Purification and cation binding properties of the recombinant human S100 calcium-binding protein A3, an EF-hand motif protein with high affinity for zinc.
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 vitro with a subunit molecular weight of 11,712. The Zn(2+)-binding parameters of S100A3 were studied by equilibrium gel filtration and yielded a stoichiometry of four Zn2+ per monomer with a [Zn2+]0.5 of 11 microM 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). Ca(2+)- and Zn(2+)-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+]0.5 of 8 microM 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+]0.5 of 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-nitro-benzoic 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. Ca(2+)-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 Zn(2+)-binding rather than a Ca(2+)-binding protein; nevertheless, very specific conformational changes are introduced by either Ca2+ or Zn2+. Since no Zn(2+)-binding motif of known structure was identified in the primary sequence of S100A3, the results are suggestive for a novel Zn(2+)-binding motif.
Föhr
U G
UG
Department of Pediatrics, University of Zurich, Switzerland.
Heizmann
C W
CW
Engelkamp
D
D
Schäfer
B W
BW
Cox
J A
JA
D001483:Base Sequence; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002412:Cations; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D008969:Molecular Sequence Data; D009282:Naphthalenesulfonates; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D009418:S100 Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013438:Sulfhydryl Compounds / Q000378:metabolism; D015032:Zinc / Q000378:metabolism*
D002135:Calcium-Binding Proteins; D002412:Cations; D017931:DNA Primers; D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D011994:Recombinant Proteins; D009418:S100 Proteins; D013438:Sulfhydryl Compounds; C009497:2-(4-toluidino)-6-naphthalenesulfonic acid; D015032:Zinc