ABSTRACT
Isothermal titration calorimetry was used to characterize the binding of calcium ion (Ca²âº) and phospholipid to the peripheral membrane-binding protein annexin a5. The phospholipid was a binary mixture of a neutral and an acidic phospholipid, specifically phosphatidylcholine and phosphatidylserine in the form of large unilamellar vesicles. To stringently define the mode of binding, a global fit of data collected in the presence and absence of membrane concentrations exceeding protein saturation was performed. A partition function defined the contribution of all heat-evolving or heat-absorbing binding states. We find that annexin a5 binds Ca²âº in solution according to a simple independent-site model (solution-state affinity). In the presence of phosphatidylserine-containing liposomes, binding of Ca²âº differentiates into two classes of sites, both of which have higher affinity compared with the solution-state affinity. As in the solution-state scenario, the sites within each class were described with an independent-site model. Transitioning from a solution state with lower Ca²âº affinity to a membrane-associated, higher Ca²âº affinity state, results in cooperative binding. We discuss how weak membrane association of annexin a5 prior to Ca²âº influx is the basis for the cooperative response of annexin a5 toward Ca²âº, and the role of membrane organization in this response.
