Human S100A5 binds Ca2+ and Cu2+ independently

Abstract

BackgroundS100A5 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).ResultsWe 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.ConclusionsS100A5 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13628-017-0040-y) contains supplementary material, which is available to authorized users.

pytc: Open-Source Python Software for Global Analyses of Isothermal Titration Calorimetry Data

“…To demonstrate pytc ’s capabilities for Bayesian global analyses, we used data from a previous publication: 25 the binding of Ca 2+ to human S100A5. This presents a particularly challenging fitting problem.…”

“…We reported these experiments previously. 25 We extracted thermodynamic parameters for a two-site binding polynomial using the Bayesian MCMC sampler in pytc . Points are integrated heats with uncertainty calculated using NITPIC.…”

Comprehensive analysis of the expression and prognosis for S100 in human ovarian cancer

“…[ 40 , 41 ] S100A5 is expressed in very restricted regions of the adult brain and regulates electrolyte metabolism. [ 42 ] S100A7A was demonstrated to be involved in the innate immune system and acts as a defense against epidermal differentiation and inflammation. However, its role in cancer has rarely been explored.…”

pytc: a python package for analysis of Isothermal Titration Calorimetry experiments

“…These packages allow for global fitting of exceedingly complex models (6,8). 23 While these packages are powerful, it remains difficult for users to extend these packages with new fitting 24 models. They also require proprietary software: either Windows (SEDPHAT) or Matlab (ITCsy).…”

“…onto human S100A5 at four different titrant/titrate ratios: 8× (blue), 10× (purple), 15× (red), and 18× 172 (green). We reported these experiments previously (23). We extracted thermodynamic parameters for 173 a two-site binding polynomial using the Bayesian MCMC sampler in pytc.…”