Integrating Gaussian Accelerated Molecular Dynamics, β-Variational Autoencoders, and Markov State Models Reveals the...

Fibroblast growth factor receptor 3 (FGFR3) kinase plays a crucial role in cellular differentiation, proliferation, and skeletal development. Activation of the kinase domain through phosphorylation regulates the downstream signaling cascades, and its dysregulation leads to human dwarfism, skeletal dysplasia, and multiple cancers. However, the mechanistic basis of FGFR3 regulation by phosphorylation and ligand binding remains unclear. Here, we introduce an integrated GaMD-βVAE-MSM pipeline, a hybrid physics-ML framework for studying kinase activation. Gaussian-accelerated molecular dynamics (GaMD) enhances simulations by reducing the energy barrier. The variational autoencoder with tunable regularization was used for nonlinear dimension reduction and identifying slow collective variables. The Markov state model (MSM) was used for identifying metastable states spanning active-inactive conformations. Our results show that phosphorylation drives pronounced domain rearrangement and activation-loop dynamics. The coordinated movement of the molecular brake and regulatory spine governs the kinase activation. Ligand interaction stabilizes the active phosphorylated state with a free energy of -19 kcal/mol. Protein structure network analysis uncovers the long-range allosteric pathways. The potential regulatory sites involving αCβ4, β34, and β45 loops can be further exploited for allosteric intervention. Overall, this study highlights that phosphorylation and ligand binding dual regulate FGFR3 kinase through allosteric communication networks. The integrated GaMD-βVAE-MSM pipeline provides mechanistic insights into FGFR3 activation and may help the rational design of next-generation FGFR3-targeted therapeutics resistant to gatekeeper mutants.