Binding of Ca2+ and Zn2+ to human nuclear S100A2 and mutant proteins.
The Ca2+-binding protein S100A2 is an unusual member of the S100 family, characterized by its nuclear localization and down-regulated expression in tumorigenic cells. In this study, we investigated the properties of human recombinant S100A2 (wtS100A2) and of two mutants in which the amino-terminal Ca2+-binding site I (N mutant) and in addition the carboxyl-terminal site II (NC mutant) were replaced by the canonical loop (EF-site) of alpha-parvalbumin. Size exclusion chromatography and circular dichroism showed that, irrespective of the state of cation binding, wtS100A2 and mutants are dimers and rich in alpha-helical structure. Flow dialysis revealed that wtS100A2 binds four Ca2+ atoms per dimer with pronounced positive cooperativity. Both mutants also bind four Ca2+ atoms but with a higher affinity than wtS100A2 and with negative cooperativity. The binding of the first two Ca2+ ions to the N mutant occurred with 100-fold higher affinity than in wtS100A2 and a 2-fold increase for the last two Ca2+ ions. A further 2-3-fold increase of affinity was observed for respective binding steps of the NC mutant. The Hummel-Dryer method demonstrated that the wild type and mutants bind four Zn2+ atoms per dimer with similar affinity. Fluorescence and difference spectrophotometry showed that the binding of Ca2+ and Zn2+ induces considerable conformational changes, mostly attributable to changes in the microenvironment of Tyr76 located in site II. Fluorescence enhancement of 4,4'-dianilino-1, 1'-binaphthyl-5,5'-disulfonic acid clearly indicated that Ca2+ and Zn2+ binding induce a hydrophobic patch at the surface of wtS100A2, which, as in calmodulin, may be instrumental for the regulatory role of S100A2 in the nucleus.
Franz
C
C
Department of Pediatrics, Division of Clinical Chemistry and Biochemistry, University of Zürich, CH-8032 Zürich, Switzerland.
Durussel
I
I
Cox
J A
JA
Schäfer
B W
BW
Heizmann
C W
CW
D000595:Amino Acid Sequence; D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002630:Chemotactic Factors / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D005456:Fluorescent Dyes / Q000378:metabolism; D005470:Fluorometry; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D009418:S100 Proteins / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000378:metabolism; D015032:Zinc / Q000378:metabolism*
D000817:Anilino Naphthalenesulfonates; D002630:Chemotactic Factors; D005456:Fluorescent Dyes; D009418:S100 Proteins; C098997:S100A2 protein, human; D014443:Tyrosine; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); D015032:Zinc; D002118:Calcium