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

  • Michael L. Reese
    Footnotes
    Affiliations
    Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 94305-5124
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  • John C. Boothroyd
    Correspondence
    To whom correspondence should be addressed: Fairchild Building, Rm. D305, 299 Campus Dr., Stanford, CA 94305-5124. Tel.: 650-723-7984; Fax: 650-725-6757
    Affiliations
    Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 94305-5124
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grant AI73756.
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S9.
    1 Supported, in part, by a postdoctoral fellowship from the American Cancer Society.
Open AccessPublished:June 27, 2011DOI:https://doi.org/10.1074/jbc.M111.253435
      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 ATP-binding motifs for non-catalytic signaling through allostery.

      Introduction

      Proteins involved in cellular signaling often have modular domain structure; whereas enzymatic domains catalyze the reactions that propagate a signal, the precise subcellular localization of a protein, and the recognition of partner molecules are usually accomplished by nonenzymatic domains (
      • Kuriyan J.
      • Cowburn D.
      ,
      • Pawson T.
      ). Complicating this paradigm, noncatalytic enzymatic domains such as pseudokinases have been recognized as important regulators of signaling networks, for example, by acting as molecular scaffolds for an effector and its downstream targets, or by modulating the activity of a catalytically active enzyme (
      • Boudeau J.
      • Miranda-Saavedra D.
      • Barton G.J.
      • Alessi D.R.
      ).
      The cellular signaling occurring at the interface between an intracellular pathogen and its host cell is particularly complex. Toxoplasma gondii is an obligate, intracellular parasite with the unusual ability to infect almost any nucleated cell of virtually any warm-blooded animal. During infection, Toxoplasma secretes a variety of effector molecules into its host cell to monitor and co-opt host signaling networks (
      • Boothroyd J.C.
      • Dubremetz J.F.
      ,
      • Bradley P.J.
      • Sibley L.D.
      ). Although these effectors originate in the specialized secretory organelles of the parasite, called rhoptries (and thus the designation of these effectors as “ROPs”), they carry out their various functions in the cytosol of the host cell. Three ROPs have been identified as active kinases that drastically alter host signaling (
      • Saeij J.P.
      • Coller S.
      • Boyle J.P.
      • Jerome M.E.
      • White M.W.
      • Boothroyd J.C.
      ,
      • Peixoto L.
      • Chen F.
      • Harb O.S.
      • Davis P.H.
      • Beiting D.P.
      • Brownback C.S.
      • Ouloguem D.
      • Roos D.S.
      ) and disease outcome (
      • Saeij J.P.
      • Coller S.
      • Boyle J.P.
      • Jerome M.E.
      • White M.W.
      • Boothroyd J.C.
      ,
      • Saeij J.P.
      • Boyle J.P.
      • Coller S.
      • Taylor S.
      • Sibley L.D.
      • Brooke-Powell E.T.
      • Ajioka J.W.
      • Boothroyd J.C.
      ,
      • Taylor S.
      • Barragan A.
      • Su C.
      • Fux B.
      • Fentress S.J.
      • Tang K.
      • Beatty W.L.
      • Hajj H.E.
      • Jerome M.
      • Behnke M.S.
      • White M.
      • Wootton J.C.
      • Sibley L.D.
      ). Indeed, of the ∼160 predicted kinases of Toxoplasma, at least one-third are predicted to be secreted into the host cell (
