Abstract

The 14-3-3s are a family of dimeric evolutionary conserved pSer/pThr binding proteins that play a key role in multiple biological processes by interacting with a plethora of client proteins. Giardia duodenalis is a flagellated protozoan that affects millions of people worldwide causing an acute and chronic diarrheal disease. The single giardial 14-3-3 isoform (g14-3-3), unique in the 14-3-3 family, needs the constitutive phosphorylation of Thr214 and the polyglycylation of its C-terminus to be fully functional in vivo. Alteration of the phosphorylation and polyglycylation status affects the parasite differentiation into the cyst stage. To further investigate the role of these post-translational modifications, the crystal structure of the g14-3-3 was solved in the unmodified apo form. Oligomers of g14-3-3 were observed due to domain swapping events at the protein C-terminus. The formation of filaments was supported by TEM. Mutational analysis, in combination with native PAGE and chemical cross-linking, proved that polyglycylation prevents oligomerization. In silico phosphorylation and molecular dynamics simulations supported a structural role for the phosphorylation of Thr214 in promoting target binding. Our findings highlight unique structural features of g14-3-3 opening novel perspectives on the evolutionary history of this protein family and envisaging the possibility to develop anti-giardial drugs targeting g14-3-3.

Highlights

  • Eukaryotic 14-3-3s are a family of dimeric highly conserved proteins (,30 kDa) with pSer/pThr binding property

  • We report the X-ray structure of Giardia duodenalis 14-3-3 (g14-33)

  • A structural analysis confirmed that the protein has many of the features common to the other 14-3-3 family members and highlights functional/structural roles for two peculiar g14-3-3 post-translational modifications, i.e. phosphorylation of Thr214 and polyglycylation of Glu246

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Summary

Introduction

Eukaryotic 14-3-3s are a family of dimeric highly conserved proteins (,30 kDa) with pSer/pThr binding property. The overall 14-3-3 architecture is well conserved among the different isoforms and eukaryotic kingdoms. The 14-3-3-target interaction is mediated by conserved residues in an amphipathic groove of each monomer (formed by a-helices C, E, G, and I) and requires specific pSer/pThr containing motifs on the targets. The phosphate moiety of the target binding peptide directly contacts a cluster of positively charged residues in the 14-3-3 amphipathic groove including Lys, Arg, Arg127 and Tyr128 (human 14-33f numbering), whereas the phosphopeptide backbone and side chains establish interactions with a hydrophobic patch of 14-3-3 conserved residues. The existence of an ancestral 14-3-3 that evolved independently into different isoforms in each kingdom has been proposed. The independent clustering of Metazoan epsilon isoforms strongly suggesting that the epsilon isoforms are relatively conserved and similar to the original animal 14-3-3 ancestor [1]

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