Aldolase provides an unusual binding site for thrombospondin-related anonymous protein in the invasion machinery of the malaria parasite

Abstract

An actomyosin motor located underneath the plasma membrane drives motility and host-cell invasion of apicomplexan parasites such as Plasmodium falciparum and Plasmodium vivax, the causative agents of malaria. Aldolase connects the motor actin filaments to transmembrane adhesive proteins of the thrombospondin-related anonymous protein (TRAP) family and transduces the motor force across the parasite surface. The TRAP-aldolase interaction is a distinctive and critical trait of host hepatocyte invasion by Plasmodium sporozoites, with a likely similar interaction crucial for erythrocyte invasion by merozoites. Here, we describe 2.4-A and 2.7-A structures of P. falciparum aldolase (PfAldo) obtained from crystals grown in the presence of the C-terminal hexapeptide of TRAP from Plasmodium berghei. The indole ring of the critical penultimate Trp-residue of TRAP fits snugly into a newly formed hydrophobic pocket, which is exclusively delimited by hydrophilic residues: two arginines, one glutamate, and one glutamine. Comparison with the unliganded PfAldo structure shows that the two arginines adopt new side-chain rotamers, whereas a 25-residue subdomain, forming a helix-loop-helix unit, shifts upon binding the TRAP-tail. The structural data are in agreement with decreased TRAP binding after mutagenesis of PfAldo residues in and near the induced TRAP-binding pocket. Remarkably, the TRAP- and actin-binding sites of PfAldo seem to overlap, suggesting that both the plasticity of the aldolase active-site region and the multimeric nature of the enzyme are crucial for its intriguing nonenzymatic function in the invasion machinery of the malaria parasite.

Fig. 1.

Structure of P. falciparum aldolase and bound TRAP-tail. (A) Structural overview of one PfAldo subunit (light blue ribbon). Residues binding to the TRAP-tail are highlighted in red; residues involved in binding of F1,6P in the active site are shown in blue. Key substrate binding residues are as follows: Pf-Asp-39 (HsA-Asp-33), Pf-Lys-112 (HsA-Lys-107), Pf-Lys-151 (HsA-Lys-146), Pf-Arg-153 (HsA-Arg-148), and Pf-Glu-194 (HsA-Glu-187). The main chain nitrogens of Pf-Ser-278 (HsA-Ser-271), Pf-Gly-279 (HsA-Gly-272) bind to the 1-phosphate group of F1,6P (30). Labels of PfAldo residues equivalent to rabbit aldolase amino acids implicated by site directed mutagenesis studies (43) to be involved in actin binding (i.e., R48, K112, R153, and K236) are highlighted with a gold background. (B) Difference electron densities for the DWN C-terminal residues of the TRAP-tail in two independent PfAldo:TRAP-tail structures. The σA weighted (F_o − F_c) difference densities, shown in green and contoured at 2σ, were calculated by omitting all TRAP-tail residues during the refinement procedure. The C-terminal TRAP-tail residues 604–606 are shown as sticks in magenta. The corresponding (2F_o − F_c) electron density maps of subunit D in both structures is contoured at 1σ in blue. (Upper) The 2.4-Å data set. (Lower) The 2.7-Å data set (see SI Materials and Methods and SI Table 1). (C) Close-up of the active site and TRAP-binding site with important residues highlighted. The TRAP-tail residues are shown in magenta, TRAP-binding residues in red, and active-site residues in blue.

Fig. 2.

Key interactions of the TRAP-tail with P. falciparum aldolase. (A) TRAP-tail residues D604, W605, and N606 (magenta) interacting with PfAldo residues (light blue). C_α positions are marked as spheres. Selected hydrophobic contacts are shown with red lines and distances in angstroms. The hydrogen bond between the indole nitrogen of TRAP-W605 and the carboxylate of E40, and the interactions between TRAP-N606 with R153 and K151, and of the backbone oxygen of TRAP-D604 with R48, are depicted in black. (B) Sequence alignment of the C-terminal TRAP-tail residues of three plasmodial species. Identical residues are shaded in red. All TRAP residues involved in contacts with aldolase are well conserved in several Plasmodium species.

Fig. 3.

Conformational changes of P. falciparum aldolase upon TRAP-tail binding. Shown is a stereoview of the TRAP-binding region. The TRAP-bound structure is shown in light blue, the unliganded structure in gray (29), and the TRAP-tail C-terminal tripeptide DWN in magenta. Key residues binding the penultimate Trp-indole ring are highlighted with sticks. Note the rigid body motion of helix α2, which is part of the helix–loop–helix T42-D66 subdomain shift upon TRAP-binding (see The Indole Anchor-Binding Pocket).

Fig. 4.

TRAP-tail binding interferes with substrate binding. Shown is a stereoview of the PfAldo:TRAP-tail complex superimposed onto the structure of human aldolase A complexed with F1,6P (30) (PDB ID code 4ALD). Key residues enabling the accommodation of the penultimate Trp-indole ring are highlighted as sticks. The TRAP-tail is shown in magenta, residues involved in substrate binding in blue, and human aldolase A in yellow. The substrate F1,6P are shown as sticks in green, with the two phosphate groups in orange. The C-terminal TRAP-tail residue partially occludes the substrate-binding site.

Fig. 5.

Effects of PfAldo residue substitutions on TRAP-binding. The percentage of TRAP-binding compared with WT PfAldo, as evaluated by methods described elsewhere (19), is plotted on the vertical axis. The aldolase mutants studied are plotted along the horizontal axis. The excellent correlation between the effect of the substitutions and the structure of the PfAldo:TRAP-tail complex is discussed in Mutation Studies.