Study of Structural Differences in H8-H9 Loop among 14-3-3 Protein Isoforms
E. Macakova1, J. Sykora3, V. Obsilova1, T. Obsil1,2
1Institute of Physiology Academy of Sciences of the Czech Republic, 14220 Prague, Czech Republic
2Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, 12843 Prague, Czech Republic
3J. Heyrovsky Institute of Physical Chemistry Academy of Sciences of the Czech Republic, 18223 Prague, Czech Republic
The 14-3-3 proteins are highly conserved dimeric signaling proteins expressed in all eukaryotic cells . Each monomer consists of nine antiparallel helices. It has been speculated that the loop between α-helixes H8 and H9 affects the binding properties of 14-3-3 protein isoforms. In this work we focused on barley isoform 14-3-3A and human isoform 14-3-3ζ. It has been observed that the isoform 14-3-3A binds its ligands with significantly lower affinity compared to other barley isoforms (14-3-3B and 14-3-3C) . These isoforms differ in the sequence of the loop between α-helixes H8 and H9. We measured the binding affinity of 14-3-3A WT and its mutant, which contained the same H8-H9 loop as isoforms B and C. For comparison, we also measured the binding affinity of human isoform 14-3-3ζ and its mutant, where the serine residue within the H8-H9 loop was replaced by glycine. The binding affinity of 14-3-3 protein isoforms was studied using two different fluorescence spectroscopy techniques (the fluorescence correlated spectroscopy and the quenching of the steady-state fluorescence intensity). Our results show that the mutation of H8-H9 loop decreased the binding affinity of both tested isoforms, even though their mutations were antagonistic (Gly/Ser in the case of 14-3-3A, Ser/Gly in the case of 14-3-3ξ). In conclusion, the H8-H9 loop plays an important role in the regulation of the 14-3-3 binding affinity in an isoform-specific manner.
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This work was funded by Grant P207/11/0455 of the Grant Agency of the Czech Republic, Grant IAA501110801 of the Grant Agency of the Academy of Sciences of the Czech Republic, by Research Project MSM0021620857 and by Research Project AV0Z50110509 of the Academy of Sciences of the Czech Republic.