Abstract
CBL1 is an EF hand Ca2+ binding protein from A. thaliana that is involved in the detection of cellular Ca2+ signals and the downstream signal transmission by interaction with the protein kinase CIPK23. So far, the structure and calcium ion binding affinities of CBL1 remain elusive. In this study it was observed that CBL1 tends to form higher oligomeric states due to an intrinsic hydrophobicity and the presence of the detergent BriJ35 was required for the purification of monomeric and functional protein. Functional insights into the in vitro Ca2+ binding capabilities of CBL1 were obtained by isothermal titration calorimetry (ITC) of the wildtype protein as well as single site EF hand mutants. Based on our results, a binding model of CBL1 for Ca2+in vivo is proposed. Additionally, upon both, ITC measurements and the analysis of an AlphaFold2 model of CBL1, we could gain first insights into the formation of the dimer interface. We could identify an area around EF hand 4 to be relevant for the structural and functional integrity of monomeric CBL1 and likely EF hand 1 to be involved in the dimer interface.
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