Structural basis for endotoxin neutralization by the eosinophil cationic protein.

Acute infection by Gram-negative pathogens can induce an exacerbated immune response that leads to lethal septic shock syndrome. Bacterial lipopolysaccharide (LPS) is a major pathogen-associated molecular pattern molecule that can initiate massive and lethal immune system stimulation. Therefore, the development of new and effective LPS-neutralizing agents is a top priority. The eosinophil cationic protein (ECP) is an antimicrobial protein secreted in response to infection, with a remarkable affinity for LPS. In the present study, we demonstrate that ECP is able to neutralize bacterial LPS and inhibit tumor necrosis factor-α production in human macrophages. We also characterized ECP neutralizing activity using progressively truncated LPS mutants, and conclude that the polysaccharide moiety and lipid A portions are required for LPS-mediated neutralization. In addition, we mapped the structural determinants required for the ECP-LPS interaction by nuclear magnetic resonance. Our results show that ECP is able to neutralize LPS and therefore opens a new route for developing novel therapeutic agents based on the ECP structural scaffolding.

DYNLT (Tctex-1) forms a tripartite complex with dynein intermediate chain and RagA, hence linking this small GTPase to the dynein motor.

It has been suggested that DYNLT, a dynein light chain known to bind to various cellular and viral proteins, can function as a microtubule-cargo adaptor. Recent data showed that DYNLT links the small GTPase Rab3D to microtubules and, for this to occur, the DYNLT homodimer needs to display a binding site for dynein intermediate chain together with a binding site for the small GTPase. We have analysed in detail how RagA, another small GTPase, associates to DYNLT. After narrowing down the binding site of RagA to DYNLT we could identify that a ß strand, part of the RagA G3 box involved in nucleotide binding, mediates this association. Interestingly, we show that both microtubule-associated DYNLT and cytoplasmic DYNLT are equally able to bind to the small GTPases Rab3D and RagA. Using NMR spectroscopy, we analysed the binding of dynein intermediate chain and RagA to mammalian DYNLT. Our experiments identify residues of DYNLT affected by dynein intermediate chain binding and residues affected by RagA binding, hence distinguishing the docking site for each of them. In summary, our results shed light on the mechanisms adopted by DYNLT when binding to protein cargoes that become transported alongside microtubules bound to the dynein motor.

XTACC3-XMAP215 association reveals an asymmetric interaction promoting microtubule elongation.

chTOG is a conserved microtubule polymerase that catalyses the addition of tubulin dimers to promote microtubule growth. chTOG interacts with TACC3, a member of the transforming acidic coiled-coil (TACC) family. Here we analyse their association using the Xenopus homologues, XTACC3 (TACC3) and XMAP215 (chTOG), dissecting the mechanism by which their interaction promotes microtubule elongation during spindle assembly. Using SAXS, we show that the TACC domain (TD) is an elongated structure that mediates the interaction with the C terminus of XMAP215. Our data suggest that one TD and two XMAP215 molecules associate to form a four-helix coiled-coil complex. A hybrid methods approach was used to define the precise regions of the TACC heptad repeat and the XMAP215 C terminus required for assembly and functioning of the complex. We show that XTACC3 can induce the recruitment of larger amounts of XMAP215 by increasing its local concentration, thereby promoting efficient microtubule elongation during mitosis.

Relationships between IgE/IgG4 epitopes, structure and function in Anisakis simplex Ani s 5, a member of the SXP/RAL-2 protein family.

BACKGROUND: Anisakiasis is a re-emerging global disease caused by consumption of raw or lightly cooked fish contaminated with L3 Anisakis larvae. This zoonotic disease is characterized by severe gastrointestinal and/or allergic symptoms which may misdiagnosed as appendicitis, gastric ulcer or other food allergies. The Anisakis allergen Ani s 5 is a protein belonging to the SXP/RAL-2 family; it is detected exclusively in nematodes. Previous studies showed that SXP/RAL-2 proteins are active antigens; however, their structure and function remain unknown. The aim of this study was to elucidate the three-dimensional structure of Ani s 5 and its main IgE and IgG4 binding regions. METHODOLOGY/PRINCIPAL FINDINGS: The tertiary structure of recombinant Ani s 5 in solution was solved by nuclear magnetic resonance. Mg2+, but not Ca2+, binding was determined by band shift using SDS-PAGE. IgE and IgG4 epitopes were elucidated by microarray immunoassay and SPOTs membranes using sera from nine Anisakis allergic patients. The tertiary structure of Ani s 5 is composed of six alpha helices (H), with a Calmodulin like fold. H3 is a long, central helix that organizes the structure, with H1 and H2 packing at its N-terminus and H4 and H5 packing at its C-terminus. The orientation of H6 is undefined. Regarding epitopes recognized by IgE and IgG4 immunoglobulins, the same eleven peptides derived from Ani s 5 were bound by both IgE and IgG4. Peptides 14 (L40-K59), 26 (A76-A95) and 35 (I103-D122) were recognized by three out of nine sera. CONCLUSIONS/SIGNIFICANCE: This is the first reported 3D structure of an Anisakis allergen. Magnesium ion binding and structural resemblance to Calmodulin, suggest some putative functions for SXP/RAL-2 proteins. Furthermore, the IgE/IgG4 binding regions of Ani s 5 were identified as segments localized on its surface. These data will contribute towards a better understanding of the interactions that occur between immunoglobulins and allergens and, in turn, facilitate the design of novel diagnostic tests and immunotherapeutic strategies.

