Environmental sensing and regulation of motility in Toxoplasma.

Toxoplasma and other apicomplexan parasites undergo a unique form of cellular locomotion referred to as "gliding motility." Gliding motility is crucial for parasite survival as it powers tissue dissemination, host cell invasion and egress. Distinct environmental cues lead to activation of gliding motility and have become a prominent focus of recent investigation. Progress has been made toward understanding what environmental cues are sensed and how these signals are transduced in order to regulate the machinery and cellular events powering gliding motility. In this review, we will discuss new findings and integrate these into our current understanding to propose a model of how environmental sensing is achieved to regulate gliding motility in Toxoplasma. Collectively, these findings also have implications for the understanding of gliding motility across Apicomplexa more broadly.

Internal daughter formation of Toxoplasma gondii tachyzoites is coordinated by transcription factor TgAP2IX-5.

Toxoplasma gondii rapidly propagates through endodyogeny of tachyzoites, a process in which daughter parasites divide within the cell of the mother parasite. Recent studies have revealed that transcription factors with AP2-domain participate in the process of cell division in T. gondii. However, the concise regulation of the division cycles by AP2 proteins is poorly understood. In this study, we evaluated the effect of the transcription factor TgAP2IX-5 on the daughter cell formation in T. gondii. TgAP2IX-5 is a nuclear protein and is highly expressed during the S phase of the cell cycle of tachyzoites. TgAP2IX-5-disrupted strain showed a severe defect in replication and completely blocked lytic parasite growth. Following 3-indoleacetic acid treatment or without treatment of AP2IX-5-AID-3HA tagged strain for 30 min, 1 and 2 hr, the differentially expressed genes were 8, 54 and 202, respectively. Among these genes, the significantly downregulated ones were AP2 proteins, inner membrane complex (IMC) proteins and SAG-related proteins. Interestingly, loss of TgAP2IX-5 leads to a defect in internal daughter IMC formation and abnormalities in the morphology of organelles during cell division. Together, our study suggests that TgAP2IX-5 is crucial in regulating IMC formation of daughter cells in T. gondii.

C-Mannosylation of Toxoplasma gondii proteins promotes attachment to host cells and parasite virulence.

C-Mannosylation is a common modification of thrombospondin type 1 repeats present in metazoans and recently identified also in apicomplexan parasites. This glycosylation is mediated by enzymes of the DPY19 family that transfer α-mannoses to tryptophan residues in the sequence WX2WX2C, which is part of the structurally essential tryptophan ladder. Here, deletion of the dpy19 gene in the parasite Toxoplasma gondii abolished C-mannosyltransferase activity and reduced levels of the micronemal protein MIC2. The loss of C-mannosyltransferase activity was associated with weakened parasite adhesion to host cells and with reduced parasite motility, host cell invasion, and parasite egress. Interestingly, the C-mannosyltransferase-deficient Δdpy19 parasites were strongly attenuated in virulence and induced protective immunity in mice. This parasite attenuation could not simply be explained by the decreased MIC2 level and strongly suggests that absence of C-mannosyltransferase activity leads to an insufficient level of additional proteins. In summary, our results indicate that T. gondii C-mannosyltransferase DPY19 is not essential for parasite survival, but is important for adhesion, motility, and virulence.

The apical annuli of Toxoplasma gondii are composed of coiled-coil and signalling proteins embedded in the inner membrane complex sutures.

The apical annuli are among the most intriguing and understudied structures in the cytoskeleton of the apicomplexan parasite Toxoplasma gondii. We mapped the proteome of the annuli in Toxoplasma by reciprocal proximity biotinylation (BioID), and validated five apical annuli proteins (AAP1-5), Centrin2, and an apical annuli methyltransferase. Moreover, inner membrane complex (IMC) suture proteins connecting the alveolar vesicles were also detected and support annuli residence within the sutures. Super-resolution microscopy identified a concentric organisation comprising four rings with diameters ranging from 200 to 400 nm. The high prevalence of domain signatures shared with centrosomal proteins in the AAPs together with Centrin2 suggests that the annuli are related and/or derived from the centrosomes. Phylogenetic analysis revealed that the AAPs are conserved narrowly in coccidian, apicomplexan parasites that multiply by an internal budding mechanism. This suggests a role in replication, for example, to provide pores in the mother IMC permitting exchange of building blocks and waste products. However, presence of multiple signalling domains and proteins are suggestive of additional functions. Knockout of AAP4, the most conserved compound forming the largest ring-like structure, modestly decreased parasite fitness in vitro but had no significant impact on acute virulence in vivo. In conclusion, the apical annuli are composed of coiled-coil and signalling proteins assembled in a pore-like structure crossing the IMC barrier maintained during internal budding.

