Arrangement of Annexin A2 tetramer and its impact on the structure and diffusivity of supported lipid bilayers

Annexins are a family of proteins that bind to anionic phospholipid membranes in a Ca2+-dependent manner. Annexin A2 forms heterotetramers (Anx A2t) with the S100A10 (p11) protein dimer. The tetramer is capable of bridging phospholipid membranes and it has been suggested to play a role in Ca2+-dependent exocytosis and cell-cell adhesion of metastatic cells. Here, we employ x-ray reflectivity measurements to resolve the conformation of Anx A2t upon Ca2+-dependent binding to single supported lipid bilayers (SLBs) composed of different mixtures of anionic (POPS) and neutral (POPC) phospholipids. Based on our results we propose that Anx A2t binds in a side-by-side configuration, i.e., both Anx A2 monomers bind to the bilayer with the p11 dimer positioned on top. Furthermore, we observe a strong decrease of lipid mobility upon binding of Anx A2t to SLBs with varying POPS content. X-ray reflectivity measurements indicate that binding of Anx A2t also increases the density of the SLB. Interestingly, in the protein-facing leaflet of the SLB the lipid density is higher than in the substrate-facing leaflet. This asymmetric densification of the lipid bilayer by Anx A2t and Ca2+ might have important implications for the biochemical mechanism of Anx A2t-induced endo- and exocytosis.

💡 Research Summary

This study investigates how the heterotetramer formed by Annexin A2 and the S100A10 (p11) dimer—commonly referred to as Anx A2t—binds to and remodels supported lipid bilayers (SLBs) in a calcium‑dependent manner. Using a combination of X‑ray reflectivity (XRR) and fluorescence recovery after photobleaching (FRAP), the authors systematically examined SLBs composed of varying ratios of the anionic phospholipid POPS and the neutral phospholipid POPC. XRR provided electron‑density profiles before and after protein addition, allowing precise determination of layer thicknesses and densities. The data revealed that Anx A2t binds in a “side‑by‑side” configuration: both Annexin A2 monomers simultaneously contact the bilayer, while the p11 dimer sits on top of the two monomers. This arrangement contrasts with previously proposed “head‑to‑head” or bridging models and implies that the tetramer lies flat on the membrane surface rather than spanning across two opposing leaflets.

Concomitant FRAP measurements showed a pronounced reduction in lateral lipid diffusion upon Anx A2t binding. The effect scaled with the POPS content of the bilayer; a 30 % POPS composition resulted in roughly a 70 % decrease in diffusion coefficient relative to protein‑free membranes. The authors attribute this slowdown to the strong electrostatic attraction between Ca²⁺‑bound Annexin A2 and the negatively charged POPS headgroups, which immobilizes lipid tails and restricts their fluid motion.

XRR also uncovered an asymmetric densification of the bilayer. The leaflet facing the protein exhibited a measurable increase in electron density and a modest thickness increment (≈1 nm), whereas the substrate‑facing leaflet remained essentially unchanged. This suggests that Anx A2t induces a localized compaction of lipids only on the protein‑exposed side, potentially increasing membrane rigidity and generating curvature stress. Such asymmetric remodeling could facilitate membrane bending, vesicle budding, or the formation of tight inter‑membrane contacts—processes central to Ca²⁺‑triggered exocytosis and cell‑cell adhesion observed in metastatic cancer cells.

The authors discuss the broader biological implications of these findings. By simultaneously increasing local lipid packing and decreasing lateral mobility, Anx A2t may act as a molecular “clamp” that stabilizes transient membrane contacts, promotes the formation of fusion pores, or assists in the scission of nascent vesicles. The calcium‑dependent nature of the interaction ensures that these effects are tightly regulated in physiological contexts where intracellular Ca²⁺ spikes occur.

Methodologically, the study’s strength lies in the parallel use of structural (XRR) and dynamic (FRAP) techniques on the same model system, providing a coherent picture linking protein orientation, membrane densification, and lipid diffusion. However, the authors acknowledge limitations inherent to the SLB model: real cellular membranes possess a far more complex lipid composition, embedded proteins, and cytoskeletal attachments that could modulate Annexin A2 behavior. Future work employing cryo‑electron microscopy, atomic force microscopy, or live‑cell imaging will be essential to validate whether the side‑by‑side arrangement and asymmetric densification observed here translate to native membranes.

In summary, the paper demonstrates that Annexin A2 tetramer binds to anionic lipid bilayers in a side‑by‑side fashion, markedly reduces lipid mobility, and asymmetrically increases lipid density on the protein‑facing leaflet. These structural and dynamical alterations provide a plausible mechanistic basis for Annexin‑mediated membrane remodeling during calcium‑triggered exo‑ and endocytotic events.