Michael L. Reese

Polymorphic family of injected pseudokinases is paramount in Toxoplasma virulence

Toxoplasma gondii, an obligate intracellular parasite of the phylum Apicomplexa, has the unusual ability to infect virtually any warm-blooded animal. It is an extraordinarily successful parasite, infecting an estimated 30% of humans worldwide. The outcome of Toxoplasma infection is highly dependent on allelic differences in the large number of effectors that the parasite secretes into the host cell. Here, we show that the largest determinant of the virulence difference between two of the most common strains of Toxoplasma is the ROP5 locus. This is an unusual segment of the Toxoplasma genome consisting of a family of 4–10 tandem, highly divergent genes encoding pseudokinases that are injected directly into host cells. Given their hypothesized catalytic inactivity, it is striking that deletion of the ROP5 cluster in a highly virulent strain caused a complete loss of virulence, showing that ROP5 proteins are, in fact, indispensable for Toxoplasma to cause disease in mice. We find that copy number at this locus varies among the three major Toxoplasma lineages and that extensive polymorphism is clustered into hotspots within the ROP5 pseudokinase domain. We propose that the ROP5 locus represents an unusual evolutionary strategy for sampling of sequence space in which the gene encoding an important enzyme has been (i) catalytically inactivated, (ii) expanded in number, and (iii) subject to strong positive selection. Such a strategy likely contributes to Toxoplasma’s successful adaptation to a wide host range and has resulted in dramatic differences in virulence.

Toxoplasma Rhoptry Protein 16 (ROP16) Subverts Host Function by Direct Tyrosine Phosphorylation of STAT6

The obligate intracellular parasite, Toxoplasma gondii, modulates host immunity in a variety of highly specific ways. Previous work revealed a polymorphic, injected parasite factor, ROP16, to be a key virulence determinant and regulator of host cell transcription. These properties were shown to be partially mediated by dysregulation of the host transcription factors STAT3 and STAT6, but the molecular mechanisms underlying this phenotype were unclear. Here, we use a Type I Toxoplasma strain deficient in ROP16 to show that ROP16 induces not only sustained activation but also an extremely rapid (within 1 min) initial activation of STAT6. Using recombinant wild-type and kinase-deficient ROP16, we demonstrate in vitro that ROP16 has intrinsic tyrosine kinase activity and is capable of directly phosphorylating the key tyrosine residue for STAT6 activation, Tyr641. Furthermore, ROP16 co-immunoprecipitates with STAT6 from infected cells. Taken together, these data strongly suggest that STAT6 is a direct substrate for ROP16 in vivo.

A robust methodology to subclassify pseudokinases based on their nucleotide-binding properties

Protein kinase-like domains that lack conserved residues known to catalyse phosphoryl transfer, termed pseudokinases, have emerged as important signalling domains across all kingdoms of life. Although predicted to function principally as catalysis-independent protein-interaction modules, several pseudokinase domains have been attributed unexpected catalytic functions, often amid controversy. We established a thermal-shift assay as a benchmark technique to define the nucleotide-binding properties of kinase-like domains. Unlike in vitro kinase assays, this assay is insensitive to the presence of minor quantities of contaminating kinases that may otherwise lead to incorrect attribution of catalytic functions to pseudokinases. We demonstrated the utility of this method by classifying 31 diverse pseudokinase domains into four groups: devoid of detectable nucleotide or cation binding; cation-independent nucleotide binding; cation binding; and nucleotide binding enhanced by cations. Whereas nine pseudokinases bound ATP in a divalent cation-dependent manner, over half of those examined did not detectably bind nucleotides, illustrating that pseudokinase domains predominantly function as non-catalytic protein-interaction modules within signalling networks and that only a small subset is potentially catalytically active. We propose that henceforth the thermal-shift assay be adopted as the standard technique for establishing the nucleotide-binding and catalytic potential of kinase-like domains.

A Toxoplasma gondii Pseudokinase Inhibits Host IRG Resistance Proteins

A secreted kinase from the parasitic protozoan, Toxoplasma gondii, is shown to cooperate with a phylogenetically related pseudokinase to phosphorylate and inactivate a mouse resistance protein of the IRG system.

A Helical Membrane‐Binding Domain Targets the Toxoplasma ROP2 Family to the Parasitophorous Vacuole

During invasion, the obligate intracellular pathogen, Toxoplasma gondii, secretes into its host cell a variety of effector molecules, several of which have been implicated in strain‐specific variation in disease. The largest family of these effectors, defined by the canonical member ROP2, quickly associates with the nascent parasitophorous vacuole membrane (PVM) after secretion. Here we demonstrate that the NH2‐terminal domain of the ROP2 family contains a series of amphipathic helices that are necessary and sufficient for membrane association. While each of the amphipathic helices is individually competent to bind cellular membranes, together they act to bind the PVM preferentially, possibly through sensing its strong negative curvature. This previously uncharacterized helical domain is an evolutionarily robust and energetically efficient design for membrane association.

A conserved non-canonical motif in the pseudoactive site of the ROP5 pseudokinase domain mediates its effect on Toxoplasma virulence.

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.

