title: Novel structural and regulatory features of rhoptry secretory kinases in Toxoplasma gondii.
abstract: Serine/threonine kinases secreted from rhoptry organelles constitute important virulence factors of Toxoplasma gondii. Rhoptry kinases are highly divergent and their structures and regulatory mechanism are hitherto unknown. Here, we report the X-ray crystal structures of two related pseudokinases named ROP2 and ROP8, which differ primarily in their substrate-binding site. ROP kinases contain a typical bilobate kinase fold and a novel N-terminal extension that both stabilizes the N-lobe and provides a unique means of regulation. Although ROP2 and ROP8 were catalytically inactive, they provided a template for homology modelling of the active kinase ROP18, a major virulence determinant of T. gondii. Autophosphorylation of key residues in the N-terminal extension resulted in ROP18 activation, which in turn phosphorylated ROP2 and ROP8. Mutagenesis and mass spectrometry experiments revealed that ROP18 was maximally activated when this phosphorylated N-terminus relieved autoinhibition resulting from extension of aliphatic side chains into the ATP-binding pocket. This novel means of regulation governs ROP kinases implicated in parasite virulence.
authors.affiliation: Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada.
authors.forename: Wei
authors.initials: W
authors.lastname: Qiu
authors.forename: Amy
authors.initials: A
authors.lastname: Wernimont
authors.forename: Keliang
authors.initials: K
authors.lastname: Tang
authors.forename: Sonya
authors.initials: S
authors.lastname: Taylor
authors.forename: Vladimir
authors.initials: V
authors.lastname: Lunin
authors.forename: Matthieu
authors.initials: M
authors.lastname: Schapira
authors.forename: Sarah
authors.initials: S
authors.lastname: Fentress
authors.forename: Raymond
authors.initials: R
authors.lastname: Hui
authors.forename: L David
authors.initials: LD
authors.lastname: Sibley
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010802:Phylogeny; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000378:metabolism; D015800:Protozoan Proteins / Q000737:chemistry* / Q000378:metabolism; D014122:Toxoplasma / Q000201:enzymology* / Q000378:metabolism
chemical_list: D015800:Protozoan Proteins; C109324:ROP8 protein, Toxoplasma gondii; D017346:Protein Serine-Threonine Kinases