Phosphorylation and pocket-binding ligands rewire the coupled networks in multidomain protein condensate

<jats:p>Biomolecular condensates formed by multidomain proteins are increasingly linked to disease and are emerging targets for chemical modulation. Yet most molecular models emphasize intrinsically disordered regions (IDRs), whereas many available ligands bind folded-domain pockets. How IDR modifications and folded-domain ligands are integrated to control condensate organization remains unclear. Here, we address this problem using histone deacetylase 6 (HDAC6), a disease-associated deacetylase whose S22 phosphorylation and catalytic-pocket ligands differentially regulate condensation. Using atomistic-informed Martini 3 coarse-grained simulations, we show that S22 phosphorylation enhances phase separation of the N-terminal IDR1 without compacting isolated chains. Instead, phosphorylation creates a phospho-S22/D26-centered anionic contact node that strengthens dense-phase interactions with Arg/Lys-rich patches. The HDAC6 ligands Nexturastat A and HPOB remodel this IDR network through distinct charged-contact mechanisms: Nexturastat A acts as a focused competitor for cationic patches, whereas HPOB permits partial compensation through alternative charged contacts. In full-length phospho-HDAC6, the assembled network is not IDR1-dominated but is organized by FD3, the C-terminal ZnF-UBP domain. Pocket-bound ligands redirect this folded-domain network from FD3-FD3 association toward pocket-FD3 contacts, with stronger and more persistent remodeling by Nexturastat A. These results reveal a two-layer regulatory mechanism in which phosphorylation rewires IDR electrostatics while pocket-binding ligands redirect folded-domain interactions, suggesting a strategy for modulating multidomain condensates through ligand-induced contact-network remodeling.</jats:p>