Human S100A5 binds Ca2+ and Cu2+ independently.

Background: S100A5 is a calcium binding protein found in a small subset of amniote tissues. Little is known about the biological roles of S100A5, but it may be involved in inflammation and olfactory signaling. Previous work indicated that S100A5 displays antagonism between binding of Ca²⁺ and Cu²⁺ ions-one of the most commonly cited features of the protein. We set out to characterize the interplay between Ca²⁺ and Cu²⁺ binding by S100A5 using isothermal titration calorimetry (ITC), circular dichroism spectroscopy (CD), and analytical ultracentrifugation (AUC).

Results: We found that human S100A5 is capable of binding both Cu²⁺ and Ca²⁺ ions simultaneously. The wildtype protein was extremely aggregation-prone in the presence of Cu²⁺ and Ca²⁺. A Cys-free version of S100A5, however, was not prone to precipitation or oligomerization. Mutation of the cysteines does not disrupt the binding of either Ca²⁺ or Cu²⁺ to S100A5. In the Cys-free background, we measured Ca²⁺ and Cu²⁺ binding in the presence and absence of the other metal using ITC. Saturating concentrations of Ca²⁺ or Cu²⁺ do not disrupt the binding of one another. Ca²⁺ and Cu²⁺ binding induce structural changes in S100A5, which are measurable using CD spectroscopy. We show via sedimentation velocity AUC that the wildtype protein is prone to the formation of soluble oligomers, which are not present in Cys-free samples.

Conclusions: S100A5 can bind Ca²⁺ and Cu²⁺ ions simultaneously and independently. This observation is in direct contrast to previously-reported antagonism between binding of Cu²⁺ and Ca²⁺ ions. The previous result is likely due to metal-dependent aggregation. Little is known about the biology of S100A5, so an accurate understanding of the biochemistry is necessary to make informed biological hypotheses. Our observations suggest the possibility of independent biological functions for Cu²⁺ and Ca²⁺ binding by S100A5.