Structural analysis of the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) intracellular domain reveals a conserved interaction epitope

Abstract

Plasmodium falciparum-infected red blood cells adhere to endothelial cells, thereby obstructing the microvasculature. Erythrocyte adherence is directly associated with severe malaria and increased disease lethality, and it is mediated by the PfEMP1 family. PfEMP1 clustering in knob-like protrusions on the erythrocyte membrane is critical for cytoadherence, however the molecular mechanisms behind this system remain elusive. Here, we show that the intracellular domains of the PfEMP1 family (ATS) share a unique molecular architecture, which comprises a minimal folded core and extensive flexible elements. A conserved flexible segment at the ATS center is minimally restrained by the folded core. Yeast-two-hybrid data and a novel sequence analysis method suggest that this central segment contains a conserved protein interaction epitope. Interestingly, ATS in solution fails to bind the parasite knob-associated histidine-rich protein (KAHRP), an essential cytoadherence component. Instead, we demonstrate that ATS associates with PFI1780w, a member of the Plasmodium helical interspersed sub-telomeric (PHIST) family. PHIST domains are widespread in exported parasite proteins, however this is the first specific molecular function assigned to any variant of this family. We propose that PHIST domains facilitate protein interactions, and that the conserved ATS epitope may be targeted to disrupt the parasite cytoadherence system.

FIGURE 1.

Molecular architecture of the ATS domain of PfEMP1. A, schematic representation of the ATS-FL, ATS-25, and ATS-Core constructs. Residue numbering starts at the end of the preceding transmembrane segment. Red segments correspond to the two well-structured α-helical parts of ATS-FL that, together, comprise ATS-Core. B, NMR ¹H-¹⁵N HSQC spectra of ATS-FL at 25 °C. Distinct resonances, corresponding to the structured part of this construct, are circled in red. Trp sidechain resonances are boxed. C, Kratky representation of SAXS data from ATS-FL at 20 °C. D and E, similar NMR and SAXS spectra from ATS-Core. Note the persistence of dispersed resonances (red circles) across ATS constructs.

FIGURE 2.

Structure of ATS-Core and ATS-25. A and B, schematic representation of the ATS-Core structure in two orthogonal orientations. The helical elements and protein termini are denoted. Shown in magenta and red are the segments arising from the first and second distinct α-helical regions of ATS, respectively. C, representative members from the ATS-25 structure ensemble calculated from NMR restraints and based on ATS-Core. The structured core is shown in red, the central flexible segment in gold, and the construct N terminus in blue. The flexible segments of this construct do not adopt a unique structure but, rather, explore a wide range of conformations and a large volume of space, as illustrated in this ensemble.

FIGURE 3.

Solution studies of the ATS interactions. A, schematic representation of KAHRP fragments. Shown here is full-length KAHRP from P. falciparum 3D7 (red bar) as well as the relative sizes and locations of the KAHRP subfragments (K1, K2, K3, K1A, and K2A). The residue boundaries of each construct are indicated. B, overlay of NMR spectra of ¹⁵N-labeled ATS-25 alone (red) or in the presence of unlabeled K1A (green). Both spectra were recorded in NMR buffer and 25 °C. C, similar overlay of NMR spectra for ATS-25 alone or with K2A. Neither K1A nor K2A produce significant perturbations in the ATS-25 spectra. D, fluorescence anisotropy titrations of labeled ATS-FL with KAHRP fragments, GST, or PFI1780w. None of the KAHRP fragments nor GST produced strong anisotropy changes in concentrations up to 300 μm (50 μm for K1 due to limited solubility). PFI1780w titrations yield a binding isotherm that can be fit (solid line) to a single site model with a K_d of ∼150 μm. Error bars derive from five replicates.

FIGURE 4.

The ATS central flexible segment is a hotspot for protein interactions. A, top: schematic representation of the central flexible segment (orange box) with respect to ATS-FL. The thirteen P. falciparum 3D7 genes found to interact with ATS variants by yeast-two-hybrid methods (21) are noted, and the ATS region of interaction in each case is shown (blue boxes). B, per-residue protein interaction index for ATS-FL. The central flexible segment is highlighted in light orange color.

FIGURE 5.

ATS interacts with PFI1780w. A, overlay of NMR spectra of ¹⁵N-labeled ATS-FL alone (red) or in the presence of unlabeled PFI1780w (green). Both spectra were recorded in NMR buffer and 25 °C. B and C, similar overlay of NMR spectra for ATS-25 (B) or ATS-Core (C) alone or in the presence of unlabeled PFI1780w. Note the significant loss of signal intensity upon titration observed for many resonances in the ATS-FL and ATS-25 spectra, but not when using ATS-Core. A number of ATS resonances originating from the structured protein core are enclosed in a dashed box; resonances originating from specific residues of the central flexible element are labeled.