The crystal structure of Giardia duodenalis 14-3-3 in the apo form: when protein post-translational modifications make the difference

Annarita Fiorillo; Daniele di Marino; Lucia Bertuccini; Allegra Via; Edoardo Pozio; Serena Camerini; Andrea Ilari; Marco Lalle

doi:10.1371/journal.pone.0092902

The crystal structure of Giardia duodenalis 14-3-3 in the apo form: when protein post-translational modifications make the difference

PLoS One. 2014 Mar 21;9(3):e92902. doi: 10.1371/journal.pone.0092902. eCollection 2014.

Authors

Annarita Fiorillo¹, Daniele di Marino², Lucia Bertuccini³, Allegra Via², Edoardo Pozio⁴, Serena Camerini⁵, Andrea Ilari⁶, Marco Lalle⁴

Affiliations

¹ Department of Biochemical Sciences "A. Rossi-Fanelli", University of Rome "Sapienza", Rome, Italy; Institute of Molecular Biology and Pathology, CNR, Rome, Italy and Institute Pasteur Cenci-Bolognetti Foundation at Department of Biochemical Sciences "A. Rossi-Fanelli", University of Rome "Sapienza", Rome, Italy.
² Department of Physics, University of Rome "Sapienza", Rome, Italy.
³ Department of Health and Technology, Istituto Superiore di Sanità, Rome, Italy.
⁴ Department of Infectious, Parasitic and Immunomediated Diseases, Istituto Superiore di Sanità, Rome, Italy.
⁵ Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Rome, Italy.
⁶ Institute of Molecular Biology and Pathology, CNR, Rome, Italy and Institute Pasteur Cenci-Bolognetti Foundation at Department of Biochemical Sciences "A. Rossi-Fanelli", University of Rome "Sapienza", Rome, Italy.

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

14-3-3 Proteins / chemistry*
14-3-3 Proteins / metabolism
Amino Acid Sequence
Amino Acid Substitution
Binding Sites
Crystallography, X-Ray
Giardia lamblia / chemistry*
Giardia lamblia / metabolism
Models, Molecular*
Molecular Dynamics Simulation
Molecular Sequence Data
Phosphorylation
Protein Binding
Protein Conformation
Protein Interaction Domains and Motifs
Protein Multimerization
Protein Processing, Post-Translational
Sequence Alignment

Substances

14-3-3 Proteins

Grants and funding

This study has been partially supported by the Italian Ministero dell'Istruzione, dell'Università e della Ricerca (FIRB grant RBFR08F41U), Istituto Superiore di Sanità (1APC/1303/2011), Fondazione Istituto Italiano di Tecnologia (IIT), (grant “Tecniche di Bioinformatica strutturale: Modellizzazione di proteine, docking e simulazione di dinamica molecolare), and the King Abdullah University of Science and Technology (KAUST, grant KUK-I1-012-43). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.