Noncanonical G recognition mediates KSRP regulation of let-7 biogenesis.

Let-7 is an important tumor-suppressive microRNA (miRNA) that acts as an on-off switch for cellular differentiation and regulates the expression of a set of human oncogenes. Binding of the human KSRP protein to let-7 miRNA precursors positively regulates their processing to mature let-7, thereby contributing to control of cell proliferation, apoptosis and differentiation. Here we analyze the molecular basis for KSRP-let-7 precursor selectivity and show how the third KH domain of the protein recognizes a G-rich sequence in the pre-let-7 terminal loop and dominates the interaction. The structure of the KH3-RNA complex explains the protein recognition of this noncanonical KH target sequence, and we demonstrate that the specificity of this binding is crucial for the functional interaction between the protein and the miRNA precursor.

Structural and functional characterization of Nrf2 degradation by the glycogen synthase kinase 3/ß-TrCP axis.

The transcription factor NF-E2-related factor 2 (Nrf2) is a master regulator of a genetic program, termed the phase 2 response, that controls redox homeostasis and participates in multiple aspects of physiology and pathology. Nrf2 protein stability is regulated by two E3 ubiquitin ligase adaptors, Keap1 and ß-TrCP, the latter of which was only recently reported. Here, two-dimensional (2D) gel electrophoresis and site-directed mutagenesis allowed us to identify two serines of Nrf2 that are phosphorylated by glycogen synthase kinase 3ß (GSK-3ß) in the sequence DSGISL. Nuclear magnetic resonance studies defined key residues of this phosphosequence involved in docking to the WD40 propeller of ß-TrCP, through electrostatic and hydrophobic interactions. We also identified three arginine residues of ß-TrCP that participate in Nrf2 docking. Intraperitoneal injection of the GSK-3 inhibitor SB216763 led to increased Nrf2 and heme oxygenase-1 levels in liver and hippocampus. Moreover, mice with hippocampal absence of GSK-3ß exhibited increased levels of Nrf2 and phase 2 gene products, reduced glutathione, and decreased levels of carbonylated proteins and malondialdehyde. This study establishes the structural parameters of the interaction of Nrf2 with the GSK-3/ß-TrCP axis and its functional relevance in the regulation of Nrf2 by the signaling pathways that impinge on GSK-3.

LC8 dynein light chain (DYNLL1) binds to the C-terminal domain of ATM-interacting protein (ATMIN/ASCIZ) and regulates its subcellular localization.

LC8 dynein light chain (now termed DYNLL1 and DYNLL2 in mammals), a dimeric 89 amino acid protein, is a component of the dynein multi-protein complex. However a substantial amount of DYNLL1 is not associated to microtubules and it can thus interact with dozens of cellular and viral proteins that display well-defined, short linear motifs. Using DYNLL1 as bait in a yeast two-hybrid screen of a human heart library we identified ATMIN, an ATM kinase-interacting protein, as a DYNLL1-binding partner. Interestingly, ATMIN displays at least 18 SQ/TQ motifs in its sequence and DYNLL1 is known to bind to proteins with KXTQT motifs. Using pepscan and yeast two-hybrid techniques we show that DYNLL1 binds to multiple SQ/TQ motifs present in the carboxy-terminal domain of ATMIN. Recombinant expression and purification of the DYNLL1-binding region of ATMIN allowed us to obtain a polypeptide with an apparent molecular mass in gel filtration close to 400 kDa that could bind to DYNLL1 in vitro. The NMR data-driven modelled complexes of DYNLL1 with two selected ATMIN peptides revealed a similar mode of binding to that observed between DYNLL1 and other peptide targets. Remarkably, co-expression of mCherry-DYNLL1 and GFP-ATMIN mutually affected intracellular protein localization. In GFP-ATMIN expressing-cells DNA damage induced efficiently nuclear foci formation, which was partly impeded by the presence of mCherry-DYNLL1. Thus, our results imply a potential cellular interference between DYNLL1 and ATMIN functions.