Crossing of the Cystic Barriers of Toxoplasma gondii by the Fluorescent Coumarin Tetra-Cyclopeptide.

FR235222 is a natural tetra-cyclopeptide with a strong inhibition effect on histone deacetylases, effective on mammalian cells as well as on intracellular apicomplexan parasites, such as Toxoplasma gondii, in the tachyzoite and bradyzoite stages. This molecule is characterized by two parts: the zinc-binding group, responsible for the binding to the histone deacetylase, and the cyclic tetrapeptide moiety, which plays a crucial role in cell permeability. Recently, we have shown that the cyclic tetrapeptide coupled with a fluorescent diethyl-amino-coumarin was able to maintain properties of cellular penetration on human cells. Here, we show that this property can be extended to the crossing of the Toxoplasma gondii cystic cell wall and the cell membrane of the parasite in its bradyzoite form, while maintaining a high efficacy as a histone deacetylase inhibitor. The investigation by molecular modeling allows a better understanding of the penetration mechanism.

Two essential Thioredoxins mediate apicoplast biogenesis, protein import, and gene expression in Toxoplasma gondii.

Apicomplexan parasites are global killers, being the causative agents of diseases like toxoplasmosis and malaria. These parasites are known to be hypersensitive to redox imbalance, yet little is understood about the cellular roles of their various redox regulators. The apicoplast, an essential plastid organelle, is a verified apicomplexan drug target. Nuclear-encoded apicoplast proteins traffic through the ER and multiple apicoplast sub-compartments to their place of function. We propose that thioredoxins contribute to the control of protein trafficking and of protein function within these apicoplast compartments. We studied the role of two Toxoplasma gondii apicoplast thioredoxins (TgATrx), both essential for parasite survival. By describing the cellular phenotypes of the conditional depletion of either of these redox regulated enzymes we show that each of them contributes to a different apicoplast biogenesis pathway. We provide evidence for TgATrx1's involvement in ER to apicoplast trafficking and TgATrx2 in the control of apicoplast gene expression components. Substrate pull-down further recognizes gene expression factors that interact with TgATrx2. We use genetic complementation to demonstrate that the function of both TgATrxs is dependent on their disulphide exchange activity. Finally, TgATrx2 is divergent from human thioredoxins. We demonstrate its activity in vitro thus providing scope for drug screening. Our study represents the first functional characterization of thioredoxins in Toxoplasma, highlights the importance of redox regulation of apicoplast functions and provides new tools to study redox biology in these parasites.

Lytic Cycle of Toxoplasma gondii: 15 Years Later.

Toxoplasmosis is the clinical and pathological consequence of acute infection with the obligate intracellular apicomplexan parasite Toxoplasma gondii. Symptoms result from tissue destruction that accompanies lytic parasite growth. This review updates current understanding of the host cell invasion, parasite replication, and eventual egress that constitute the lytic cycle, as well as the ways T. gondii manipulates host cells to ensure its survival. Since the publication of a previous iteration of this review 15 years ago, important advances have been made in our molecular understanding of parasite growth and mechanisms of host cell egress, and knowledge of the parasite's manipulation of the host has rapidly progressed. Here we cover molecular advances and current conceptual frameworks that include each of these topics, with an eye to what may be known 15 years from now.

Bioassay-based detection of Toxoplasma gondii in free-range chickens from Khorramabad, Iran: comparison of direct microscopy and semi-nested PCR.

This study aimed to investigate the occurrence of anti-Toxoplasma gondii antibodies in free-range chickens from Khorramabad, western Iran, and also to compare the performance of direct microscopy and semi-nested PCR in mice bioassayed with tissues from seropositive chickens. We investigated 97 serum samples from free-range chickens, using the modified agglutination test (MAT). Tissues from all seropositive chickens (MAT ≥ 1:10) were bioassayed in mice. All inoculated mice were examined by direct microscopy and a semi-nested PCR targeting the 529 bp repeat element (RE) of the parasite. Anti-T. gondii antibodies were detected in 21.6% of chicken sera. Eighteen of 21 (85.7%) seropositive chickens were positive in mouse bioassay using molecular DNA detection. However, biological forms of the parasite were isolated only from 11 (52.3%) seropositive chickens. Compared with semi-nested PCR, the sensitivity of direct microscopy was 62.1%. It can be concluded that although direct microscopy is a rapid and specific method for the detection of T. gondii, it does not detect the parasite in all experimentally infected mice. The low sensitivity of direct microscopy highlights the need for molecular techniques, such as RE-based semi-nested PCR, to increase the sensitivity of the mouse bioassay.

