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Aberrant activation of the epidermal growth factor receptor (EGFR) by mutations has been implicated in a variety of human cancers. Elucidation of the structure of the full-length receptor is essential to understand the molecular mechanisms underlying its activation. Unlike previously anticipated, here, we report that purified full-length EGFR adopts a homodimeric form in vitro before and after activation. Cryo-electron tomography analysis of the purified receptor also showed that the extracellular domains of the receptor dimer, which are conformationally flexible before activation, are stabilised by ligand binding. Consistently, optical single-molecule observation also demonstrated that binding of only one ligand activates the receptor dimer on the cell surface. Based on these results, we propose an allosteric model for the activation of EGFR dimers by ligand binding. Our results demonstrate how oncogenic mutations spontaneously activate the receptor and shed light on the development of novel cancer therapies.

Proliferation and differentiation inside erythrocytes are important steps in the life cycle of Plasmodium spp. To achieve these, the parasites export polypeptides to the surface of infected erythrocytes; for example, to import nutrients and to bind to other erythrocytes and the host microvasculature. Binding is mediated by the adhesive polypeptides Plasmodium falciparum-encoded repetitive interspersed families of polypeptides (RIFINs), subtelomeric variant open reading frame (STEVOR) and P. falciparum erythrocyte membrane protein 1 (PfEMP1), which are encoded by multigene families to ensure antigenic variation and evasion of host immunity. These variant surface antigens are suggested to mediate the sequestration of infected erythrocytes in the microvasculature and block the blood flow when binding is excessive. In this Review, we discuss the multigene families of surface variant polypeptides and highlight their roles in causing severe malaria.

Malaria parasite UIS3 (up-regulated in infective sporozoites gene 3) is essential for sporozoite development in infected hepatocytes. UIS3 encodes for a membrane protein that is localized to the parasite parasitophorous vacuolar membrane in infected hepatocytes. We describe here 2.5-A resolution crystal structure of Plasmodium falciparum UIS3 soluble domain (PfUIS3(130-229)) in complex with the lipid phosphatidylethanolamine (PE). PfUIS3(130-229) is a novel, compact, and all alpha-helical structure bound to one molecule of PE. The PfUIS3(130-229)-PE complex structure reveals a novel binding site with specific interactions between PfUIS3(130-229) and the PE head group. One acyl chain of PE wraps around part of PfUIS3(130-229) and docks onto a hydrophobic channel. We additionally provide new structural and biochemical evidence of PfUIS3(130-229) interactions with lipids (phosphatidylethanolamine), with phospholipid liposomes, and with the human liver fatty acid-binding protein. The direct interaction of PfUIS3(130-229) with liver fatty acid-binding protein most likely provides the parasite with a conduit for importing essential fatty acids/lipids. Therefore, our analyses have implications for lipid transport into the parasite during the rapid growth phases of sporozoites. Given that PfUIS3 is essential for establishment of liver stage infection by P. falciparum, our data provide a new target for abrogating parasite development within liver cells before typical symptoms of malaria can manifest.

SummaryPlasmodium falciparum virulence is associated with sequestration of infected erythrocytes. Microvascular binding mediated by PfEMP1 in complex with non-immune immunoglobulin M (IgM) is common among parasites that cause both severe childhood malaria and pregnancy-associated malaria. Here, we present cryo-molecular electron tomography structures of human IgM, PfEMP1 and their complex. Three-dimensional reconstructions of IgM reveal that it has a dome-like core, randomly oriented Fab2s units, and the overall shape of a turtle. PfEMP1 is a C- shaped molecule with a flexible N terminus followed by an arc-shaped backbone and a bulky C terminus that interacts with IgM. Our data demonstrate that the PfEMP1 binding pockets on IgM overlap with those of C1q, and the bulkiness of PfEMP1 limits the capacity of IgM to interact with PfEMP1. We suggest that P. falciparum exploits IgM to cluster PfEMP1 into an organized matrix to augment its affinity to host cell receptors.

Plasmodium falciparum encodes approximately 5300 proteins of which approximately 35% have repeats of amino acids, significantly higher than in other fully sequenced eukaryotes. The proportion of proteins with amino acid homorepeats varies from 4 to 54% amongst different functional classes of proteins. These homorepeats are dominated by asparagines, which are selected over lysines despite equivalent AT codon content. Surprisingly, asparagine repeats are absent from the variant surface antigen protein families of PfEMP1s, Stevors and Rifins. The PfEMP1 protein family is instead rich in recurrences of glutamates, similar to human cell surface proteins. Structural mapping of homorepeats suggests that these segments are likely to form surface exposed structures that protrude from the main protein cores. We also found an abundance of asparagine-rich prion-like domains in P. falciparum, significantly larger than in any other eukaryote. Domains rich in glutamines and asparagines have an innate predisposition to form self-propagating amyloid fibers, which are involved both in prion-based inheritance and in human neurodegenerative disorders. Nearly 24% (1302 polypeptides) of P. falciparum proteins contain prion-forming or prion-inducing domains, in comparison to Drosophila (approximately 3.4%) which to date showed the highest number of prion-like proteins. The unexpected properties of P. falciparum revealed here open new avenues for investigating parasite biology.

