular contexts.

biochemistry↗