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 marks neurodegenerative disease. The intrinsically disordered, prion-like C-terminal domain (CTD) drives liquid-liquid phase separation (LLPS). Using residue-level coarse-grained simulations, we systematically examine 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. Both crowder types preserve correlations between single-chain compaction, dimerization propensity, and macroscopic phase separation, yet act through different mechanisms: repulsive crowders promote condensation via excluded-volume entropic stabilization, whereas attractive crowders modulate assembly through competitive enthalpic interactions. Region-specific spatial and orientation analyses reveal a robust internal architecture in which α-helices are enriched in the condensate interior (aligned roughly parallel to the interface) while intrinsically disordered regions (IDRs) populate the surface with broader, outward-facing orientations. Beyond these baseline trends, our region-specific orientation maps and contact-relaxation metrics show that the character of crowding actively resculpts condensate organization: repulsive crowders compact and centralize the helical network into a single dense core layer, whereas attractive crowders redistribute helices toward the interface by sequestering contacts. This establishes a structure-dynamics decoupling with strong but short-lived helix-helix contacts versus weaker yet more persistent IDR-IDR contacts, reconciling core stabilization with interfacial fluidity. Our results define crowding class as a tunable control knob for region-specific redistribution and dynamics, suggesting testable readouts and offering mechanistic links to physiological modulators such as RNA stoichiometry, ATP levels, chaperone engagement, and client size/permeability. Collectively, our findings 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.