Mechanism of annexin I-mediated membrane aggregation

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 inv estigated the properties of the inducible secondary interaction site implic ated in membrane aggregation. X-ray specular reflectivity measurements show ed that the thickness of annexin I layer bound to the phospholipid monolaye r was 31 +/- 2 Angstrom, indicating that annexin I binds membranes as a pro tein monomer or monolayer. Surface plasmon resonance measurements of annexi n I, V, and mutants, which allowed evaluation of membrane aggregation activ ity of annexin I separately from its membrane binding, revealed direct corr elation 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-li nking of membrane-bound annexin I showed that a significant degree of later al association of annexin I molecules precedes its membrane aggregation. Ta ken together, these results support a hypothetical model of annexin I-media ted membrane aggregation, in which a laterally aggregated monolayer of memb rane-bound annexin I directly interacts with a secondary membrane via its i nduced hydrophobic interaction site.