The crystal structure of Echinococcus granulosus fatty-acid-binding protein 1

We describe the 1.6 Å crystal structure of the fatty-acid-binding protein EgFABP1 from the parasitic platyhelminth Echinococcus granulosus. E. granulosus causes hydatid disease, which is a major zoonosis. EgFABP1 has been implicated in the acquisition, storage, and transport of lipids, and may be important to the organism since it is incapable of synthesising most of its lipids de novo. Moreover, EgFABP1 is a promising candidate for a vaccine against hydatid disease.The crystal structure reveals that EgFABP1 has the expected 10-stranded β-barrel fold typical of the family of intracellular lipid-binding proteins, and that it is structurally most similar to P2 myelin protein. We describe the comparison of the crystal structure of EgFABP1 with these proteins and with an older homology model for EgFABP1.The electron density reveals the presence of a bound ligand inside the cavity, which we have interpreted as palmitic acid. The carboxylate group of the fatty acid interacts with the protein's P2 motif, consisting of a conserved triad R…R-x-Y. The hydrophobic tail of the ligand assumes a fairly flat, U-shaped conformation and has relatively few interactions with the protein.We discuss some of the structural implications of the crystal structure of EgFABP1 for related platyhelminthic FABPs.     

Direct Interaction between EgFABP1, a Fatty Acid Binding Protein from Echinococcus granulosus,and Phospholipid Membranes

Background

Growth and maintenance of hydatid cysts produced by Echinococcus granulosus have a high requirement for host lipids for biosynthetic processes, membrane building and possibly cellular and developmental signalling. This requires a high degree of lipid trafficking facilitated by lipid transporter proteins. Members of the fatty acid binding protein (FABP) family have been identified in Echinococcus granulosus, one of which, EgFABP1 is expressed at the tegumental level in the protoscoleces, but it has also been described in both hydatid cyst fluid and secretions of protoscoleces. In spite of a considerable amount of structural and biophysical information on the FABPs in general, their specific functions remain mysterious.

Methodology/Principal Findings

We have investigated the way in which EgFABP1 may interact with membranes using a variety of fluorescence-based techniques and artificial small unilamellar vesicles. We first found that bacterial recombinant EgFABP1 is loaded with fatty acids from the synthesising bacteria, and that fatty acid binding increases its resistance to proteinases, possibly due to subtle conformational changes induced on EgFABP1. By manipulating the composition of lipid vesicles and the ionic environment, we found that EgFABP1 interacts with membranes in a direct contact, collisional, manner to exchange ligand, involving both ionic and hydrophobic interactions. Moreover, we observed that the protein can compete with cytochrome c for association with the surface of small unilamellar vesicles (SUVs).

Conclusions/Significance

This work constitutes a first approach to the understanding of protein-membrane interactions of EgFABP1. The results suggest that this protein may be actively involved in the exchange and transport of fatty acids between different membranes and cellular compartments within the parasite.     

Binding properties of Echinococcus granulosus fatty acid binding protein.

EgFABP1 is a developmentally regulated intracellular fatty acid binding protein characterized in the larval stage of parasitic platyhelminth Echinococcus granulosus. It is structurally related to the heart group of fatty acid binding proteins (H-FABPs). Binding properties and ligand affinity of recombinant EgFABP1 were determined by fluorescence spectroscopy using cis- and trans-parinaric acid. Two binding sites for cis- and trans-parinaric acid were found (K(d(1)) 24+/-4 nM, K(d(2)) 510+/-60 nM for cis-parinaric acid and K(d(1)) 32+/-4 nM, K(d(2)) 364+/-75 nM for trans-parinaric). A putative third site for both fatty acids is discussed. Binding preferences were determined using displacement assays. Arachidonic and oleic acids presented the highest displacement percentages for EgFABP1. The Echinococcus FABP is the unique member of the H-FABP group able to bind two long chain fatty acid molecules with high affinity. Structure-function relationships and putative roles for EgFABP1 in E. granulosus metabolism are discussed.     

Genomic structure and expression of a gene coding for a new fatty acid binding protein from Echinococcus granulosus

This work describes a new gene coding for a fatty acid binding protein (FABP) in the parasite Echinococcus granulosus, named EgFABP2. The complete gene structure, including the promoter sequence, is reported. The genomic coding domain organisation of the previously reported E. granulosus FABP gene (EgFABP1) has been also determined. The corresponding polypeptide chains share 76% of identical residues and an overall 96% of similarity. The two EgFABPs present the highest amino acid homologies with the mammalian FABP subfamily containing heart-FABPs (H-FABPs). The coding sequences of both genes are interrupted by a single intron located in the position of the third intron reported for vertebrate FABP genes. Both genes are expressed in the protoscolex stage of the parasite. The promoter region of EgFABP2 presents several consensus putative cis-acting elements found in other members of the family, suggesting interesting possible mechanisms involved in the host-parasite adaptation.     

