Phosphorylation mutants elucidate the mechanism of annexin IV-mediated membrane aggregation

Site-directed mutagenesis, electron microscopy, and X-ray crystallography w ere used to probe the structural basis of annexin IV-induced membrane aggre gation and the inhibition of this property by protein kinase C phosphorylat ion. Site-directed mutants that either mimic (Thr6Asp, T6D) or prevent (Thr 6Ala, T6A) phosphorylation of threonine 6 were produced for these studies a nd compared with wildtype annexin IV. In vitro assays showed that unmodifie d wild-type annexin IV and the T6A mutant, but not PKC-phosphorylated wild- type or the T6D mutant, promote vesicle aggregation. Electron crystallograp hic data of wild-type and T6D annexin IV revealed that, similar to annexin V, the annexin IV proteins form 2D trimer-based ordered arrays on phospholi pid monolayers. Cryo-electron microscopic images of junctions formed betwee n lipid vesicles in the presence of wild-type annexin IV indicated a separa tion distance corresponding to the thickness of two layers of membrane-boun d annexin IV. In this orientation, a single layer of WT annexin IV, attache d to the outer leaflet of one vesicle, would undergo face-to-face self-asso ciation with the annexin layer of a second vesicle. The 2.0-Angstrom resolu tion crystal structure of the T6D mutant showed that the mutation causes re lease of the N-terminal tail from the protein core. This change would precl ude the face to-face annexin self-association required to aggregate vesicle s. The data suggest that reversible complex formation through phosphorylati on and dephosphorylation could occur in vivo and play a role in the regulat ion of vesicle trafficking following changes in physiological states.