      • Peixoto L.
      • Chen F.
      • Harb O.S.
      • Davis P.H.
      • Beiting D.P.
      • Brownback C.S.
      • Ouloguem D.
      • Roos D.S.
      ), suggesting an active role in pathogenesis.
      Intriguingly, the kinome of the Toxoplasma, especially its secreted effectors, is highly enriched in pseudokinases. We and others previously identified a closely related family of these secreted pseudokinases, the ROP5 family, as absolutely critical to pathogenesis in mice; ablation of the locus results in a complete loss of virulence in mice (
      • Reese M.L.
      • Zeiner G.M.
      • Saeij J.P.
      • Boothroyd J.C.
      • Boyle J.P.
      ,
      • Behnke M.S.
      • Khan A.
      • Wootton J.C.
      • Dubey J.P.
      • Tang K.
      • Sibley L.D.
      ). The ROP5 family is encoded by a locus of tandemly duplicated genes that are highly polymorphic (supplemental Fig. S1). Allelic variation of these isoforms is concentrated within the pseudokinase domains and is responsible for a >105 difference in virulence (LD50) in a mouse model of disease (
      • Reese M.L.
      • Zeiner G.M.
      • Saeij J.P.
      • Boothroyd J.C.
      • Boyle J.P.
      ).
      There is an abundance of structural information on active protein kinases, which has led to a detailed understanding both of the reaction mechanism of phosphoryl transfer and various methods of regulation by diverse actors such as small molecules (
      • Knight Z.A.
      • Shokat K.M.
      ) and both cis- (
      • Kannan N.
      • Haste N.
      • Taylor S.S.
      • Neuwald A.F.
      ) and trans- acting protein-protein interactions (
      • Pellicena P.
      • Kuriyan J.
      ). Pseudokinases, however, remain something of a functional mystery. Recent structures of the pseudokinase domains of HER3 (
      • Jura N.
      • Shan Y.
      • Cao X.
      • Shaw D.E.
      • Kuriyan J.
      ), ILK (
      • Fukuda K.
      • Gupta S.
      • Chen K.
      • Wu C.
      • Qin J.
      ), and STRADα (
      • Zeqiraj E.
      • Filippi B.M.
      • Deak M.
      • Alessi D.R.
      • van Aalten D.M.
      ) have suggested these proteins act as “smart” scaffolds that allosterically regulate their binding partners. Other studies have indicated that some proteins originally predicted to be pseudokinases may catalyze phosphoryl transfer through atypical means (
      • Min X.
      • Lee B.H.
      • Cobb M.H.
      • Goldsmith E.J.
      ,
      • Mukherjee K.
      • Sharma M.
      • Urlaub H.
      • Bourenkov G.P.
      • Jahn R.
      • Südhof T.C.
      • Wahl M.C.
      ,
      • Shi F.
      • Telesco S.E.
      • Liu Y.
      • Radhakrishnan R.
      • Lemmon M.A.
      ).
      Most studies of pseudokinases to date have concentrated on those residues that are missing from the canonical kinase motifs. The ROP5 family is a closely related family of proteins (up to 10 paralogous ROP5 genes exist within a given strain) under extraordinary selective pressure, and thus provides an opportunity to examine the functional relevance of conserved substitutions within the canonical kinase motifs (i.e. things conserved in pseudokinases that are divergent from an active kinase). Here, we present such an examination, guided by the crystal structure of the ROP5 pseudokinase domain, and demonstrate the in vivo relevance of a key substitution in the pseudoactive site to function for ROP5 in determining the outcome of Toxoplasma infection.