Silent mutations affect in vivo protein folding in Escherichia coli

As an approach to investigate the molecular mechanism of in vivo protein folding and the role of translation kinetics on specific folding pathways, we made codon substitutions in the EgFABP1 (Echinococcus granulosus fatty acid binding protein1) gene that replaced five minor codons with their synonymous major ones. The altered region corresponds to a turn between two short alpha helices. One of the silent mutations of EgFABP1 markedly decreased the solubility of the protein when expressed in Escherichia coli. Expression of this protein also caused strong activation of a reporter gene designed to detect misfolded proteins, suggesting that the turn region seems to have special translation kinetic requirements that ensure proper folding of the protein. Our results highlight the importance of codon usage in the in vivo protein folding.     

In silico studies of Echinococcus granulosus FABPs

Fatty acid (FA) binding proteins are small intracellular proteins whose members exhibit great diversity and low similarity at the primary structure level, but a highly conserved three-dimensional structure. Characterised by a high-affinity non-covalent binding of hydrophobic ligands, these proteins have a molecular mass of 14–15?kDa with a characteristic β-barrel structure. Members of this family have been identified along the zoological scale, with Platyhelminthes being the more primitive organisms where they have been reported. Two FA binding proteins (FABPs), EgFABP1 and EgFABP2, with 88% similarity have been identified in Echinococcus granulosus. In an effort to understand why two such similar proteins are expressed by this organism, we performed an in silico analysis of the binding capabilities of both proteins. The crystallographic structure of EgFABP1 was utilised as a template to model EgFABP2, and both were docked against palmitate, oleate, linoleate and arachidonate. The docked structures were submitted to 4?ns molecular dynamics simulations, and their protein–ligand interaction energies were measured. The collected data demonstrated that linoleate and arachidonate had the higher interaction energies when bound to EgFABP1 and that palmitate and linoleate had the higher interaction energies when bound to EgFABP2. External and internal binding surfaces were analysed, showing differences at both levels. Internal surface compositions suggested that both proteins could have preferences for certain FAs. Comparisons of the holo and apo forms of each protein indicated that the ligand imposed subtle, but specific modifications that could trigger surface signals. The differences found between the proteins under study suggest that they could have functional uniqueness in the parasite's metabolism.     

Proteomic analysis of the larval stage of the parasite Echinococcus granulosus: causative agent of cystic hydatid disease

We describe the preparation of Echinococcus granulosus metacestode protein extracts for two-dimensional electrophoresis (2-DE). Protoscoleces and hydatid fluid were prepared by precipitation using trichloroacetic acid (TCA) to remove nonprotein contaminants. Compared to the untreated control, TCA precipitation improved the 2-DE gel profile of the protoscoleces proteins. Comparison of 2-DE gels from insoluble and soluble fractions of the protoscoleces protein extract showed that most proteins are insoluble after lysis by sonication. Host serum proteins, especially albumin and globulins, caused horizontal streaking problems on the hydatid fluid 2-DE gels due to their high content in this sample. Even after the preparation of a hydatid fluid parasite enriched fraction, the high amount of bovine serum albumin and globulins made parasite-specific proteins difficult to detect by 2-DE. Despite the absence of an E. granulosus genome sequencing or expressed sequence tag (EST) projects, it was possible to identify 15 prominent protein spots from a whole protein protoscoleces 2-DE gel by peptide mass fingerprinting. These include actins, tropomyosin, paramyosin, thioredoxin reductase, antigen P-29, cyclophilin, and the heat shock proteins hsp70 and hsp20. This work demonstrates that 2-DE and PMF are important tools to identify proteins from the hydatid fluid and protoscoleces and for the comparative analysis of cysts from different hosts or between active and resting cysts.     