Dynamic protein S-palmitoylation mediates parasite life cycle progression and diverse mechanisms of virulence.

Eukaryotic parasites possess complex life cycles and utilize an assortment of molecular mechanisms to overcome physical barriers, suppress and/or bypass the host immune response, including invading host cells where they can replicate in a protected intracellular niche. Protein S-palmitoylation is a dynamic post-translational modification in which the fatty acid palmitate is covalently linked to cysteine residues on proteins by the enzyme palmitoyl acyltransferase (PAT) and can be removed by lysosomal palmitoyl-protein thioesterase (PPT) or cytosolic acyl-protein thioesterase (APT). In addition to anchoring proteins to intracellular membranes, functions of dynamic palmitoylation include - targeting proteins to specific intracellular compartments via trafficking pathways, regulating the cycling of proteins between membranes, modulating protein function and regulating protein stability. Recent studies in the eukaryotic parasites - Plasmodium falciparum, Toxoplasma gondii, Trypanosoma brucei, Cryptococcus neoformans and Giardia lamblia - have identified large families of PATs and palmitoylated proteins. Many palmitoylated proteins are important for diverse aspects of pathogenesis, including differentiation into infective life cycle stages, biogenesis and tethering of secretory organelles, assembling the machinery powering motility and targeting virulence factors to the plasma membrane. This review aims to summarize our current knowledge of palmitoylation in eukaryotic parasites, highlighting five exemplary mechanisms of parasite virulence dependent on palmitoylation.

Checkpoints of apicomplexan cell division identified in Toxoplasma gondii.

The unusual cell cycles of Apicomplexa parasites are remarkably flexible with the ability to complete cytokinesis and karyokinesis coordinately or postpone cytokinesis for several rounds of chromosome replication, and are well recognized. Despite this surprising biology, the molecular machinery required to achieve this flexibility is largely unknown. In this study, we provide comprehensive experimental evidence that apicomplexan parasites utilize multiple Cdk-related kinases (Crks) to coordinate cell division. We determined that Toxoplasma gondii encodes seven atypical P-, H-, Y- and L- type cyclins and ten Crks to regulate cellular processes. We generated and analyzed conditional tet-OFF mutants for seven TgCrks and four TgCyclins that are expressed in the tachyzoite stage. These experiments demonstrated that TgCrk1, TgCrk2, TgCrk4 and TgCrk6, were required or essential for tachyzoite growth revealing a remarkable number of Crk factors that are necessary for parasite replication. G1 phase arrest resulted from the loss of cytoplasmic TgCrk2 that interacted with a P-type cyclin demonstrating that an atypical mechanism controls half the T. gondii cell cycle. We showed that T. gondii employs at least three TgCrks to complete mitosis. Novel kinases, TgCrk6 and TgCrk4 were required for spindle function and centrosome duplication, respectively, while TgCrk1 and its partner TgCycL were essential for daughter bud assembly. Intriguingly, mitotic kinases TgCrk4 and TgCrk6 did not interact with any cyclin tested and were instead dynamically expressed during mitosis indicating they may not require a cyclin timing mechanism. Altogether, our findings demonstrate that apicomplexan parasites utilize distinctive and complex mechanisms to coordinate their novel replicative cycles.

Calmodulin-like proteins localized to the conoid regulate motility and cell invasion by Toxoplasma gondii.

Toxoplasma gondii contains an expanded number of calmodulin (CaM)-like proteins whose functions are poorly understood. Using a combination of CRISPR/Cas9-mediated gene editing and a plant-like auxin-induced degron (AID) system, we examined the roles of three apically localized CaMs. CaM1 and CaM2 were individually dispensable, but loss of both resulted in a synthetic lethal phenotype. CaM3 was refractory to deletion, suggesting it is essential. Consistent with this prediction auxin-induced degradation of CaM3 blocked growth. Phenotypic analysis revealed that all three CaMs contribute to parasite motility, invasion, and egress from host cells, and that they act downstream of microneme and rhoptry secretion. Super-resolution microscopy localized all three CaMs to the conoid where they overlap with myosin H (MyoH), a motor protein that is required for invasion. Biotinylation using BirA fusions with the CaMs labeled a number of apical proteins including MyoH and its light chain MLC7, suggesting they may interact. Consistent with this hypothesis, disruption of MyoH led to degradation of CaM3, or redistribution of CaM1 and CaM2. Collectively, our findings suggest these CaMs may interact with MyoH to control motility and cell invasion.