Chemical genetic screen identifies Toxoplasma DJ-1 as a regulator of parasite secretion, attachment, and invasion

Toxoplasma gondii is a member of the phylum Apicomplexa that includes several important human pathogens, such as Cryptosporidium and Plasmodium falciparum, the causative agent of human malaria. It is an obligate intracellular parasite that can cause severe disease in congenitally infected neonates and immunocompromised individuals. Despite the importance of attachment and invasion to the success of the parasite, little is known about the underlying mechanisms that drive these processes. Here we describe a screen to identify small molecules that block the process of host cell invasion by the T. gondii parasite. We identified a small molecule that specifically and irreversibly blocks parasite attachment and subsequent invasion of host cells. Using tandem orthogonal proteolysis–activity-based protein profiling, we determined that this compound covalently modifies a single cysteine residue in a poorly characterized protein homologous to the human protein DJ-1. Mutation of this key cysteine residue in the native gene sequence resulted in parasites that were resistant to inhibition of host cell attachment and invasion by the compound. Further analysis of the invasion phenotype confirmed that modification of Cys127 on TgDJ-1 resulted in a block of microneme secretion and motility, even in the presence of direct stimulators of calcium release. Together, our results suggest that TgDJ-1 plays an important role that is likely downstream of the calcium flux required for microneme secretion, parasite motility, and subsequent invasion of host cells.

Clathrin light and heavy chain interface: α‐helix binding superhelix loops via critical tryptophans

Clathrin light chain subunits (LCa and LCb) contribute to regulation of coated vesicle formation to sort proteins during receptor‐mediated endocytosis and organelle biogenesis. LC binding to clathrin heavy chain (HC) was characterized by genetic and structural approaches. The core interactions were mapped to HC residues 1267–1522 (out of 1675) and LCb residues 90–157 (out of 228), using yeast two‐hybrid assays. The C‐termini of both subunits also displayed interactions extending beyond the core domains. Mutations to helix breakers within the LCb core disrupted HC association. Further suppressor mutagenesis uncovered compensatory mutations in HC (K1415E or K1326E) capable of rescuing the binding defects of LCb mutations W127R or W105R plus W138R, thereby pinpointing contacts between HC and LCb. Mutant HC K1415E also rescued loss of binding by LCa W130R, indicating that both LCs interact similarly with HC. Based on circular dichroism data, mapping and mutagenesis, LCa and LCb were represented as α‐helices, aligned along the HC and, using molecular dynamics, a structural model of their interaction was generated with novel implications for LC control of clathrin assembly.

The guanylate kinase domain of the MAGUK PSD-95 binds dynamically to a conserved motif in MAP1a.

The postsynaptic density protein PSD-95 and related membrane-associated guanylate kinases are scaffolding proteins, whose modular interaction motifs organize protein complexes at cell junctions. The signature guanylate kinase domain (GK) contains elements of the proteins GMP-binding site but does not bind nucleotide. Instead, the GK domain has evolved from an enzyme to a protein-protein interaction motif. Here, we show that this canonical GMP-binding region interacts with microtubule-associated protein-1a (MAP1a) and we present a structural model. We determine the consensus GK-binding sequence in MAP1a and demonstrate that PSD-95 can use a similar interaction mode to bind diverse protein partners. Furthermore, we show that PSD-95 GK has adopted the conformational flexibility of the ancestral enzyme to bind its varied ligands, which suggests a mechanism of regulation.

The Toxoplasma pseudokinase ROP5 is an allosteric inhibitor of the immunity-related GTPases

Background: Competition between pathogens and their hosts drives the evolution of molecules that give either organism an edge. Results: Structural and biochemical data show how the parasite pseudokinase ROP5 inhibits the murine GTPase IRGa6. Conclusion: The surfaces of both ROP5 and IRG proteins that interact in the complex are under strong selective pressure. Significance: This highlights an extreme case of evolutionary competition. The Red Queen hypothesis proposes that there is an evolutionary arms race between host and pathogen. One possible example of such a phenomenon could be the recently discovered interaction between host defense proteins known as immunity-related GTPases (IRGs) and a family of rhoptry pseudokinases (ROP5) expressed by the protozoan parasite, Toxoplasma gondii. Mouse IRGs are encoded by an extensive and rapidly evolving family of over 20 genes. Similarly, the ROP5 family is highly polymorphic and consists of 4–10 genes, depending on the strain of Toxoplasma. IRGs are known to be avidly bound and functionally inactivated by ROP5 proteins, but the molecular basis of this interaction/inactivation has not previously been known. Here we show that ROP5 uses a highly polymorphic surface to bind adjacent to the nucleotide-binding domain of an IRG and that this produces a profound allosteric change in the IRG structure. This has two dramatic effects: 1) it prevents oligomerization of the IRG, and 2) it alters the orientation of two threonine residues that are targeted by the Toxoplasma Ser/Thr kinases, ROP17 and ROP18. ROP5s are highly specific in the IRGs that they will bind, and the fact that it is the most highly polymorphic surface of ROP5 that binds the IRG strongly supports the notion that these two protein families are co-evolving in a way predicted by the Red Queen hypothesis.