Identification of membrane-bound and secreted proteins from Echinococcus granulosus by signal sequence trap

The signal sequence trap technique was applied to identify genes coding for secreted and membrane bound proteins from Echinococcus granulosus, the etiologic agent of cystic hydatid disease. An E. granulosus protoscolex cDNA library was constructed in the AP-PST vector such that randomly primed cDNAs were fused with a placental alkaline phosphatase reporter gene lacking its endogenous signal peptide. E. granulosus cDNAs encoding a functional signal peptide were selected by their ability to rescue secretion of alkaline phosphatase by COS-7 cells that had been transfected with the cDNA library. Eighteen positive clones were identified and sequenced. Their deduced amino acid sequences showed significant similarity with amino acid transporters, Krebs cycle intermediates transporters, presenilins and vacuolar protein sorter proteins. Other cDNAs encoded secreted proteins without homologues. Three sequences were transcribed antisense to E. granulosus expressed sequence tags. All the mRNAs were expressed in protoscoleces and adult worms, but some of them were not found in oncospheres. The putative E. granulosus secreted and membrane bound proteins identified are likely to play important roles in the metabolism, development and survival in the host and represent potential targets for diagnosis, drugs and vaccines against E. granulosus.     

Fructose-bisphosphate aldolase and enolase from Echinococcus granulosus: genes, expression patterns and protein interactions of two potential moonlighting proteins

Glycolytic enzymes, such as fructose-bisphosphate aldolase (FBA) and enolase, have been described as complex multifunctional proteins that may perform non-glycolytic moonlighting functions, but little is known about such functions, especially in parasites. We have carried out in silico genomic searches in order to identify FBA and enolase coding sequences in Echinococcus granulosus, the causative agent of cystic hydatid disease. Four FBA genes and 3 enolase genes were found, and their sequences and exon-intron structures were characterized and compared to those of their orthologs in Echinococcus multilocularis, the causative agent of alveolar hydatid disease. To gather evidence of possible non-glycolytic functions, the expression profile of FBA and enolase isoforms detected in the E. granulosus pathogenic larval form (hydatid cyst) (EgFBA1 and EgEno1) was assessed. Using specific antibodies, EgFBA1 and EgEno1 were detected in protoscolex and germinal layer cells, as expected, but they were also found in the hydatid fluid, which contains parasite's excretory-secretory (ES) products. Besides, both proteins were found in protoscolex tegument and in vitro ES products, further suggesting possible non-glycolytic functions in the host-parasite interface. EgFBA1 modeled 3D structure predicted a F-actin binding site, and the ability of EgFBA1 to bind actin was confirmed experimentally, which was taken as an additional evidence of FBA multifunctionality in E. granulosus. Overall, our results represent the first experimental evidences of alternative functions performed by glycolytic enzymes in E. granulosus and provide relevant information for the understanding of their roles in host-parasite interplay.     

Structural and Functional Characterization of Human Peripheral Nervous System Myelin Protein P2

The myelin sheath is a tightly packed multilayered membrane structure insulating selected axons in the central and the peripheral nervous systems. Myelin is a biochemically unique membrane, containing a specific set of proteins. In this study, we expressed and purified recombinant human myelin P2 protein and determined its crystal structure to a resolution of 1.85 Å. A fatty acid molecule, modeled as palmitate based on the electron density, was bound inside the barrel-shaped protein. Solution studies using synchrotron radiation indicate that the crystal structure is similar to the structure of the protein in solution. Docking experiments using the high-resolution crystal structure identified cholesterol, one of the most abundant lipids in myelin, as a possible ligand for P2, a hypothesis that was proven by fluorescence spectroscopy. In addition, electrostatic potential surface calculations supported a structural role for P2 inside the myelin membrane. The potential membrane-binding properties of P2 and a peptide derived from its N terminus were studied. Our results provide an enhanced view into the structure and function of the P2 protein from human myelin, which is able to bind both monomeric lipids inside its cavity and membrane surfaces.     

Molecular characterization of Echinococcus granulosus in Tunisia and Mauritania by mitochondrial rrnS gene sequencing

Cystic hydatid disease is a zoonotic parasitic disease caused by the cestode Echinococcus granulosus and represents a major public health problem in many countries around the world, including North Africa. E. granulosus exists as a series of genetic variants or strains which differ in a wide variety of criteria that impact on the epidemiology, pathology and control of cystic hydatid disease. Nucleotide sequencing of the mitochondrial rrnS gene was here used to characterize 38 E. granulosus isolates collected from different regions and hosts in Tunisia and Mauritania. The results obtained reveal a significant genetic differentiation between E. granulosus hydatid cysts identified as belonging to the G1 genotype and to the G6/G7 cluster using the rrnS gene as marker, and indicate the circulation of the common sheep strain (G1) in all host species from Tunisia and the camel/pig strain cluster (G6/G7) in camel from Mauritania. Other investigations, using this method, are necessary for further genetic analysis of a wider range of isolates from different host species in order to more fully understand the genetic structure of E. granulosus populations and their transmission dynamics in this and neighbouring African countries.     