Structural models of DYNLL1 with interacting partners: African swine fever virus protein p54 and postsynaptic scaffolding protein gephyrin.

DYNLL1, the smallest dynein light chain, interacts with different cargos facilitating their cellular transport. Usually the sequence recognized in the targets is homologous to the GIQVD or the KXTQT motifs with a glutamine that is important for binding. Here we add two new examples of DYNLL1 targets that can be classified into these two groups: ASFV p54 and gephyrin. Using NMR we demonstrate the direct interaction between DYNLL1 and two peptides derived from their interacting sequences. We model the structure of both complexes and show that the overall binding mode is preserved as in other complexes despite differences at the residue-specific interactions.

NMR structural determinants of eosinophil cationic protein binding to membrane and heparin mimetics.

Eosinophil cationic protein (ECP) is a highly stable, cytotoxic ribonuclease with the ability to enter and disrupt membranes that participates in innate immune defense against parasites but also kills human cells. We have used NMR spectroscopy to characterize the binding of ECP to membrane and heparin mimetics at a residue level. We believe we have identified three Arg-rich surface loops and Trp(35) as crucial for membrane binding. Importantly, we have provided evidence that the interaction surface of ECP with heparin mimetics is extended with respect to that previously described (fragment 34-38). We believe we have identified new sites involved in the interaction for the first time, and shown that the N-terminal alpha-helix, the third loop, and the first and last beta-strands are key for heparin binding. We have also shown that a biologically active ECP N-terminal fragment comprising the first 45 residues (ECP1-45) retains the capacity to bind membrane and heparin mimetics, thus neither the ECP tertiary structure nor its high conformational stability are required for cytotoxicity.

The sequence selectivity of KSRP explains its flexibility in the recognition of the RNA targets.

K-homology (KH) splicing regulator protein (KSRP) is a multi-domain RNA-binding protein that regulates different steps of mRNA metabolism, from mRNA splicing to mRNA decay, interacting with a broad range of RNA sequences. To understand how KSRP recognizes its different RNA targets it is necessary to define the general rules of KSRP-RNA interaction. We describe here a complete scaffold-independent analysis of the RNA-binding potential of the four KH domains of KSRP. The analysis shows that KH3 binds to the RNA with a significantly higher affinity than the other domains and recognizes specifically a G-rich target. It also demonstrates that the other KH domains of KSRP display different sequence preferences explaining the broad range of targets recognized by the protein. Further, KSRP shows a strong negative selectivity for sequences containing several adjacent Cytosines limiting the target choice of KSRP within single-stranded RNA regions. The in-depth analysis of the RNA-binding potential of the KH domains of KSRP provides us with an understanding of the role of low sequence specificity domains in RNA recognition by multi-domain RNA-binding proteins.

Scaffold-independent analysis of RNA-protein interactions: the Nova-1 KH3-RNA complex.

We describe a method to analyze the sequence specificity of an RNA-binding domain. The method, which we have named scaffold-independent analysis, reports on the specificity for each nucleotide position within an RNA target, uncoupled from the surrounding structural and sequence context. We expect this information to improve our understanding of protein-RNA interfaces in ssRNA binding domains (e.g., KH or RRM domains) and to be useful to the design of novel protein-RNA recognition surfaces. Our NMR binding assays using the third KH domain of the Nova-1 protein provide a proof-of-principle for the method and novel information on the specificity of this domain for its RNA targets.

The structure of the C-terminal KH domains of KSRP reveals a noncanonical motif important for mRNA degradation.

The AU-rich element (ARE) RNA-binding protein KSRP (K-homology splicing regulator protein) contains four KH domains and promotes the degradation of specific mRNAs that encode proteins with functions in cellular proliferation and inflammatory response. The fourth KH domain (KH4) is essential for mRNA recognition and decay but requires the third KH domain (KH3) for its function. We show that KH3 and KH4 behave as independent binding modules and can interact with different regions of the AU-rich RNA targets of KSRP. This provides KSRP with the structural flexibility needed to recognize a set of different targets in the context of their 3'UTR structural settings. Surprisingly, we find that KH4 binds to its target AREs with lower affinity than KH3 and that KSRP's mRNA binding, and mRNA degradation activities are closely associated with a conserved structural element of KH4.

pH-Dependent conformational stability of the ribotoxin alpha-sarcin and four active site charge substitution variants.

