Characterization of the Ca2+ Binding Affinity and Coordination Site of the LIN-12/Notch-Repeat

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Notch receptors are transmembrane glycoproteins of a highly conserved signaling pathway that regulate cell growth, differentiation, and death in multicellular organisms. Notch activation requires two successive ligand-induced proteolytic cleavages that enable the intracellular Notch to translocate to the nucleus and regulate gene transcription. Notch proteins exhibit a highly conserved modular architecture, which includes three tandem LIN-12/Notch-Repeats (LNRs) responsible for maintaining the receptor in its resting conformation prior to ligand binding. These highly conserved modules contain a characteristic arrangement of three disulfide bonds and a group of aspartate/asparagine residues that coordinate a Ca2+ ion, essential for the correct folding of an LNR. Outside of the Notch family of proteins, LNR modules also exist in proteins such as the PAPP-A and the stealth proteins. In our previous work, we had recombinantly expressed, purified, and refolded the first repeat of human Notch1 and used it as a model system to characterize the binding specificity and affinity of different metals to an LNR via isothermal titration calorimetry (ITC). In this work, we used a combination of computer modeling and experimental approaches to characterize and compare the Ca2+ binding affinities and coordination geometries of various LNR sequences from different proteins. We expect this work to elucidate the basis for Ca2+ ion selectivity by the LNRs that is integral for their structural integrity and is required for the proper regulation of the Notch signaling pathway.