Construction and validation of an atomic model for bacterial TSPO from electron microscopy density,evolutionary constraints,and biochemical and biophysical data

The 18 kDa protein TSPO is a highly conserved transmembrane protein found in bacteria, yeast, animals and plants. TSPO is involved in a wide range of physiological functions, among which the transport of several molecules. The atomic structure of monomeric ligand-bound mouse TSPO in detergent has been published recently. A previously published low-resolution structure of Rhodobacter sphaeroides TSPO, obtained from tubular crystals with lipids and observed in cryo-electron microscopy, revealed an oligomeric structure without any ligand. We analyze this electron microscopy density in view of available biochemical and biophysical data, building a matching atomic model for the monomer and then the entire crystal. We compare its intra- and inter-molecular contacts with those predicted by amino acid covariation in TSPO proteins from evolutionary sequence analysis. The arrangement of the five transmembrane helices in a monomer of our model is different from that observed for the mouse TSPO. We analyze possible ligand binding sites for protoporphyrin, for the high-affinity ligand PK 11195, and for cholesterol in TSPO monomers and/or oligomers, and we discuss possible functional implications.     

Structural and biochemical characterization of CIB1 delineates a new family of EF-hand-containing proteins

CIB1 (CIB) is an EF-hand-containing protein that binds multiple effector proteins, including the platelet alphaIIbbeta3 integrin and several serine/threonine kinases and potentially modulates their function. The crystal structure for Ca(2+)-bound CIB1 has been determined at 2.0 A resolution and reveals a compact alpha-helical protein containing four EF-hands, the last two of which bind calcium ions in the standard fashion seen in many other EF-hand proteins. CIB1 shares high structural similarity with calcineurin B and the neuronal calcium sensor (NCS) family of EF-hand-containing proteins. Most importantly, like calcineurin B and NCS proteins, which possess a large hydrophobic pocket necessary for ligand binding, CIB1 contains a hydrophobic pocket that has been implicated in ligand binding by previous mutational analysis. However, unlike several NCS proteins, Ca(2+)-bound CIB1 is largely monomeric whether bound to a relevant peptide ligand or ligand-free. Differences in structure, oligomeric state, and phylogeny define a new family of CIB1-related proteins that extends from arthropods to humans.     

DNA damage, RAD9 and fertility/infertility of Echinococcus granulosus hydatid cysts

Hydatidosis, caused by the larval stage of the platyhelminth parasite Echinococcus granulosus, affects human and animal health. Hydatid fertile cysts are formed in intermediate hosts (human and herbivores) producing protoscoleces, the infective form to canines, at their germinal layers. Infertile cysts are also formed, but they are unable to produce protoscoleces. The molecular mechanisms involved in hydatid cysts fertility/infertility are unknown. Nevertheless, previous work from our laboratory has suggested that apoptosis is involved in hydatid cyst infertility and death. On the other hand, fertile hydatid cysts can resist oxidative damage due to reactive oxygen and nitrogen species. On these foundations, we have postulated that when oxidative damage of DNA in the germinal layers exceeds the capability of DNA repair mechanisms, apoptosis is triggered and hydatid cysts infertility occurs. We describe a much higher percentage of nuclei with oxidative DNA damage in dead protoscoleces and in the germinal layer of infertile cysts than in fertile cysts, suggesting that DNA repair mechanisms are active in fertile cysts. rad9, a conserved gene, plays a key role in cell cycle checkpoint modulation and DNA repair. We found that RAD9 of E. granulosus (EgRAD9) is expressed at the mRNA and protein levels. As it was found in other eukaryotes, EgRAD9 is hyperphosphorylated in response to DNA damage. Our results suggest that molecules involved in DNA repair in the germinal layer of fertile hydatid cysts and in protoscoleces, such as EgRAD9, may allow preserving the fertility of hydatid cysts in the presence of ROS and RNS.     

