Copper binding affinity of S100A13, a key component of the FGF-1 nonclassical copper-dependent release complex

by Sivaraja, Vaithiyalingam; Kumar, Thallapuranam Krishnaswamy Suresh; Dakshinamurthy, Rajalingam; Graziani, Irene; Prudovsky, Igor; Yu, Chin

S100A13 is a member of the S100 protein family that is involved in the copper-dependent nonclassical secretion of signal peptideless proteins fibroblast growth factor 1 and interleukin 1 alpha. In this study, we investigate the effects of interplay of Cu2+ and Ca2+ on the structure of S100A13 using a variety of biophysical techniques, including multi-dimensional NMR spectroscopy. Results of the isothermal titration calorimetry experiments show that S100A13 can bind independently to both Ca2+ and Cu2+ with almost equal affinity (Kd in the micromolar range). Terbium binding and isothermal titration calorimetry data reveal that two atoms of Cu2+/Ca2+ bind per subunit of S100A13. Results of the thermal denaturation experiments monitored by far-ultraviolet circular dichroism, limited trypsin digestion, and hydrogen-deuterium exchange (using H-1-N-15 heteronuclear single quantum coherence spectra) reveal that Ca2+ and Cu2+ have opposite effects on the stability of S100A13. Binding of Ca2+ stabilizes the protein, but the stability of the protein is observed to decrease upon binding to Cu2+. H-1-N-15 chemical shift perturbation experiments indicate that S100A13 can bind simultaneously to both Ca2+ and Cu2+ and the binding of the metal ions is not mutually exclusive. The results of this study suggest that the Cu2+- binding affinity of S100A13 is important for the formation of the FGF-1 homodimer and the subsequent secretion of the signal peptideless growth factor through the nonclassical release pathway.

Biophysical Journal
Start Page
1542-0086; 0006-3495