The inner membrane complex sub-compartment proteins critical for replication of the apicomplexan parasite Toxoplasma gondii adopt a pleckstrin homology fold

Abstract

Toxoplasma gondii, an apicomplexan parasite prevalent in developed nations, infects up to one-third of the human population. The success of this parasite depends on several unique structures including an inner membrane complex (IMC) that lines the interior of the plasma membrane and contains proteins important for gliding motility and replication. Of these proteins, the IMC sub-compartment proteins (ISPs) have recently been shown to play a role in asexual T. gondii daughter cell formation, yet the mechanism is unknown. Complicating mechanistic characterization of the ISPs is a lack of sequence identity with proteins of known structure or function. In support of elucidating the function of ISPs, we first determined the crystal structures of representative members TgISP1 and TgISP3 to a resolution of 2.10 and 2.32 Å, respectively. Structural analysis revealed that both ISPs adopt a pleckstrin homology fold often associated with phospholipid binding or protein-protein interactions. Substitution of basic for hydrophobic residues in the region that overlays with phospholipid binding in related pleckstrin homology domains, however, suggests that ISPs do not retain phospholipid binding activity. Consistent with this observation, biochemical assays revealed no phospholipid binding activity. Interestingly, mapping of conserved surface residues combined with crystal packing analysis indicates that TgISPs have functionally repurposed the phospholipid-binding site likely to coordinate protein partners. Recruitment of larger protein complexes may also be aided through avidity-enhanced interactions resulting from multimerization of the ISPs. Overall, we propose a model where TgISPs recruit protein partners to the IMC to ensure correct progression of daughter cell formation.

Keywords: Apicomplexa; Cell Division; Endodyogeny; Inner Membrane Complex; Parasite; Pleckstrin Homology Fold; Protein-Protein Interactions; Structural Biology; Toxoplasma gondii; X-ray Crystallography.

FIGURE 1.

Construct design and gel filtration profiles for TgISPs from both apicomplexan ISP clades show complex molecular organization. A, schematic of the distribution of ISPs along the IMC of T. gondii (adapted from Ref 14). B, phylogenetic analysis of apicomplexan ISPs shows a bifurcated clustering with either TgISP1/ISP2 or TgISP3. C, top, two constructs were subcloned for characterization of TgISP1: TgISP1-1 and TgISP1-2. Bottom, size exclusion chromatograms for TgISP1-1 (black line, 19.5 kDa) and TgISP1-2 (purple line; 13.9 kDa) from a Superdex75 16/60 column. Vertical lines represent the peak centers for a set of globular standards. D, top, TgISP2: TgISP2-1 and TgISP2–2. Bottom, size exclusion chromatograms for TgISP2-1 (black line; 17.3 kDa), TgISP2–2 (blue line, 14.5 kDa). E, top, TgISP3: TgISP3-1, TgISP3-2. Bottom, size exclusion chromatograms for TgISP3-1 (black line; 18.3 kDa), TgISP3-2 (green line, 15.0 kDa).

FIGURE 2.

Structural characterization of TgISP1-2 and TgISP3-1 reveals conservation of a similar fold across both apicomplexan ISP phylogenetic clades. A, orthogonal views of TgISP1-2 shown as a purple schematic with the disulfide bond shown in ball-and-stick representation. B, orthogonal views of TgISP3-1 shown as a green schematic; dotted lines indicate connectivity of disordered loops. C, overlay of TgISP1-2 (purple schematic) on TgISP3-1 (green schematic). Although the core fold is clearly conserved, arrows indicate the two major regions of divergence between the two proteins. Solid arrow, β2-β3 disulfide bond of TgISP1-2. Dashed arrow, β5-β6 loop and N-term positioning. D, structure-based sequence alignment of TgISP1, TgISP2 (based on homology model), and TgISP3. Triangles indicate the crystallized constructs; boxed Gly/Cys residues are lipidated; the divergent β5-β6 loop is underlined.

FIGURE 3.

The ISPs adopt a pleckstrin homology fold. A, top, secondary structure depiction of TgISP1-2 colored in rainbow from N (blue) to C (red). Note: the extended ordering of the N terminus in TgISP3 leads to a shift in the rainbow throughout the secondary structure elements. The black arrow indicates the single disulfide bond. Bottom, topology diagram for the PH fold of TgISP1-2; common features with TgISP3-1 are shown as black outlined arrows (β-strands) and rectangles (α-helices) with black connectors. Features specific to TgISP1-2 are shown in gray. B, top, secondary structure depiction of TgISP3-1 colored as in A. Dotted lines indicate the two unmodeled loops in the crystal structure. Bottom, topology diagram as described in A. C, top, secondary structure depiction of phospholipase C-δ 1 (PDB ID 1MAI-A). Bottom, topology diagram.

FIGURE 4.

Surface analysis and basic side chain distribution support the lack of a phospholipid binding role for the TgISPs. A, chimera (34) coulombic-colored surface representation of the PH domain of phospholipase Cδ1 (PLCδ1_PH; PDB ID 1MAI) shows a concentration of basic charge around the inositol-(1,4,5)-trisphosphate (Ins(1,4,5)P3, green ball-and-stick representation) binding site at the bottom of the molecule open end (top), which is due to a clustering of basic residues forming specific interactions with the ligand (bottom). B, TgISP1-2; coulombic-colored surface shows a strong hydrophobic patch and just two basic residues forming separate small basic patches (top); a sulfate ion is seen coordinated predominately by Arg-110 near the top of the open end, and the constraining disulfide bond is indicated by a black arrow (bottom). C, TgISP3-1; coulombic-colored surface shows both basic and hydrophobic character (top), but the residues are dispersed (bottom).

FIGURE 5.

The region corresponding to lipid binding functionality observed for other PH domains is highly conserved in the apicomplexan ISPs and capable of coordinating a polypeptide structure in TgISP1. A, surfaces on PH domains identified to mediate interactions with phospholipid headgroups (left) or proteins/peptides (right) are mapped in purple onto TgISP1. B, mapping of conserved (burgundy) and variable (teal) residues onto the TgISP1core using ConSurf (19) shows a clear bias for conservation on one side of the protein. Viewpoint in (B, left) is aligned with RnPLC-δ1 of Fig. 4A. C, left, the purple surface of TgISP1-2 shows a deep groove (black line) that partially overlays with the phospholipid binding region of other PH domains. Bottom, in every chain of the AU in two different space groups, the N terminus of a neighboring chain (gray schematic; SMASPQV sequence is shown as in ball and stick representation) buries into the surface groove. Inset, the positioning of the sulfate ion coordinated by the highly conserved Arg-110 is consistent with an acidic or phosphorylated amino acid at the A position (gray dotted line).

FIGURE 6.

Multimeric state may play a role in ISP function. A schematic representation of experimentally supported organizations of the ISPs on the inner membrane complex of T. gondii is shown. ISP structures and homology models are shown as a colored schematic backbone with a semi-transparent gray surface. Purple ovals represent predicted N-terminal helices of TgISP1. Orange starbursts indicate phosphorylation sites. TgISP1 core and TgISP3 full were structurally determined, whereas TgISP2 full-length monomer and dimer are homology models.