The annexin A3-membrane interaction is modulated by an N-terminal tryptophan

The crystal structure of annexin A3 (human annexin III) solved recently rev ealed a well-ordered folding of its N-terminus with the side chain of trypt ophan 5 interacting with residues at the extremity of the central pore. Sin ce the pore of annexins has been suggested as the ion pathway involved in m embrane permeabilization by these proteins, we investigated the effect of t he N-terminal tryptophan on the channel activity of annexin A3 by a compara tive study of the wild-type and the W5A mutant in structural and functional aspects. Calcium influx and patch-clamp recordings revealed that the mutan t exhibited an enhanced membrane permeabilization activity as compared to t he wild-type protein. Analysis of the phospholipid binding behavior of wild -type and mutant protein was carried out by cosedimentation with lipids and inhibition of PLA(2) activity, Both methods reveal a much stronger binding of the mutant to phospholipids. The structure is very similar for the wild -type and the mutant protein. The exchange of the tryptophan for an alanine results in a disordered N-terminal segment. Urea-induced denaturation of t he wild-type and mutant monitored by intrinsic fluorescence indicates a sep arate unfolding of the N-terminal region which occurs at lower urea concent rations than unfolding of the protein core. We therefore conclude that the N-terminal domain of annexin A3, and especially tryptophan 5, is involved i n the modulation of membrane binding and permeabilization by annexin A3.