Hydration-Mediated Energy Landscapes Govern Rotational Flexibility in Membrane-Bound Annexin V Assemblies

Interfacial water organization and dynamics govern protein stability and function across molecular to supramolecular scales. Annexin V (AnxA5), a membrane repair protein, forms 2D assemblies on lipid membranes, yet the hydration role in repair remains unexplored. Combining three-dimensional atomic force microscopy (3D-AFM) and molecular dynamics (MD) simulations, we resolve the 3D hydration architecture of AnxA5 assemblies at molecular resolution. AnxA5 is enveloped by a continuous, nonlayered hydration network extending 1.5-2 nm into bulk solvent, exhibiting quasi-periodic lateral organization across crystalline and noncrystalline trimer domains. MD simulations indicate this network forms dynamic hydrogen-bonded bridges that may stabilize interdomain junctions, thereby modulating the local energy landscape. This hydration-dependent configurational flexibility, coupled with thermal fluctuations, drives stochastic, reversible trimer rotation, potentially modulating membrane interactions and Ca²⁺ coordination. Our findings establish interfacial water as a key mediator of supramolecular organization and stabilization, proposing a mechanism for hydration-mediated conformational flexibility during Annexin-driven membrane repair.