title: The crystal structure of Giardia duodenalis 14-3-3 in the apo form: when protein post-translational modifications make the difference.
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.
authors.affiliation: 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.
authors.forename: Annarita
authors.initials: A
authors.lastname: Fiorillo
authors.affiliation: Department of Physics, University of Rome "Sapienza", Rome, Italy.
authors.forename: Daniele
authors.initials: D
authors.lastname: di Marino
authors.affiliation: Department of Health and Technology, Istituto Superiore di Sanità, Rome, Italy.
authors.forename: Lucia
authors.initials: L
authors.lastname: Bertuccini
authors.affiliation: Department of Physics, University of Rome "Sapienza", Rome, Italy.
authors.forename: Allegra
authors.initials: A
authors.lastname: Via
authors.affiliation: Department of Infectious, Parasitic and Immunomediated Diseases, Istituto Superiore di Sanità, Rome, Italy.
authors.forename: Edoardo
authors.initials: E
authors.lastname: Pozio
authors.affiliation: Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Rome, Italy.
authors.forename: Serena
authors.initials: S
authors.lastname: Camerini
authors.affiliation: 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.
authors.forename: Andrea
authors.initials: A
authors.lastname: Ilari
authors.affiliation: Department of Infectious, Parasitic and Immunomediated Diseases, Istituto Superiore di Sanità, Rome, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Lalle
mesh_terms: D048948:14-3-3 Proteins / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D018360:Crystallography, X-Ray; D016829:Giardia lamblia / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D011499:Protein Processing, Post-Translational; D016415:Sequence Alignment
chemical_list: D048948:14-3-3 Proteins