A proteomic analysis unravels novel CORVET and HOPS proteins involved in Toxoplasma gondii secretory organelles biogenesis.

Apicomplexans use the endolysosomal system for the biogenesis of their secretory organelles, namely, micronemes, rhoptries, and dense granules. In Toxoplasma gondii, our previous in silico search identified the HOPS tethering but not the CORVET complex and demonstrated a role of Vps11 (a common component for both complexes) in its secretory organelle biogenesis. Herein, we performed Vps11-GFP-Trap pull-down assays and identified by proteomic analysis, not only the CORVET-specific subunit Vps8 but also a BEACH domain-containing protein (BDCP) conserved in eukaryotes. We show that knocking-down Vps8 affects targeting of dense granule proteins, transport of rhoptry proteins, and the localization of the cathepsin L protease vacuolar compartment marker. Only a subset of micronemal proteins are affected by the absence of Vps8, shedding light on at least two trafficking pathways involved in microneme maturation. Knocking-down BDCP revealed a restricted and particular role of this protein in rhoptry and vacuolar compartment biogenesis. Moreover, depletion of BDCP or Vps8 abolishes parasite virulence in vivo. This study identified BDCP as a novel CORVET/HOPS-associated protein, playing specific roles and acting in concert during secretory organelle biogenesis, an essential process for host cell infection. Our results open the hypothesis for a role of BDCP in the vesicular trafficking towards lysosome-related organelles in mammals and yeast.

Excretion of Toxoplasma gondii oocysts from Feral Cats in Korea.

Sporulated oocysts from the feces of infected cats with Toxoplasma gondii can cause detrimental disease in both humans and animals. To investigate the prevalence of feral cats that excrete T. gondii oocysts in the feces, we examined fecal samples of 563 feral cats over a 3-year period from 2009 to 2011. Oocysts of T. gondii excreted into the feces were found from 4 of 128 cats in 2009 (3.1%) and one of 228 (0.4%) in 2010 while none of the 207 cats in 2010 were found positive with oocysts in their feces, resulting in an overall prevalence rate of 0.89% (5/563) between 2009 and 2011. Among the 5 cats that tested positive with T. gondii oocysts, 4 of the cats were male and 1 was a female with an average body weight of 0.87 kg. Numerous tissue cysts of 60 µm in diameter with thin (<0.5 µm) cyst walls were found in the brain of one of the 5 cats on necropsy 2 months after the identification of oocysts in the feces. A PCR amplification of the T. gondii-like oocysts in the feces of the positive cats using the primer pairs Tox-5/Tox-8 and Hham34F/Hham3R confirmed the presence of T. gondii oocysts in the feces. This study provides a good indication of the risk assessment of feral cats in the transmission of T. gondii to humans in Korea.

A plant/fungal-type phosphoenolpyruvate carboxykinase located in the parasite mitochondrion ensures glucose-independent survival of Toxoplasma gondii.

Toxoplasma gondii is considered to be one of the most successful intracellular pathogens, because it can reproduce in varied nutritional milieus, encountered in diverse host cell types of essentially any warm-blooded organism. Our earlier work demonstrated that the acute (tachyzoite) stage of T. gondii depends on cooperativity of glucose and glutamine catabolism to meet biosynthetic demands. Either of these two nutrients can sustain the parasite survival; however, what determines the metabolic plasticity has not yet been resolved. Here, we reveal two discrete phosphoenolpyruvate carboxykinase (PEPCK) enzymes in the parasite, one of which resides in the mitochondrion (TgPEPCKmt), whereas the other protein is not expressed in tachyzoites (TgPEPCKnet). Parasites with an intact glycolysis can tolerate genetic deletions of TgPEPCKmt as well as of TgPEPCKnet, indicating their nonessential roles for tachyzoite survival. TgPEPCKnet can also be ablated in a glycolysis-deficient mutant, while TgPEPCKmt is refractory to deletion. Consistent with this, the lytic cycle of a conditional mutant of TgPEPCKmt in the glycolysis-impaired strain was aborted upon induced repression of the mitochondrial isoform, demonstrating its essential role for the glucose-independent survival of parasites. Isotope-resolved metabolomics of the conditional mutant revealed defective flux of glutamine-derived carbon into RNA-bound ribose sugar as well as metabolites associated with gluconeogenesis, entailing a critical nodal role of PEPCKmt in linking catabolism of glucose and glutamine with anabolic pathways. Our data also suggest a homeostatic function ofTgPEPCKmt in cohesive operation of glycolysis and the tricarboxylic acid cycle in a normal glucose-replete milieu. Conversely, we found that the otherwise integrative enzyme pyruvate carboxylase (TgPyC) is dispensable not only in glycolysis-competent but also in glycolysis-deficient tachyzoites despite a mitochondrial localization. Last but not least, the observed physiology of T. gondii tachyzoites appears to phenocopy cancer cells, which holds promise for developing common therapeutics against both threats.

