Exploring the interactome of EF-hand proteins

Abstract: EF-hand proteins are central in many cellular processes being the main group of Ca2+-sensing and buffering proteins in the cell. In the work presented in this thesis we study ligand interactions of three EF-hand proteins; calmodulin, calbindin D28k and secretagogin. We have developed a screening method using proteins arrays and validation of targets with surface plasmon resonance. With have applied this method to the two proteins calmodulin and secretagogin. In a study of calmodulin interacting proteins we find 72 novel human calmodulin targets and determine the strength of their interaction with calmodulin. The relevance of the interactions is confirmed for four of the novel targets with co-immunoperciptations using cultured brain cells. For four of proteins we also determine the calmodulin binding sites. One of the calmodulin binding proteins found in the screen is the protein STIM1 which is essential for sensing and refilling the ER calcium stores. We show that the C-terminal of this protein binds to calmodulin and hypothesize that this process could be important in the termination of the refilling process. Using the same screening method in a study of secretagogin interacting proteins we identify nine novel target proteins and also show the interaction of three of them in brain tissue with GST-pull downs. The fact that six of the novel targets are proteins which are important in vesicle handling processes corroborates the theory that secretagogin is involved in secretion. We also study the protein calbindin D28k and show that the protein can bind three Zn2+-ions. The binding is associated with a small conformational change and we show that histidine 80 is involved in the coordination of one of the ions. We make the hypothesis that the biological role of the Zn2+-binding is to buffer the ions to protect the cell from high concentrations which can trigger apoptosis.