Alpha-sarcin is an exquisitely specific ribonuclease that binds and cleaves a single phosphodiester bond in the large rRNA of the eukaryotic ribosome, inactivating it. To better understand this remarkable activity, the contributions of the active site residues (His 50, Glu 96, and His 137) to the conformational stability have been determined as a function of pH using variant proteins containing uncharged substitutes. Wild-type alpha-sarcin and the variants are maximally stable near pH 5.5, coinciding with the pH of optimal activity. A comparison of the stability vs pH profiles determined by thermal denaturation experiments to those calculated on the basis of pKa values shows that the charged forms of Glu 96 and His 137 compromise the enzyme's stability, lowering it. In contrast to barnase, there is little evidence for significant electrostatic interactions in the denatured states of alpha-sarcin or its active site variants between pH 3.5 and pH 8.5. Alpha-sarcin contains a long beta-hairpin and surface loops which are highly positively charged and which play key roles in membrane translocation and in ribosome binding. These positive charges decrease the stability of alpha-sarcin, particularly below pH 5. Hydrogen exchange measurements have been performed at pH 5.5 and reveal that the catalytic residues are firmly anchored in highly stable elements of secondary structure. Significant, though lower, levels of protection are observed for many amide protons in the positively charged beta-hairpin and long loops.

Refined NMR structure of alpha-sarcin by 15N-1H residual dipolar couplings.

(15)N-(1)H residual dipolar couplings (RDC) have been used as additional restraints to refine the solution structure of the ribotoxin alpha-sarcin. The RDC values were obtained by partial alignment of alpha-sarcin in the binary mixture of n-dodecyl hexa(ethylene glycol)/hexanol. A total of 131 RDCs were measured and 106 were introduced in the final steps of the calculation protocol following the main calculation based on nuclear Overhauser enhancements and torsion angle restraints. A homogeneous family of 81 conformers was obtained. The resulting average pairwise root-mean-square deviation corresponding to the superposition of the 20 best structures is 0.69+/-0.12 A for the backbone and 1.29+/-0.14 A for all heavy atoms. The new structural features derived from the refined structure, compared with the non-refined structure of alpha-sarcin, consist of new hydrogen bonds and a better definition of the backbone conformation. In particular, the loop segment spanning Gly 60 to Lys 70 shows a single conformation, corresponding to the most populated family of conformers observed in the unrefined structure. The information derived from the analysis of the refined structure and the comparison with the homologous protein restrictocin could help in establishing further structure-function relationships concerning alpha-sarcin which can be reasonably extrapolated to other members of the ribotoxin family.

NMR solution structure of Ole e 6, a major allergen from olive tree pollen.

Ole e 6 is a pollen protein from the olive tree (Olea europaea) that exhibits allergenic activity with a high prevalence among olive-allergic individuals. The three-dimensional structure of Ole e 6 has been determined in solution by NMR methods. This is the first experimentally determined structure of an olive tree pollen allergen. The structure of this 50-residue protein is based on 486 upper limit distance constraints derived from nuclear Overhauser effects and 24 torsion angle restraints. The global fold of Ole e 6 consists of two nearly antiparallel alpha-helices, spanning residues 3-19 and 23-33, that are connected by a short loop and followed by a long, unstructured C-terminal tail. Viewed edge-on, the structured N terminus has a dumbbell-like shape with the two helices on the outside and with the hydrophobic core, mainly composed of 3 aromatic and 6 cysteine residues, on the inside. All the aromatic rings lie on top of and pack against the three disulfide bonds. The lack of thermal unfolding, even at 85 degrees C, indicates a high conformational stability. Based on the analysis of the molecular surface, we propose five plausible epitopes for IgE recognition. The results presented here provide the structural foundation for future experiments to verify the antigenicity of the proposed epitopes, as well as to design novel hypoallergenic forms of the protein suitable for diagnosis and treatment of type-I allergies. In addition, three-dimensional structure features of Ole e 6 are discussed to provide a basis for future functional studies.

Leucine 145 of the ribotoxin alpha-sarcin plays a key role for determining the specificity of the ribosome-inactivating activity of the protein.

Secreted fungal RNases, represented by RNase T1, constitute a family of structurally related proteins that includes ribotoxins such as alpha-sarcin. The active site residues of RNase T1 are conserved in all fungal RNases, except for Phe 100 that is not present in the ribotoxins, in which Leu 145 occupies the equivalent position. The mutant Leu145Phe of alpha-sarcin has been recombinantly produced and characterized by spectroscopic methods (circular dichroism, fluorescence spectroscopy, and NMR). These analyses have revealed that the mutant protein retained the overall conformation of the wild-type alpha-sarcin. According to the analyses performed, Leu 145 was shown to be essential to preserve the electrostatic environment of the active site that is required to maintain the anomalous low pKa value reported for the catalytic His 137 of alpha-sarcin. Enzymatic characterization of the mutant protein has revealed that Leu 145 is crucial for the specific activity of alpha-sarcin on ribosomes.