Abstract
In solution, S100B protein is a noncovalent homodimer composed of two subunits associated in an antiparallel manner. Upon calcium binding, the conformation of S100B changes dramatically, leading to the exposure of hydrophobic residues at the surface of S100B. The residues in the C-terminal domain of S100B encompassing Phe(87) and Phe(88) have been implicated in interaction with target proteins. In this study, we used two-hybrid technology to identify specific S100B target proteins. Using S100B as bait, we identify S100A6 and S100A11 as specific targets for S100B. S100A1, the closest homologue of S100B, is capable of interaction with S100B but does not interact with S100A6 or S100A11. S100B, S100A6, and S100A11 isoforms are co-regulated and co-localized in astrocytoma U373 cells. Furthermore, co-immunoprecipitation experiments demonstrated that Ca(2+)/Zn(2+) stabilizes S100B-S100A6 and S100B-S100A11 heterocomplexes. Deletion of the C-terminal domain or mutation of Phe(87) and Phe(88) residues has no effect on S100B homodimerization and heterodimerization with S100A1 but drastically decreases interaction between S100B and S100A6 or S100A11. Our data suggest that the interaction between S100B and S100A6 or S100A11 should not be viewed as a typical S100 heterodimerization but rather as a model of interaction between S100B and target proteins.
Highlights
In solution, S100B protein is a noncovalent homodimer composed of two subunits associated in an antiparallel manner
Interaction specificity was retested on YC-UWLH medium and filter X-gal test after mating assays using L40 yeast strain transformed with either the original bait LexADBDS100B, the LexADBD-S100A1, or an unrelated fusion protein LexADBD-lamin
The results showed that S100B/ A1Cter fused either with LexADBD or Gal4AD still interacts with S100A6 or S100A11
Summary
Materials—T4 DNA ligase and Taq polymerase were purchased from Life Technologies, Inc. The beads were pelleted and washed in TBS-T, and the immunoprecipitated proteins were analyzed by Western blot using a polyclonal antibody directed against GFP. Immunoprecipitated proteins were analyzed by Western blot using the mouse anti-S100A6 monoclonal antibody. PLexADBD-S100B bait construct was transformed singly into L40, and cells were grown on YC-UW medium for 2 days. A large scale transformation was performed using a human brain cDNA library constructed in pGAD10 plasmid (Matchmaker). Each cDNA was retested in a mating assay against pLexAlamin, pLexA-S100A1, and the original bait construct using AMR70 yeast strain as described above. The pLexA9 and pGAD positive transformants were grown for 18 h in 5 ml of YC-UW or YC-UL liquid medium, respectively. L40 containing LexADBD-S100B, -S100A1, or -lamin fusion proteins were mated to AMR70 transformed yeast as detailed at the bottom.
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