TRAP (thrombospondin-related anonymous protein) is a sporozoite surface protein that plays a central role in hepatocyte invasion. We have developed procedures for recombinant production of the entire ECD (extracellular domain) and A domain of TRAP using bacterial- and baculovirus-expression systems respectively. The ECD and A domain were purified to homogeneity and migrated on gel-filtration columns as non-aggregated, monomeric proteins. These adhesive modules bound to HepG2 cells in a dose-dependent and bivalent cation-independent manner. The binding of ECD and the A domain to HepG2 cells was inhibited poorly by an excess of sulphatide analogues, suggesting the presence of as yet unidentified receptors for the A domain on hepatocytes. Using surface-plasmon-resonance-based sensor technology (Biacore), we demonstrate that TRAP ECD has higher affinity for heparin (KD=40 nM) compared with the A domain (KD=79 nM). We also present a three-dimensional structure of the A domain based on the crystal structure of the homologous von Willebrand factor A1 domain. The TRAP A domain shows two spatially distinct ligand-binding surfaces. One surface on the A domain contains the MIDAS (metal-ion-dependent adhesion site) motif, where point mutations of Thr131 and Asp162 correlate with impairment of cell infectivity by sporozoites. The other surface contains a putative heparin-binding site and consists of a basic residue cluster. Our studies suggest that TRAP interacts with multiple receptors during the hepatocyte invasion process. Our results also pave the way for inclusion of these high-quality recombinant TRAP domains in subunit-based vaccines against malaria.

Rosetting is a virulent Plasmodium falciparum phenomenon associated with severe malaria. Here we demonstrate that P. falciparum-encoded repetitive interspersed families of polypeptides (RIFINs) are expressed on the surface of infected red blood cells (iRBCs), where they bind to RBCs--preferentially of blood group A--to form large rosettes and mediate microvascular binding of iRBCs. We suggest that RIFINs have a fundamental role in the development of severe malaria and thereby contribute to the varying global distribution of ABO blood groups in the human population.

We have conducted a survey of proline-rich (PxxP) motifs in the proteomes of human, mouse, yeast, Mycobacterium tuberculosis and Plasmodium falciparum. Our analyses reveal a strikingly high occurrence of these motifs in each organism, suggesting a wide dependence on protein-protein interaction networks in cellular systems. All proteomes considered have an abundance of PxxP motifs which can potentially participate in binding to SH3 domain-containing proteins. A large fraction of these motifs can be assigned to structurally conserved types of class I and class II sequences. We propose that while maintaining the primary biochemical function, many proteins are likely to participate in additional interactions involving molecular cross-talk with other proteins using proline-rich and other motifs. We have also identified PxxP-containing motifs that are unique to P.falciparum and M.tuberculosis. These sequences may serve as leads for the development of peptidomimics that specifically target these organisms. We propose a novel drug target selection strategy where shared PxxP-containing motifs can be used to direct the development of inhibitors that focus on multiple targets in the cell. Screening for such unique PxxP-containing motifs in the P.falciparum proteome yielded highly conserved sequences in the variant surface antigen family that can be used to initiate design of peptidomimics that may potentially abrogate parasite cytoadherence during malaria infections.

Background Thrombospondin-related anonymous protein (TRAP) is essential for sporozoite motility and for liver cell invasion. TRAP is a type 1 membrane protein that possesses multiple adhesive domains in its extracellular region. Methods Plasmodium falciparum TRAP (PfTRAP) and its subdomains were expressed in a mammalian expression system, and eleven different mutants generated to study interaction of PfTRAP with liver cells. Binding studies between HepG2 cell extracts and PfTRAP were performed using co-immunoprecipitation protocols. Results Five different amino acid residues of PfTRAP that are involved in liver cell binding have been identified. These PfTRAP mutants bound to heparin like the wild type PfTRAP thereby suggesting a non-heparin mediated binding of PfTRAP to liver cells. Three Src family proteins -Lyn, Lck and CrkL which interact with PfTRAP are also identified. Liver cell extracts and immunoprecipitated Src family kinases phosphorylated PfTRAP at multiple sites. An analysis of multiple TRAP sequences revealed Src homology 3 domain (SH3) binding motifs. Conclusion Binding of PfTRAP to SH3-domain containing proteins like Src-family kinases and their ability to phosphorylate PfTRAP suggests a novel role for PfTRAP in cell signaling during sporozoite invasion and homing inside the liver cells. These data shed new light on TRAP-liver cell interactions.