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

Abstract

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.

Graphical abstract

Figure 1

Structural examination of Net-1 in complex with metal ions. (A) X-ray diffraction structure model of Net-1 in complex with Sm³⁺ ions. Three Sm³⁺ ions are coordinated through crystallographic contacts (indicated with asterisk ^∗), while one Sm³⁺ ion is bound to the Ca²⁺ binding loop of Net-1 (PDB: 8SNP). (B) Amino acid metal coordination of Sm³⁺ in the Ca²⁺ binding loop of Net-1. The ion is coordinated by side chain and backbone functional groups of the residues F107, D110, N112, T118, and S277. The electron density level of the 2Fo-Fc map is set to 0.522 e/ų (8 sigma contour level). (C) Ca²⁺ coordination in the Ca²⁺ binding loop of Net-1 (PDB: 4OVE) (31) with the indirect coordination of Ca²⁺ by D278 through a hydrogen bonded water molecule. To see this figure in color, go online.

Figure 2

Characterization of the biophysical properties of the LN domain Ca²⁺ binding site. Thermal shift assay characterization of Net-1 (A) and Lam-γ (B) in 20 mM HEPES (pH 7.3) and 150 mM NaCl. Melt curve of holo-protein at a physiological extracellular Ca²⁺ concentration of 1.4 mM shown in a purple short dashed line (Net-1 T_m of 50.6°C, Lam-γ T_m of 52.8°C). Melt curve of Chelex-100-treated apo-protein shown in solid black lines and in the presence of 1.6–50 mM EDTA shown in orange dotted lines (Net-1 T_m of 38.8°C, Lam-γ T_m of 42.6°C), demonstrating Chelex-100 treatment results in the complete removal of Ca²⁺ to produce apo-protein. Melt curve before treatment with Chelex-100 resin (untreated) shown in a gray long-dashed line (Net-1 T_m of 44.2°C, Lam-γ T_m of 48.2°C). Isothermal calorimetric titration of apo-Net-1 with Ca²⁺ (C). apo-Net-1 (29.9 μM) in 20 mM HEPES (pH 7.3) and 150 mM NaCl was titrated with 250 μM CaCl₂ at 25°C. The binding constant (K_a) is measured to be 1.46 ± 0.20 × 10⁷ M⁻¹ with a stoichiometry of 0.942 ± 0.022. The enthalpy change (ΔH) is −7040 ± 200 cal mol⁻¹ and the entropy change (ΔS) is 9.16 ± 0.96 cal mol⁻¹ K⁻¹. ITC values are reported as the mean ± 1 standard deviation of three experimental replicates. To see this figure in color, go online.

Figure 3

Molecular dynamics simulation analysis of the Ca²⁺ binding loop of Net-1. (A) Structural model of the Net-1 Ca²⁺ binding loop bound to Ca²⁺. Interacting residues and water over the 100 ns molecular dynamics trajectory are shown. (B) MMPBSA residue decomposition analysis of the ligand interaction. D110 is the major contributor to the interaction while L108 is only minimally involved via its carbonyl backbone group. Error bars are based on the standard deviation of the mean energy for each residue. (C) Sequence logo showing conservation of the Ca²⁺ binding interface for 831 Net-1 sequences from various species. The residue positions that contribute to the Ca²⁺ binding through their side chain functional groups are highly conserved. To see this figure in color, go online.