Characterization of a cytoplasmic glucosyltransferase that extends the core trisaccharide of the Toxoplasma Skp1 E3 ubiquitin ligase subunit.

Skp1 is a subunit of the SCF (Skp1/Cullin 1/F-box protein) class of E3 ubiquitin ligases that are important for eukaryotic protein degradation. Unlike its animal counterparts, Skp1 from Toxoplasma gondii is hydroxylated by an O2-dependent prolyl-4-hydroxylase (PhyA), and the resulting hydroxyproline can subsequently be modified by a five-sugar chain. A similar modification is found in the social amoeba Dictyostelium, where it regulates SCF assembly and O2-dependent development. Homologous glycosyltransferases assemble a similar core trisaccharide in both organisms, and a bifunctional α-galactosyltransferase from CAZy family GT77 mediates the addition of the final two sugars in Dictyostelium, generating Galα1, 3Galα1,3Fucα1,2Galß1,3GlcNAcα1-. Here, we found that Toxoplasma utilizes a cytoplasmic glycosyltransferase from an ancient clade of CAZy family GT32 to catalyze transfer of the fourth sugar. Catalytically active Glt1 was required for the addition of the terminal disaccharide in cells, and cytosolic extracts catalyzed transfer of [3H]glucose from UDP-[3H]glucose to the trisaccharide form of Skp1 in a glt1-dependent fashion. Recombinant Glt1 catalyzed the same reaction, confirming that it directly mediates Skp1 glucosylation, and NMR demonstrated formation of a Glcα1,3Fuc linkage. Recombinant Glt1 strongly preferred the full core trisaccharide attached to Skp1 and labeled only Skp1 in glt1Δ extracts, suggesting specificity for Skp1. glt1-knock-out parasites exhibited a growth defect not rescued by catalytically inactive Glt1, indicating that the glycan acts in concert with the first enzyme in the pathway, PhyA, in cells. A genomic bioinformatics survey suggested that Glt1 belongs to the ancestral Skp1 glycosylation pathway in protists and evolved separately from related Golgi-resident GT32 glycosyltransferases.

Apicoplast fatty acid synthesis is essential for pellicle formation at the end of cytokinesis in Toxoplasma gondii.

The apicomplexan protozoan Toxoplasma gondii, the causative agent of toxoplasmosis, harbors an apicoplast, a plastid-like organelle with essential metabolic functions. Although the FASII fatty acid biosynthesis pathway located in the apicoplast is essential for parasite survival, the cellular effects of FASII disruption in T. gondii had not been examined in detail. Here, we combined light and electron microscopy techniques - including focused ion beam scanning electron microscopy (FIB-SEM) - to characterize the effect of FASII disruption in T. gondii, by treatment with the FASII inhibitor triclosan or by inducible knockdown of the FASII component acyl carrier protein. Morphological analyses showed that FASII disruption prevented cytokinesis completion in T. gondii tachyzoites, leading to the formation of large masses of 'tethered' daughter cells. FIB-SEM showed that tethered daughters had a mature basal complex, but a defect in new membrane addition between daughters resulted in incomplete pellicle formation. Addition of exogenous fatty acids to medium suppressed the formation of tethered daughter cells and supports the notion that FASII is essential to generate lipid substrates required for the final step of parasite division.

The Conoid Associated Motor MyoH Is Indispensable for Toxoplasma gondii Entry and Exit from Host Cells.

