Mechanism of Annexin I-Mediated Membrane Aggregation

Abstract

It has been proposed that annexin I has two separate interaction sites that are involved in membrane binding and aggregation, respectively. To better understand the mechanism of annexin I-mediated membrane aggregation, we investigated the properties of the inducible secondary interaction site implicated in membrane aggregation. X-ray specular reflectivity measurements showed that the thickness of annexin I layer bound to the phospholipid monolayer was 31 ( 2 Å, indicating that annexin I binds membranes as a protein monomer or monolayer. Surface plasmon resonance measurements of annexin I, V, and mutants, which allowed evaluation of membrane aggregation activity of annexin I separately from its membrane binding, revealed direct correlation between the relative membrane aggregation activity and the relative affinity of the secondary interaction site for the secondary membrane. The secondary binding was driven primarily by hydrophobic interactions, unlike calcium-mediated electrostatic primary membrane binding. Chemical cross-linking of membrane-bound annexin I showed that a significant degree of lateral association of annexin I molecules precedes its membrane aggregation. Taken together, these results support a hypothetical model of annexin I-mediated membrane aggregation, in which a laterally aggregated monolayer of membrane-bound annexin I directly interacts with a secondary membrane via its induced hydrophobic interaction site.

The N-terminal Domain of Annexin 2 Serves as a Secondary Binding Site during Membrane Bridging

“…These N-terminal interactions stabilize a complex between two AnxA2 molecules that form the Ca 2ϩ -mediated membrane bridges, consistent with previous observations by cryo-electron microscopy (11) and with our aggregation experiments in the present study. Our model for H ϩ -dependent membrane bridging, consistent with cryo-electron microscopy studies (26), proposes that these bridges are formed with only one AnxA2 molecule located between two juxtaposed membranes, with the core interacting with one membrane surface on its convex side, and the N-terminal domain interacting with the second membrane surface on the concave side, as previously suggested for AnxA1 (22,43) (Fig. 7B).…”

“…Direct interactions of the N terminus with the membrane, as previously described for AnxA1 (22,(43)(44)(45) may underlie these observations. To determine the extent of AnxA2 acryl -membrane interactions, quenching experiments were performed with LUV (PC/PS 75/25) containing n-doxyl PC at different mol fractions (10, 20, and 30) (40,46).…”

“…Other factors such as ionic strength or temperature may be involved in determining the organization of Anx membrane bridges. In both cases, core-core contacts may contribute to the stabilization of the structure as shown by cross-linking of AnxA1 (43) and AnxA2 (53). Two possible orientations of the AnxA2 core are schematized.…”

A Calcium-driven Conformational Switch of the N-terminal and Core Domains of Annexin A1

“…Interestingly, this secondary binding site exhibits neither calcium dependency nor lipid specificity. 9,13,[18][19][20] These data suggest that the N-terminal domain of annexin 1 alone might provide a second membrane-binding site. This hypothesis is supported by a publication from Lee and co-workers in which they present data that a peptide comprising residues 1-26 of annexin 1 alone is able to form an α-helix in 50% trifluorethanol (TFE)/water and 10 mM sodium dodecyl sulfate (SDS), suggesting membrane binding activity.…”

“…This model is supported by our x-ray structure of full-length annexin 1 and by results published by other groups over the last ten years. 13,18,20,21,24 In this model, the structure of full-length annexin 1 in the absence of calcium ions reflects the starting point of the reaction cascade that eventually causes membrane aggregation. This reaction cascade would start with calcium-dependent binding of one annexin 1 monomer to a membrane containing negatively charged phospholipids.…”