Annexin A11 C-terminal domain reveals calcium-dependent co-binding to RNA and lipid vesicles

Intracellular RNA transport is a key process for neuronal function, yet the molecular mechanisms underlying RNA tethering to organelles remain poorly understood. Annexin A11 (ANXA11) has been proposed as a tether linking RNA granules to lysosomes, but the specific role of its C-terminal domain (Ct) has remained unclear. Here, we provide direct biochemical evidence that the ANXA11 Ct directly binds lipid vesicles and RNA simultaneously. Using a combination of pull-down assays, microscale thermophoresis, and fluorescence-based approaches, we quantified the binding affinities of the Ct for phospholipid vesicles and RNA and demonstrated that these interactions are strictly Ca²⁺-dependent. Small-angle X-ray scattering and molecular dynamics simulations reveal that Ca²⁺ induces conformational changes in the Ct, modulating its conformational heterogeneity and redistributing surface properties, thereby exposing a membrane-binding face opposite to the Ca²⁺-coordinating RNA-binding loops. Pull-down assays with neutral micelles suggested that membrane engagement requires both hydrophobic and ionic interactions. These findings challenge the classical view that RNA binding is restricted to the N-terminal domain and that the Ct engages membrane exclusively. Our data establish the Ct as a Ca²⁺-regulated domain, capable of simultaneously coordinating RNA and membrane interactions, providing a mechanistic framework for ANXA11-mediated RNA hitchhiking and intracellular RNA trafficking in neurons.