Annexin XII E105K crystal structure: identification of a pH-dependent switch for mutant hexamerization.
Annexins are a family of calcium- and phospholipid-binding proteins involved with numerous cellular processes including membrane fusion, ion channel activity, and heterocomplex formation with other proteins. The annexin XII (ANXB12) crystal structure presented evidence that calcium mediates the formation of a hexamer through a novel intermolecular calcium-binding site [Luecke et al. (1995) Nature 378, 512-515]. In an attempt to disrupt hexamerization, we mutated a conserved key ligand in the intermolecular calcium-binding site, Glu105, to lysine. Despite its occurrence in a new spacegroup, the 1.93 A resolution structure reveals a hexamer with the Lys105 epsilon-amino group nearly superimposable with the original intermolecular calcium position. Our analysis shows that the mutation is directly involved in stabilizing the hexamer. The local residues are reoriented to retain affinity between the two trimers via a pH-dependent switch residue, Glu76, which is now protonated, allowing it to form tandem hydrogen bonds with the backbone carbonyl and nitrogen atoms of Thr103 located across the trimer interface. The loss of the intermolecular calcium-binding site is recuperated by extensive hydrogen bonding favoring hexamer stabilization. The presence of this mutant structure provides further evidence for hexameric annexin XII, and possible in vivo roles are discussed.
Cartailler
J P
JP
Department of Molecular Biology & Biochemistry, Program in Macromolecular Structure, and Department of Physiology and Biophysics, University of California, Irvine, California 92697-3900, USA.
Haigler
H T
HT
Luecke
H
H
D000818:Animals; D017302:Annexins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D017124:Conserved Sequence; D003460:Crystallization; D018698:Glutamic Acid / Q000235:genetics*; D006829:Hydra; D006863:Hydrogen-Ion Concentration; D008239:Lysine / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D055672:Static Electricity
D017302:Annexins; D018698:Glutamic Acid; D008239:Lysine; D002118:Calcium