Identifying the molecular basis of Laminin N-terminal domain Ca(2+) binding using a hybrid approach.

Ca(2+) 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(2+) 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(2+) binding domain and in many cases requires Ca(2+) to perform its biological function. Here, we conduct a detailed examination of the molecular basis of the LN domain Ca(2+) 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(2+) interaction and identify a conserved sequence present in Ca(2+) binding LN domains. These findings enable a sequence-based prediction of LN domain Ca(2+) binding ability. We use thermal shift assays and isothermal titration calorimetry to explore the biophysical properties of the LN domain Ca(2+) interaction. We show that the netrin-1 LN domain exhibits a high affinity and specificity for Ca(2+), which structurally stabilizes the LN domain. This study elucidates the molecular foundation of the LN domain Ca(2+) binding interaction and provides a detailed functional characterization of this essential interaction, advancing our understanding of protein-Ca(2+) dynamics within the context of the LN domain.