Repulsive vs. Attractive Crowding Distinctly Regulate TDP-43 Condensates through Region-Specific Structural Dynamics

TAR DNA-binding protein 43 (TDP-43) is crucial for RNA processing and nucleocytoplasmic transport, and its pathological aggregation is a hallmark of neurodegenerative disorders such as amyotrophic lateral sclerosis and frontotemporal dementia. The intrinsically disordered, prion-like C-terminal domain (CTD) of TDP-43 drives its liquid-liquid phase separation (LLPS), a process fundamental to both normal cellular function and disease-associated aggregation. Using coarse-grained molecular simulations, we systematically explored how distinct macromolecular crowding environments, including repulsive (steric) and attractive (interaction-based) crowding conditions, influence the phase behavior and internal organization of TDP-43 CTD condensates. Our findings reveal that both repulsive and attractive crowders maintain strong correlations between single-chain compaction, dimerization propensity, and macroscopic phase separation, yet operate via distinct physical mechanisms: repulsive crowders drive condensation through volume-exclusion-mediated entropic stabilization, while attractive crowders modulate condensate formation via competitive enthalpic interactions. Spatial structural analysis identifies a robust region-specific internal architecture, with α-helices enriched at the condensate core and aligned parallel to the interface, and intrinsically disordered regions (IDRs) preferentially localized near the condensate surface in orientations nearly perpendicular to the interface. Dynamically, α-helical regions form strong yet transient interaction hubs, whereas IDRs establish weaker but more persistent contacts, thereby maintaining structural fluidity. Collectively, our results uncover a regulatory principle by which macromolecular crowding modulates TDP-43 condensation through distinct entropic and enthalpic contributions, offering key mechanistic insights into condensate formation and dysregulation relevant to neurodegenerative diseases.