CALCIUM-DRIVEN PHASE SEPARATION AND STRUCTURAL MODULATION OF ANNEXIN A11

Annexins are a family of 12 calcium-binding proteins in vertebrates that play   essential roles in cell signaling, membrane trafficking, and repair. All annexins share a conserved C-terminal domain (annexin repeat) that mediates calcium-dependent binding to negatively charged membranes. In contrast, their N-terminal regions vary widely and are thought to determine functional specificity. Annexin A11 (AnxA11) stands out in the family due to its unusually long and intrinsically disordered N-terminal domain, which enables it to form biomolecular condensates and has been linked to various pathological conditions such as systemic autoimmune diseases and ALS. However, how calcium affects AnxA11’s structure and phase behavior remains largely unexplored. This project addresses this gap by revealing how calcium modulates both the structural organization and condensation properties of AnxA11, uncovering a potential mechanism by which the protein senses cellular calcium changes to regulate its function. Circular dichroism analysis revealed that AnxA11 undergoes significant structural changes upon calcium binding, increasing its α-helix content and its thermal stability. Microscopy studies further demonstrated that AnxA11 condensates are sensitive to the effect of molecular crowding and the salt concentration, with high salt levels inhibiting condensate formation, while molecular crowding was initially shown to be essential for phase separation. However, we observe that calcium can modulate AnxA11’s propensity to undergo phase separation: at low concentrations, it enhances condensate formation (even at low protein concentrations and in the absence of crowding agents), while at higher levels induce a dynamic and reversible liquid-to-solid phase transition. This study will further explore how ALS-associated mutations affect calcium sensitivity, structural changes, and condensate dynamics, providing novel insights into the calcium-dependent regulation of AnxA11 and its potential role in ALS pathogenesis.

This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP # 2024/10159-1) and by University of São Paulo.