Intrinsic Disorder and Liquid-Liquid Phase Separation Potential of the Human Acrosomal Proteome

Intrinsic disorder is attributed to protein regions that lack a fixed three-dimensional structure under physiological conditions, enabling conformational plasticity. This flexibility allows for diverse functions, including transient interactions, signaling, and phase separation via disorder-to-order transitions upon binding. The acrosome, a sperm-specific organelle essential for fertilization, has a number of proteins whose functions may rely on intrinsic disorder. We analyzed 250 human acrosomal proteins to understand the roles of intrinsic disorder and liquid-liquid phase separation (LLPS) in their functional diversity. Intrinsic disorder was predicted using multiple computational models, which yielded 97 proteins that exhibited high disorder levels (>30%). Functional enrichment analysis then identified associations between disordered regions overlapping with SCOP domains and critical acrosomal processes, including vesicle trafficking, membrane fusion, and enzymatic activation. Examples of disordered SCOP domains include the PLC-like phosphodiesterase domain, the t-SNARE domain, and P-domain of calnexin/calreticulin. Next, protein-protein interaction networks revealed acrosomal proteins as hubs in tightly interconnected systems, emphasizing their functional importance. The acrosomal proteome was also analyzed for phase separation propensity, revealing that over 30% of these proteins are high-probability LLPS drivers (>60%), underscoring their role in dynamic compartmentalization. Proteins such as myristoylated alanine-rich C-kinase substrate (MARCKS) and nuclear transition protein 2 (TNP2) exhibited both high LLPS propensities and high levels of structural disorder. A significant relationship (p < 0.0001, R² = 0.649) was observed between level of intrinsic disorder and LLPS propensity, showing the role of disorder in facilitating phase separation. Overall, these findings provide insights into how intrinsic disorder and LLPS contribute to the structural adaptability and functional precision required for fertilization, with implications for understanding disorders associated with the human acrosome reaction.