Metal ions modulate the folding and stability of the tumor suppressor protein S100A2.
The EF-hand protein S100A2 is a cell cycle regulator involved in tumorigenesis, acting through regulation of the p53 activation state. Metal ion-free S100A2 is homodimeric and contains two Ca(2+)-binding sites and two Zn(2+)-binding sites per subunit, whereby the Zn(2+) ion binding to one of the sites is coordinated by residues from two homodimers. The effect of selective binding of these metal ions was investigated using site-specific mutants which lacked one or both zinc sites. CD analysis of secondary structure changes on metallation showed that Zn(2+) binding was associated with a decrease in the secondary structure content, whereas Ca(2+) had the opposite effect in two of the three S100A2 mutants studied. The energy of unfolding (DeltaG(U)) of the apo wild-type S100A2 was determined to be 89.9 kJ mol(-1), and the apparent midpoint transition temperature (T(m)(app))) was 58.4 degrees C. In addition, a detailed study of the urea and thermal unfolding of the S100A2 mutants in different metallation states (apo, Zn(2+) and Ca(2+)) was performed. Thermal denaturation experiments showed that Zn(2+) acts as a destabilizer and Ca(2+) as a stabilizer of the protein conformation. This suggests a synergistic effect between metal binding, protein stability and S100A2 biological activity, according to which Ca(2+) activates and stabilizes the protein, the opposite being observed on Zn(2+) binding.
Botelho
Hugo M
HM
Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Portugal.
Koch
Michael
M
Fritz
Günter
G
Gomes
Cláudio M
CM
D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002630:Chemotactic Factors / Q000737:chemistry / Q000378:metabolism*; D002942:Circular Dichroism; D006801:Humans; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D009418:S100 Proteins / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D017550:Spectroscopy, Fourier Transform Infrared; D013816:Thermodynamics; D015032:Zinc / Q000378:metabolism*
D002630:Chemotactic Factors; D009418:S100 Proteins; C098997:S100A2 protein, human; D015032:Zinc; D002118:Calcium