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Copper Binding Affinity of S100A13, a Key Component of the FGF-1 Nonclassical Copper-Dependent Release Complex

Vaithiyalingam Sivaraja ^*, Thallapuranam Krishnaswamy Suresh Kumar ^*, Dakshinamurthy Rajalingam ^*, Irene Graziani , Igor Prudovsky  and Chin Yu ^* 

^* Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, Arkansas; Center for Molecular Medicine, Maine Medical Center Research Institute, Scarborough, Maine; and Department of Chemistry, National Tsing Hua University, Hsinchu, Taiwan

Correspondence: Address reprint requests to T. K. S. Kumar, Dept. of Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR 72701. Tel.: 479-5755646; Fax: 479-5754049; E-mail: sthalla{at}uark.edu; or to Chin Yu, Dept. of Chemistry, National Tsing Hua University, Hsinchu, Taiwan 300. Tel.: 886-35-721524; Fax: 886-35-711082; E-mail: cyu{at}mx.nthu.edu.tw.

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. 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 (K_d 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.