title: Calcium-dependent binding of annexin 12 to phospholipid bilayers: stoichiometry and implications.
abstract: Annexins (ANXs) are a superfamily of proteins whose functional hallmark is Ca2+-dependent binding to anionic phospholipids. Their core domains are usually composed of a 4-fold repeat of a conserved amino acid sequence, with each repeat containing a type II Ca2+ binding site that is generally thought to mediate Ca2+-dependent binding to the membrane. We now report that ANX12 binding to phospholipid vesicles is highly cooperative with respect to Ca2+ concentration (Hill constant approximately 7), thereby suggesting that more than the four well-characterized type II Ca2+ binding sites are involved in phospholipid binding. Two independent approaches, a novel 45Ca2+ copelleting assay and isothermal titration calorimetry, indicate a stoichiometry of approximately 12 mol of Ca2+/mol of ANX12 for binding to phospholipid vesicles. On the basis of the "low-affinity" Ca2+-binding sites in a number of ANX X-ray crystal structures, we propose a model for ANX12 bilayer binding that involves three types of Ca2+ sites in each of the four repeats. In this model, there is a complementarity between the spacing of the ANX12 Ca2+ binding sites and the spacing of the phospholipid headgroups in bilayers. We tested the implications of the model by manipulating the physical state of vesicles composed of phospholipids with saturated acyl chains with temperature and measuring its influence on ANX12 binding. ANX12 bound to vesicles in a Ca2+-dependent manner when the vesicles were in the liquid crystal phase but not when the phospholipid was in the gel phase. Furthermore, ANX12 bound initially to fluid bilayers remained bound when cooled to 4 degrees C, a temperature that should induce the gel phase transition. Overall, these studies suggest that ANX12 is well suited to being a Ca2+ sensor for rapid all-or-none intercellular membrane-related events.
authors.affiliation: Department of Physiology and Biophysics, University of California, Irvine, California 92697, USA.
authors.forename: D R
authors.initials: DR
authors.lastname: Patel
authors.forename: C C
authors.initials: CC
authors.lastname: Jao
authors.forename: W S
authors.initials: WS
authors.lastname: Mailliard
authors.forename: J M
authors.initials: JM
authors.lastname: Isas
authors.forename: R
authors.initials: R
authors.lastname: Langen
authors.forename: H T
authors.initials: HT
authors.lastname: Haigler
mesh_terms: D000818:Animals; D017302:Annexins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D002132:Calcium Radioisotopes / Q000378:metabolism; D002151:Calorimetry; D004134:Dimyristoylphosphatidylcholine / Q000737:chemistry; D006829:Hydra; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism; D008560:Membrane Fluidity; D008956:Models, Chemical; D008958:Models, Molecular; D010713:Phosphatidylcholines / Q000737:chemistry; D010715:Phosphatidylglycerols / Q000737:chemistry; D010743:Phospholipids / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding
chemical_list: D017302:Annexins; D002132:Calcium Radioisotopes; D008051:Lipid Bilayers; D010713:Phosphatidylcholines; D010715:Phosphatidylglycerols; D010743:Phospholipids; C069258:1-(decanoylthio)-2-decanoyl-3-phosphatidylcholine; C002773:dimyristoylphosphatidylglycerol; D002118:Calcium; D004134:Dimyristoylphosphatidylcholine