A Conserved Non-canonical Motif in the Pseudoactive Site of the ROP5 Pseudokinase Domain Mediates Its Effect on Toxoplasma Virulence

Abstract

The ROP5 family is a closely related set of polymorphic pseudokinases that are critical to the ability of Toxoplasma to cause disease. Polymorphisms in ROP5 also make it a major determinant of strain-specific differences in virulence. ROP5 possesses all of the major kinase motifs required for catalysis except for a substitution at the catalytic Asp. We show that this substitution in the catalytic loop of ROP5 is part of a motif conserved in other pseudokinases of both Toxoplasma and human origin, and that this motif is required for the full activity in vivo of ROP5. This suggests evolutionary selection at this site for a biochemical function, rather than simple drift away from catalysis. We present the crystal structures of a virulent isoform of ROP5 both in its ATP-bound and -unbound states and have demonstrated that despite maintaining the canonical ATP-binding motifs, ROP5 binds ATP in a distorted conformation mediated by unusual magnesium coordination sites that would not be predicted from the primary sequence. In addition, we have mapped the polymorphisms spread throughout the primary sequence of ROP5 to two major surfaces, including the activation segment of ROP5. This suggests that the pseudoactive site of this class of pseudokinases may have evolved to use the canonical ATPbinding motifs for non-catalytic signaling through allostery.

Dawn of the dead: protein pseudokinases signal new adventures in cell biology

“…In many cases, it appears that the inability of many pseudokinases to catalyse a phosphotransfer reaction is likely to be a moot point with respect to their cellular functions. The detailed characterization of pseudokinases, including STRADα (STE20-related adaptor α) [28,29] and ROP5B I (rhoptry protein kinase 5B I ) [30], illustrates that ATP binding is likely to serve an important role in governing protein conformation and that this, rather than any vestigial catalytic activity, underlies in vivo function. In other cases, as vividly illustrated by studies of ILK (integrin-linked kinase) [19,31,32], ATP binding does not modulate protein conformation, suggesting that an ATP-binding conformation, rather than ATP binding itself, is crucial to the scaffolding function of some pseudokinases.…”

Genetic Mapping of Pathogenesis Determinants inToxoplasma gondii

“…duplication of genes in tandem arrays such that their apparent copy number varies (greater than 1) when compared to the genome as a whole Positive selection: divergence from the expected random ratio of mutations such that nonsynonymous changes occur at an elevated frequency relative to expected values a kinase domain and binds ATP, it adopts an altered conformation and is not known to catalyze phosphate transfer (96).…”

TheToxoplasma gondiirhoptry protein ROP18 is an Irga6‐specific kinase and regulated by the dense granule protein GRA7

“…Although not apparent for an IRG‐specific function of ROP5 in our own experiments (Fleckenstein et al ., 2012), an interaction of ROP5 with ROP18 was reported to control T. gondii virulence by up‐regulation of ROP18 activity (Behnke et al ., 2012). The rop5 locus is represented by a cluster of tandemly repeated genes encoding three different ROP5 isoforms, ROP5A, ROP5B and ROP5C, each containing multiple copies, and all isoforms are predicted to be catalytically inactive (El Hajj et al ., 2006; Reese and Boothroyd, 2011). Individual sequences of these rop5 paralogues are almost identical within strains but differ considerably between T. gondii types I, II and III strains (Behnke et al ., 2011; Reese et al ., 2011; Niedelman et al ., 2012).…”