Many members of the phylum of Apicomplexa have adopted an obligate intracellular life style and critically depend on active invasion and egress from the infected cells to complete their lytic cycle. Toxoplasma gondii belongs to the coccidian subgroup of the Apicomplexa, and as such, the invasive tachyzoite contains an organelle termed the conoid at its extreme apex. This motile organelle consists of a unique polymer of tubulin fibres and protrudes in both gliding and invading parasites. The class XIV myosin A, which is conserved across the Apicomplexa phylum, is known to critically contribute to motility, invasion and egress from infected cells. The MyoA-glideosome is anchored to the inner membrane complex (IMC) and is assumed to translocate the components of the circular junction secreted by the micronemes and rhoptries, to the rear of the parasite. Here we comprehensively characterise the class XIV myosin H (MyoH) and its associated light chains. We show that the 3 alpha-tubulin suppressor domains, located in MyoH tail, are necessary to anchor this motor to the conoid. Despite the presence of an intact MyoA-glideosome, conditional disruption of TgMyoH severely compromises parasite motility, invasion and egress from infected cells. We demonstrate that MyoH is necessary for the translocation of the circular junction from the tip of the parasite, where secretory organelles exocytosis occurs, to the apical position where the IMC starts. This study attributes for the first time a direct function of the conoid in motility and invasion, and establishes the indispensable role of MyoH in initiating the first step of motility along this unique organelle, which is subsequently relayed by MyoA to enact effective gliding and invasion.

Surface attachment, promoted by the actomyosin system of Toxoplasma gondii is important for efficient gliding motility and invasion.

BACKGROUND: Apicomplexan parasites employ a unique form of movement, termed gliding motility, in order to invade the host cell. This movement depends on the parasite's actomyosin system, which is thought to generate the force during gliding. However, recent evidence questions the exact molecular role of this system, since mutants for core components of the gliding machinery, such as parasite actin or subunits of the MyoA-motor complex (the glideosome), remain motile and invasive, albeit at significantly reduced efficiencies. While compensatory mechanisms and unusual polymerisation kinetics of parasite actin have been evoked to explain these findings, the actomyosin system could also play a role distinct from force production during parasite movement. RESULTS: In this study, we compared the phenotypes of different mutants for core components of the actomyosin system in Toxoplasma gondii to decipher their exact role during gliding motility and invasion. We found that, while some phenotypes (apicoplast segregation, host cell egress, dense granule motility) appeared early after induction of the act1 knockout and went to completion, a small percentage of the parasites remained capable of motility and invasion well past the point at which actin levels were undetectable. Those act1 conditional knockout (cKO) and mlc1 cKO that continue to move in 3D do so at speeds similar to wildtype parasites. However, these mutants are virtually unable to attach to a collagen-coated substrate under flow conditions, indicating an important role for the actomyosin system of T. gondii in the formation of attachment sites. CONCLUSION: We demonstrate that parasite actin is essential during the lytic cycle and cannot be compensated by other molecules. Our data suggest a conventional polymerisation mechanism in vivo that depends on a critical concentration of G-actin. Importantly, we demonstrate that the actomyosin system of the parasite functions in attachment to the surface substrate, and not necessarily as force generator.

Structures of the Toxoplasma gliding motility adhesin.

Micronemal protein 2 (MIC2) is the key adhesin that supports gliding motility and host cell invasion by Toxoplasma gondii. With a von Willebrand factor A (VWA) domain and six thrombospondin repeat domains (TSR1-6) in its ectodomain, MIC2 connects to the parasite actomyosin system through its cytoplasmic tail. MIC2-associated protein (M2AP) binds noncovalently to the MIC2 ectodomain. MIC2 and M2AP are stored in micronemes as proforms. We find that the MIC2-M2AP ectodomain complex is a highly elongated 1:1 monomer with M2AP bound to the TSR6 domain. Crystal structures of N-terminal fragments containing the VWA and TSR1 domains for proMIC2 and MIC2 reveal a closed conformation of the VWA domain and how it associates with the TSR1 domain. A long, proline-rich, disulfide-bonded pigtail loop in TSR1 overlaps the VWA domain. Mannose α-C-linked to Trp-276 in TSR1 has an unusual (1)C4 chair conformation. The MIC2 VWA domain includes a mobile α5-helix and a 22-residue disordered region containing two disulfide bonds in place of an α6-helix. A hydrophobic residue in the prodomain binds to a pocket adjacent to the α7-helix that pistons in opening of the VWA domain to a putative high-affinity state.