Annexin XII E105K crystal structure: Identification of a pH-dependent switch for mutant hexamerization

Annexins are a family of calcium- and phospholipid-binding proteins involve d with numerous cellular processes including membrane fusion, ion channel a ctivity, and heterocomplex formation with other proteins. The annexin XII ( ANXB12) crystal structure presented evidence that calcium mediates the form ation of a hexamer through a novel intermolecular calcium-binding site [Lue cke et al. (1995) Nature 378, 512-515]. In an attempt to disrupt hexameriza tion, we mutated a conserved key ligand in the intermolecular calcium-bindi ng site, Glu105, to lysine. Despite its occurrence in a new spacegroup, the 1.93 Angstrom resolution structure reveals a hexamer with the Lys105 epsil on-amino group nearly superimposable with the original intermolecular calci um position. Our analysis shows that the mutation is directly involved in s tabilizing the hexamer. The local residues are reoriented to retain affinit y between the two trimers via a pH-dependent switch residue, Glu76, which i s now protonated, allowing it to form tandem hydrogen bonds with the backbo ne carbonyl and nitrogen atoms of Thr103 located across the trimer interfac e. The loss of the intermolecular calcium-binding site is recuperated by ex tensive hydrogen bonding favoring hexamer stabilization. The presence of th is mutant structure provides further evidence for hexameric annexin XII, an d possible in vivo roles are discussed.