Copper binding affinity of S100A13, a key component of the FGF-1 nonclassical copper-dependent release complex.
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 lpha. 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 1H-15N 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+. 1H-15N 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.
Sivaraja
Vaithiyalingam
V
Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, Arkansas 72701, USA.
Kumar
Thallapuranam Krishnaswamy Suresh
TK
Rajalingam
Dakshinamurthy
D
Graziani
Irene
I
Prudovsky
Igor
I
Yu
Chin
C
D001665:Binding Sites; D003198:Computer Simulation; D003300:Copper / Q000032:analysis / Q000737:chemistry*; D016220:Fibroblast Growth Factor 1 / Q000737:chemistry*; D008956:Models, Chemical; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D009418:S100 Proteins / Q000737:chemistry*
D009418:S100 Proteins; D016220:Fibroblast Growth Factor 1; D003300:Copper