PHOSPHOLIPID DETERMINANTS FOR ANNEXIN-V BINDING-SITES AND THE ROLE OF TRYPTOPHAN-187

Annexin V is part of a family of Ca2+-dependent phospholipid-binding proteins, whose purported functions are related to their interactions with biological membranes. While Ca2+-dependent binding to phospholipids is well-established, the specific structural interactions within the phospholipid-binding sites have only been inferred to resemble those of phospholipase A(2), with no direct structural evidence. In this study, the binding avidity of various phospholipid analogs, with variations at the headgroup or sn-2 acyl chain, was monitored in a C(12)E(8) detergent micelle system using the increase in fluorescence of tryptophan 187. Micelles also contained excess negative surface charge to saturate 8 nonspecific component of the binding. The Ca2+ and phospholipid concentrations required for the binding of annexin V to various phospholipid headgroups were very similar, except for the relatively weak binding to phosphatidylinositol (PI). The unique close proximity of the PI sugar ring to the phosphate group may lead to steric hindrance in this case. Binding was also strongly dependent on the presence of an sn-3 phosphate group and an sn-2 acyl chain, as previously observed. The relatively shallow nature of the annexin V phospholipid-binding sites was reflected by the nearly equivalent binding of D and L versions of phospholipids, i.e., a large shift in the position of the sn-1 acyl chain is accommodated in this case. Binding of annexin V does not specifically require an ester carbonyl oxygen, as it occurs with eher-linked, amide-linked, and phosphonate-linked sn-2 hydrocarbon chains, under these conditions. Binding to an analog without an electronegative group at the sn-2 position (hexadecylphosphocholine) was observed to be of intermediate avidity, indicating the importance of the hydrophobic nature of the sn-2 acyl chain as well as electronegative groups at the linkage position. Further support for hydrophobic interactions came from the dependence of binding on the lengths of the sn-2 acyl chains of other analogs. Consistent with such limited hydrophobic interactions is the observation that the maximal fluorescence of tryptophan 187 was greatly affected by the nature of the sn-2 acyl chain. Competitive binding between known specific ligands and a phospholipid derivative with an sn-2 quenching group further suggests that tryptophan 187 makes contact with specifically bound phospholipids, probably at the sn-2 acyl chain. Thus, hydrophobic interactions at the sn-2 acyl chain, along with interactions at the phosphate group and possibly the ester carbonyl oxygen, appear to account for the major phospholipid determinants for annexin V binding.