Identifying the molecular basis of Laminin N-terminal domain Ca²⁺ binding using a hybrid approach

Ca²⁺ is a highly abundant ion involved in numerous biological processes, particularly in multicellular eukaryotic organisms where it exerts many of these functions through interactions with Ca²⁺ binding proteins. The laminin N-terminal (LN) domain is found in members of the laminin and netrin protein families where it plays a critical role in the function of these proteins. The LN domain of laminins and netrins is a Ca²⁺ binding domain and in many cases requires Ca²⁺ to perform its biological function. Here, we conduct a detailed examination of the molecular basis of the LN domain Ca²⁺ interaction combining structural, computational, bioinformatics, and biophysical techniques. By combining computational and bioinformatic techniques with x-ray crystallography we explore the molecular basis of the LN domain Ca²⁺ interaction and identify a conserved sequence present in Ca²⁺ binding LN domains. These findings enable a sequence-based prediction of LN domain Ca²⁺ binding ability. We use thermal shift assays and isothermal titration calorimetry to explore the biophysical properties of the LN domain Ca²⁺ interaction. We show that the netrin-1 LN domain exhibits a high affinity and specificity for Ca²⁺, which structurally stabilizes the LN domain. This study elucidates the molecular foundation of the LN domain Ca²⁺ binding interaction and provides a detailed functional characterization of this essential interaction, advancing our understanding of protein-Ca²⁺ dynamics within the context of the LN domain.