Structure of the 14-3-3/RGS3 complex: New details on architecture of complexes formed by 14-3-3 proteins
1Faculty of Science, Charles University, 12843 Prague, Czech Republic
2Institute of Physiology, Academy of Sciences of the Czech Republic, 14220 Prague 4, Czech Republic
14-3-3 proteins are a family of highly conserved dimeric regulatory molecules that are expressed in all eukaryotic cells and specifically bind to the phosphoserine (or phosphothreonine)-containing motifs in a sequence-specific manner. To date more than 300 binding partners of 14-3-3 proteins have been identified. It remains largely unresolved how 14-3-3 proteins exhibit such a diverse assortment of ligands involved in so many different signaling pathways. Not all ligands show equal affinity for different 14-3-3 isoforms, despite the relatively high sequence homology that all 14-3-3s share. Despite structural knowledge of the 14-3-3 protein alone, there is only restricted information about the structure of 14-3-3 proteins in complex with their binding partners .Exactly this information is of paramount importance for mechanistic understanding by which 14-3-3 proteins regulate signal transduction, metabolic pathways, cell cycle, apoptosis etc. We were particularly interested in 14-3-3 ζ /RGS3 protein complex. RGS proteins bind specifically to the GTP-bound forms of Gα and significantly stimulate their GTPase activity by stabilizing the transition state. Upon phosphorylation the RGS protein interacts with the 14-3-3 protein. Phosphorylation-dependent binding of 14-3-3 acts as a molecular switch that controls the GAP activity keeping a substantial fraction of RGS proteins in a dormant stay .
To map the interaction between 14-3-3 ζ and RGS3 protein we performed a wide range of biophysical measurements: H/D exchange and cross link experiments coupled to mass spectrometry were used to determine the interacting surface, FRET (Förster resonance energy transfer) time-resolved fluorescence experiments were used to measure distances between AEDANS-labeled RGS3 molecule and fluorescein-labeled 14-3-3 ζ. The stoichiometry of 14-3-3 ζ /RGS3 protein complex was elucidated using the analytical ultracentrifugation. The crystal structure of RGS domain of RGS3 at 2.3Å resolution was solved. Time resolved tryptophan fluorescence spectroscopy was employed to characterize the conformational changes of RGS3 induced by 14-3-3ζ protein binding .SAXS (small angle X-ray scattering) measurement was used to obtain molecular envelope of 14-3-3/RGS3 protein complex. Based on all these results we build 3D model of 14-3-3 ζ /RGS3 protein complex.
Our model revealed new details on architecture of complex formed by 14-3-3 proteins. To date all known structure of 14-3-3 proteins complexes suggests that the ligand is docked in the central channel of 14-3-3 protein. Our results indicate that the RGS domain of RGS3 protein is located outside the central channel of 14-3-3ζ protein interacting with less-conserved residues of 14-3-3ζ. This could explain why different ligands show different affinity for different 14-3-3 isoforms.
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This work was funded by Grant IAA501110801 of the Grant Agency of the Academy of Sciences of the Czech Republic, by Grant P305/11/0708 of the Czech Science Foundation, by Grant 28510 of the Grant Agency of the Charles University, by Research Project MSM0021620857 and Centre of Neurosciences LC554 of the Ministry of Education, Youth, and Sports of the Czech Republic,and by Research Project AV0Z50110509 of the Academy of Sciences of the Czech Republic.