Identifying the molecular basis of Laminin N-terminal domain Ca binding using a hybrid approach.

Ca 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 Ca 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 Ca binding domain and in many cases requires Ca to perform its biological function. Here, we conduct a detailed examination of the molecular basis of the LN domain Ca 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 Ca interaction and identify a conserved sequence present in Ca binding LN domains. These findings enable a sequence-based prediction of LN domain Ca binding ability. We use thermal shift assays and isothermal titration calorimetry to explore the biophysical properties of the LN domain Ca interaction. We show that the netrin-1 LN domain exhibits a high affinity and specificity for Ca, which structurally stabilizes the LN domain. This study elucidates the molecular foundation of the LN domain Ca binding interaction and provides a detailed functional characterization of this essential interaction, advancing our understanding of protein-Ca dynamics within the context of the LN domain.