      DISCUSSION

      We have solved the structure of the pseudokinase domain of ROP5, a protein that is critical for the ability of Toxoplasma to cause disease and is the major determinant of strain-specific variation in virulence (
      • Reese M.L.
      • Zeiner G.M.
      • Saeij J.P.
      • Boothroyd J.C.
      • Boyle J.P.
      ,
      • Behnke M.S.
      • Khan A.
      • Wootton J.C.
      • Dubey J.P.
      • Tang K.
      • Sibley L.D.
      ). The rhoptry kinase family is specific to coccidian parasites, and is highly enriched in proteins that are predicted to be secreted (
      • Peixoto L.
      • Chen F.
      • Harb O.S.
      • Davis P.H.
      • Beiting D.P.
      • Brownback C.S.
      • Ouloguem D.
      • Roos D.S.
      ), implicating the family as a focal point in the interaction between Toxoplasma and its varied hosts. Recent work demonstrated that the ROP5 pseudokinase domain contains many polymorphic residues that appear to be under strong positive selection (
      • Reese M.L.
      • Zeiner G.M.
      • Saeij J.P.
      • Boothroyd J.C.
      • Boyle J.P.
      ). Building on this, we have shown that these polymorphic residues throughout the primary sequence are concentrated within a major surface. Our results indicate a strong selective pressure has operated on this region of the protein, which is adjacent to the hinge between the N- and C-lobes of the kinase fold. Taken together, these data suggest that ROP5 mediates its effect on virulence through interaction with and dysregulation of its target or targets using this surface. Although we cannot yet know whether the binding partners are of host or parasite origin, the fact that the ROP5 proteins reside on the parasitophorous vacuolar membrane and are exposed to the host cytosol (
      • El Hajj H.
      • Demey E.
      • Poncet J.
      • Lebrun M.
      • Wu B.
      • Galéotti N.
      • Fourmaux M.N.
      • Mercereau-Puijalon O.
      • Vial H.
      • Labesse G.
      • Dubremetz J.F.
      ,
      • Reese M.L.
      • Boothroyd J.C.
      ,
      • El Hajj H.
      • Lebrun M.
      • Fourmaux M.N.
      • Vial H.
      • Dubremetz J.F.
      ), suggests that the potential binding partner(s) derive from the host.
      The structure of ROP5 is especially interesting in its relationship with current thinking of both kinase and pseudokinase function. Analysis of proteins with kinase folds often invokes the conservation of canonical motifs (
      • Hanks S.K.
      • Hunter T.
      ), whose contribution to catalysis has been exhaustively studied structurally, biochemically, and through simulation. Of the pseudokinases whose structures are known, ROP5 has among the greatest conservation of the canonical kinase motifs, apparently lacking only the catalytic Asp, suggesting that ROP5 should be competent to bind ATP in a canonical conformation competent for catalysis. Remarkably, in the structure of ROP5BI bound to ATP, the canonical ATP-binding residues in the ROP5 pseudoactive site have evolved to stabilize a surprisingly non-canonical conformation of ATP, which appears protected from hydrolysis. As shown here, this ATP conformation appears incompatible with catalytic activity, even with the re-introduction of the catalytic Asp by mutagenesis. Thus, it is possible that even proteins with apparently complete canonical kinase motifs may not be catalytically active, which provides an interesting corollary to the recent discovery that several proteins originally thought to be pseudokinases appear to have maintained their catalytic activities even with a non-canonical active site (
      • Mukherjee K.
      • Sharma M.
      • Urlaub H.
      • Bourenkov G.P.
      • Jahn R.
      • Südhof T.C.
      • Wahl M.C.
      ,
      • Shi F.
      • Telesco S.E.
      • Liu Y.
      • Radhakrishnan R.
      • Lemmon M.A.
      ). For instance, Wnk1 originally appeared to lack a catalytically active Lys, but instead has adapted to use a non-canonical Lys from a different position in its primary sequence (
      • Min X.
      • Lee B.H.
      • Cobb M.H.
      • Goldsmith E.J.
      ). CASK, on the other hand, whereas missing the Mg2+-coordinating DFG motif, has adapted a new set of residues to form a magnesium-independent active site (
      • Mukherjee K.
      • Sharma M.
      • Urlaub H.
      • Bourenkov G.P.
      • Jahn R.
      • Südhof T.C.
      • Wahl M.C.
      ). Interestingly, in CASK, mutagenesis of the canonical catalytic Asp and Lys did not alter its catalytic activity (
      • Mukherjee K.
      • Sharma M.
      • Urlaub H.
      • Bourenkov G.P.
      • Jahn R.
      • Südhof T.C.
      • Wahl M.C.
      ), indicating that its atypical active site truly acts independently from many of the canonical motifs. This is in contrast to our mutagenesis data of ROP5, in which substitution to restore the canonical HRD motif Asp resulted in a less functional (but, biologically, not inactive) protein in vivo. Particular care should be taken when ascribing function or non-function to novel sequences; nevertheless, and although we cannot prove unequivocally that the ROP5 proteins described here lack catalytic activity, the presence of a basic residue in place of the catalytic Asp, the altered conformation of the bound ATP, and our failure to detect any activity in biochemical assays, even using a mutant form engineered to reintroduce the Asp, makes the possibility of ROP5 being a functional kinase extremely unlikely.
      By examining the kinomes of both humans and Toxoplasma, we have identified a potential subclass of pseudokinases based on their convergent evolution of the substitution of the canonical HRD motif with an HGB motif. We confirmed the importance of this motif by testing the in vivo activity of ROP5 in which a charge-swap mutation had replaced its basic Arg to restore the canonical Asp. In vitro, such protein maintained its ability to bind ATP, although it was still not catalytically active. In vivo, R389D mutant ROP5 was able to partially complement the virulence of a Δrop5 phenotype, but to a significantly lesser extent as compared with complementation with the wild-type ROP5. This is consistent with the proposed role of ROP5 as a molecular scaffold rather than as an atypical, active enzyme. By extension, our data implicate the conserved non-canonical HGB motif as important to the function of multiple other pseudokinases in a variety of organisms. Hence, pseudokinases may not be the outliers as they first appeared; just as diligent research has elucidated general principles that govern a catalytic activity of the kinase, with further study the principles that govern the functioning of specific families of pseudokinases should soon be determinable.

      Acknowledgments

      We thank Arvin Dar for suggesting the ATP-Sepharose pull-down experiment, Mark Wilson and Tim Fenn for crystallographic software assistance, the Brünger lab for crystallization space, members of the Boothroyd lab for thoughtful suggestions, and Eva LaDow, Kara Norman, Selena Sagan, Mark Wilson, and Rich Yu for critical comments on the manuscript.

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