| Abstrakt: |
The calcium-binding protein S100A3 is an unusual member of the S100 family, characterized by its very high content of Cys. In order to study the biochemical, cation-binding, and conformational properties, we produced and purified the recombinant human protein in Escherichia coli. The recombinant protein forms noncovalent homodimers, tetramers, and polymers in vitrowith a subunit molecular weight of 11,712. The Zn2+-binding parameters of S100A3 were studied by equilibrium gel filtration and yielded a stoichiometry of four Zn2+per monomer with a [Zn2+]0.5of 11 μM and a Hill coefficient of 1.4 at physiological ionic strength. The affinity for Ca2+is too low to be determined by direct methods (KCa> 10 mM). Ca2+- and Zn2+-binding can be followed by optical methods: the Trp-45 fluorescence is high in the metal-free form and addition of Zn2+and Ca2+, but not of Mg2+, leads to a 4-fold quenching. Ca2+and Zn2+promote also quite similar conformational changes in the Tyr and Trp environment as monitored by difference spectrophotometry. Fluorescence titrations with Zn2+confirmed that there is one set of high affinity binding sites with a [Zn2+]0.5of 8 μM and a Hill coefficient of 1.3. Binding of Zn2+to a second set of low affinity sites induces protein precipitation. Fluorescence titrations with Ca2+confirmed the very low affinity of S100A3 for this ion with a [Ca2+]0.5of 30 mM and slight negative cooperativity. Mg2+has no effect on this binding curve. Of the 10 Cys residues in S100A3, 5 only are free thiols, and accessible to 5,5′-dithiobis(2-nitrobenzoic acid); they display a high reactivity in the metal-free and Ca2+form, but a 20-fold lowered reactivity in the Zn2+form of S100A3. Ca2+-binding promotes the formation of a solvent-accessible hydrophobic surface as monitored by the 60-fold fluorescence increase of 2-p-toluidinylnaphthalene-6-sulfonate, whereas Zn2+has no noticeable influence. Our data indicate that Ca2+and Zn2+do not bind to the same sites and that under physiological conditions S100A3 is a Zn2+-binding rather than a Ca2+-binding protein; nevertheless, very specific conformational changes are introduced by either Ca2+or Zn2+. Since no Zn2+-binding motif of known structure was identified in the primary sequence of S100A3, the results are suggestive for a novel Zn2+-binding motif. |
