Investigating Membrane and Actin Interactions of Annexin A2/A2t

Abstract

Annexin A2 (A2) and its heterotetrameric form, Annexin A2t (A2t), participate in membrane organization, anionic phospholipid clustering, exocytosis, and other membrane-associated cellular processes. Both proteins selectively associate with membranes containing negatively charged phospholipids, particularly phosphatidylinositol 4,5-bisphosphate (PIP2). Cholesterol (CHOL) has also been proposed to influence these interactions, although its role in regulating A2- and A2t-mediated membrane binding and lipid-domain formation has not been fully characterized. Not only are these interactions important for cellular processes, but also they are implicated in various pathologies and cancers. The goal of this study was to investigate A2 and A2t–membrane interactions over extended simulation timescales and to determine how variations in membrane CHOL content affected protein binding, PIP2 clustering, lipid mobility, preferred binding orientations, and interaction energetics. Coarse-grained conventional and umbrella sampling molecular dynamics simulations were conducted using PIP2-containing membranes with varying CHOL %. The A2 simulations demonstrated that increasing membrane CHOL content strengthened protein–membrane association. Lipid-specific distance and linear interaction-energy analyses confirmed that PIP2 was the membrane component most directly involved in A2 binding, as expected from previous studies. The A2–PIP2 interaction also became increasingly favorable as CHOL concentration increased. CHOL itself was located deeper within the membrane and did not form extensive direct contacts with A2, suggesting that it enhanced protein binding indirectly by modifying membrane structure, lipid organization, and lipid mobility. Residue-level distance analysis identified several regions along the convex membrane-binding surface of A2 that closely approached PIP2. Residues 26–30, 59–76, 183, 218–230, and 260–267 were consistently near PIP2 lipids, with K27, K60, S72, A76, R225, K261, R264, and D265 exhibiting particularly short distances. The close association of K261 and R264 was consistent with their location within the previously identified PIP-binding region of A2. A2 also promoted the aggregation of PIP2 lipids beneath the protein, as seen in PyMOL visualizations, PIP2 self-distance heat maps, and self-contact analyses, and this action was increased with increased CHOL %. Membrane-only simulations provided insight into the mechanism underlying this effect. Lipid diffusion coefficients decreased for all lipid species as CHOL concentration increased, indicating that CHOL reduced membrane fluidity and lipid mobility. This reduction in diffusion limited the ability of PIP2 lipids to move away from A2, thereby stabilizing the PIP2-rich lipid microdomains formed beneath the protein. Consistent with these observations, umbrella-sampling simulations showed that the CHOL 21% membrane produced a shorter optimal binding distance and a more energetically favorable A2–membrane interaction than the CHOL 0% and 12% membranes. The A2t simulations revealed a similar CHOL-dependent enhancement of membrane binding and PIP2 clustering. Across all starting orientations, A2t tended to adopt a membrane-bound configuration in which both A2 subunits interacted with the membrane. This indicated that simultaneous membrane association of both subunits was the preferred overall binding mode. Increasing CHOL content generally increased interaction between A2t and the membrane. The results supported a model in which A2 and A2t selectively bound PIP2-containing membranes through the convex surfaces of their A2 subunits and promoted the formation of PIP2-rich lipid microdomains beneath the proteins. Increasing CHOL % in the membrane reduced lipid diffusion, stabilized PIP2 clustering, increased protein–PIP2 contacts, decreased protein–membrane distances, and produced more energetically favorable interactions. These and further systems that look into the more complex roles of A2 and A2t in exocytosis, membrane reorganization, and as an actin-binding-protein can provide a more in-depth understanding of the interactions throughout the body that our cells rely on and that can cause the metastasis of various cancers.