Echinococcus granulosus antigen B hydrophobic ligand binding properties

Antigen B (AgB), an immunodominant component of the cestode parasite Echinococcus granulosus, presents homology to and shares apparent structural similarities with helix-rich hydrophobic ligand binding proteins (HLBPs) from other cestodes. In order to investigate the fatty acid binding properties of AgB, two of its subunit components (rAgB8/1 and rAgB8/2) were expressed in Escherichia coli and purified, and the native antigen was purified from the hydatid cyst fluid by affinity chromatography using a monoclonal antibody raised against rAgB8/1. The interaction of the purified native and recombinant proteins with the fluorescent ligands DAUDA, ANS, DACA and 16-AP was investigated. The palmitic acid derived fluorescent ligand, 16-AP, showed the greatest enhancement in fluorescence when bound to native AgB or to its recombinant subunits, and the dissociation constants for 16-AP binding were determined. Surprisingly, in contrast to HLBPs from other cestodes, interactions with other fatty acids, including palmitic acid, caused an increase in fluorescence instead of competing with 16-AP. Our results suggest that AgB might have evolved different functions in the binding of hydrophobic compounds, dependent on cestode environment.     

Crystal structure of a phosphoinositide phosphatase, MTMR2: insights into myotubular myopathy and Charcot-Marie-Tooth syndrome

Myotubularin-related proteins are a large subfamily of protein tyrosine phosphatases (PTPs) that dephosphorylate D3-phosphorylated inositol lipids. Mutations in members of the myotubularin family cause the human neuromuscular disorders myotubular myopathy and type 4B Charcot-Marie-Tooth syndrome. The crystal structure of a representative member of this family, MTMR2, reveals a phosphatase domain that is structurally unique among PTPs. A series of mutants are described that exhibit altered enzymatic activity and provide insight into the specificity of myotubularin phosphatases toward phosphoinositide substrates. The structure also reveals that the GRAM domain, found in myotubularin family phosphatases and predicted to occur in approximately 180 proteins, is part of a larger motif with a pleckstrin homology (PH) domain fold. Finally, the MTMR2 structure will serve as a model for other members of the myotubularin family and provide a framework for understanding the mechanism whereby mutations in these proteins lead to disease.     

High resolution crystal structures of Siglec-7. Insights into ligand specificity in the Siglec family

Sialic acid-binding immunoglobulin-like lectins (Siglecs) recognize sialylated glycoconjugates and play a role in cell-cell recognition. Siglec-7 is expressed on natural killer cells and displays unique ligand binding properties different from other members of the Siglec family. Here we describe the high resolution structures of the N-terminal V-set Ig-like domain of Siglec-7 in two crystal forms, at 1.75 and 1.9 A. The latter crystal form reveals the full structure of this domain and allows us to speculate on the differential ligand binding properties displayed by members of the Siglec family. A fully ordered N-linked glycan is observed, tethered by tight interactions with symmetry-related protein molecules in the crystal. Comparison of the structure with that of sialoadhesin and a model of Siglec-9 shows that the unique preference of Siglec-7 for alpha(2,8)-linked disialic acid is likely to reside in the C-C' loop, which is variable in the Siglec family. In the Siglec-7 structure, the ligand-binding pocket is occupied by a loop of a symmetry-related molecule, mimicking the interactions with sialic acid.     

Crystal structure of a protein associated with cell division from Mycoplasma pneumoniae (GI: 13508053): a novel fold with a conserved sequence motif

UPF0040 is a family of proteins implicated in a cellular function of bacteria cell division. There is no structure information available on protein of this family. We have determined the crystal structure of a protein from Mycoplasma pneumoniae that belongs to this family using X-ray crystallography. Structural homology search reveals that this protein has a novel fold with no significant similarity to any proteins of known three-dimensional structure. The crystal structures of the protein in three different crystal forms reveal that the protein exists as a ring of octamer. The conserved protein residues, including a highly conserved DXXXR motif, are examined on the basis of crystal structure.     

Crystal structures of ASK1‐inhibtor complexes provide a platform for structure‐based drug design

ASK1, a member of the MAPK Kinase Kinase family of proteins has been shown to play a key role in cancer, neurodegeneration and cardiovascular diseases and is emerging as a possible drug target. Here we describe a ‘replacement‐soaking’ method that has enabled the high‐throughput X‐ray structure determination of ASK1/ligand complexes. Comparison of the X‐ray structures of five ASK1/ligand complexes from 3 different chemotypes illustrates that the ASK1 ATP binding site is able to accommodate a range of chemical diversity and different binding modes. The replacement‐soaking system is also able to tolerate some protein flexibility. This crystal system provides a robust platform for ASK1/ligand structure determination and future structure based drug design.