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.
Department of Pediatrics, Division of Clinical Chemistry and Biochemistry, University of Zürich, CH-8032 Zürich, Switzerland.
C
C
Franz
I
I
Durussel
J A
JA
Cox
B W
BW
Schäfer
C W
CW
Heizmann
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