High-Speed Atomic Force Microscopy Reveals Disordered Region-Mediated Structural Plasticity of Anaplastic Lymphoma Kinase Fusion Proteins Induced by Inhibitors.

EML4-ALK is a key oncogenic driver in lung cancer, but variant-specific chemoresistance limits the efficacy of current ALK inhibitors. Because the N-terminus contains an intrinsically disordered region (IDR), how ALK inhibitors affect the structure and dynamics of full-length EML4-ALK remains unclear. Here, using high-speed atomic force microscopy (HS-AFM), we visualize the overall structures of three full-length EML4-ALK variants (v1, v3, and v5) at the single-molecule level. We identified a transient globular subdomain (residues 191-217) within the v3 IDR that contributes to distinct oligomerization patterns. Notably, ALK inhibitors compact the IDR subdomain and reduce oligomerization, whereas this effect is abolished by the resistance mutation ALK, suggesting that ALK inhibitors not only inhibit kinase activity but also modulate IDR dynamics. Our study reveals a structural basis linking IDR dynamics to variant-specific oligomerization in EML4-ALK, offering further insights into the regulation of oncogenic signaling and the development of targeted therapies.