Structural basis of pathogen recognition by an integrated HMA domain in a plant NLR immune receptor.

Plants have evolved intracellular immune receptors to detect pathogen proteins known as effectors. How these immune receptors detect effectors remains poorly understood. Here we describe the structural basis for direct recognition of AVR-Pik, an effector from the rice blast pathogen, by the rice intracellular NLR immune receptor Pik. AVR-PikD binds a dimer of the Pikp-1 HMA integrated domain with nanomolar affinity. The crystal structure of the Pikp-HMA/AVR-PikD complex enabled design of mutations to alter protein interaction in yeast and in vitro, and perturb effector-mediated response both in a rice cultivar containing Pikp and upon expression of AVR-PikD and Pikp in the model plant Nicotiana benthamiana. These data reveal the molecular details of a recognition event, mediated by a novel integrated domain in an NLR, which initiates a plant immune response and resistance to rice blast disease. Such studies underpin novel opportunities for engineering disease resistance to plant pathogens in staple food crops.

The crystal structure of PEBP-2, a homologue of the PEBP/RKIP family.

Proteins from the PEBP (phosphatidylethanolamine-binding protein) family have been identified in a wide variety of species and are thought to regulate a range of intracellular signalling cascades. The rat homologue (known as RKIP; Raf-1 kinase inhibitor protein) has been shown to negatively regulate the MAP kinase pathway through formation of inhibitory complexes with Raf-1 and MEK. The crystal structure of a new, murine member of the PEBP family, termed mPEBP-2, has been determined. On the basis of amino-acid homology, mPEBP-2 belongs to a distinct subset of the mammalian PEBP proteins. Nonetheless, mPEBP-2 is seen to be very similar in structure to other PEBP proteins from human, bovine and plant sources. Regions of distinctive sequence associated with the PEBP-2 subset are discussed with reference to this structure.

Specificity in Trk receptor:neurotrophin interactions: the crystal structure of TrkB-d5 in complex with neurotrophin-4/5.

BACKGROUND: The binding of neurotrophin ligands to their respective Trk cellular receptors initiates intracellular signals essential for the growth and survival of neurons. The site of neurotrophin binding has been located to the fifth extracellular domain of the Trk receptor, with this region regulating both the affinity and specificity of Trk receptor:neurotrophin interaction. Neurotrophin function has been implicated in a number of neurological disorders, including Alzheimer's disease and Parkinson's disease. RESULTS: We have determined the 2.7 A crystal structure of neurotrophin-4/5 bound to the neurotrophin binding domain of its high-affinity receptor TrkB (TrkB-d5). As previously seen in the interaction of nerve growth factor with TrkA, neurotrophin-4/5 forms a crosslink between two spatially distant receptor molecules. The contacts formed in the TrkB-d5:neurotrophin-4/5 complex can be divided into a conserved area similar to a region observed in the TrkA-d5:NGF complex and a second site-unique in each ligand-receptor pair-formed primarily by the ordering of the neurotrophin N terminus. CONCLUSIONS: Together, the structures of the TrkB-d5:NT-4/5 and TrkA-d5:NGF complexes confirm a consistent pattern of recognition in Trk receptor:neurotrophin complex formation. In both cases, the N terminus of the neurotrophin becomes ordered only on complex formation. This ordering appears to be directed largely by the receptor surface, with the resulting complementary surfaces providing the main determinant of receptor specificity. These features provide an explanation both for the limited crossreactivity observed between the range of neurotrophins and Trk receptors and for the high-affinity binding associated with respective ligand-receptor pairs.

Structure of HisF, a histidine biosynthetic protein from Pyrobaculum aerophilum.

HisF (imidazole glycerol phosphate synthase) is an important branch-point enzyme in the histidine biosynthetic pathway of microorganisms. Because of its potential relevance for structure-based drug design, the crystal structure of HisF from the hyperthermophilic archaeon Pyrobaculum aerophilum has been determined. The structure was determined by molecular replacement and refined at 2.0 A resolution to a crystallographic R factor of 20.6% and a free R of 22.7%. The structure adopts a classic (beta/alpha)(8) barrel fold and has networks of surface salt bridges that may contribute to thermostability. The active site is marked out by the presence of two bound phosphate ions and two glycerol molecules that delineate a long groove at one end of the (beta/alpha)(8) barrel. The two phosphate ions, 17 A apart, are bound to sequence-conserved structural motifs that seem likely to provide much of the specificity for the two phosphate groups of the HisF substrate. The two glycerol molecules bind in the vicinity of other sequence-conserved residues that are likely to be involved in binding and/or catalysis. Comparisons with the homologous HisF from Thermatoga maritima reveal a displaced loop that may serve as a lid over the active site.

Effects of whole wheat dilution v. substitution on coccidiosis in broiler chickens.

To test the hypothesis that inclusion of whole cereals in the diet of broiler chickens reduces the severity of a coccidial infection, the effects were investigated, in birds infected with Eimeria acervulina, of feeding a complete pelleted control food, the control food diluted with whole wheat (400 g/kg), or a diet in which ground wheat in the pellets was substituted with whole wheat (400 g/kg) so as to achieve the same composition as the control diet. In the weeks prior to and after infection, (days 14--20 and days 21--27), no significant differences in performance were observed between birds fed the complete pellet and substituted feeds. Birds fed the diluted feeds had significantly lower gains per unit of feed and numerically lower weight gains than the birds fed the control and substituted feeds during this period. No significant differences in performance were observed between treatments from days 28--34. Diluting feeds with whole wheat (400 g/kg) significantly increased ileal digesta viscosity levels, compared with feeding the control and substituted feeds in birds dissected on day 21, although no significant differences in digesta viscosity levels were observed in birds dissected on day 35. Whole wheat feeding, either by dilution or substitution, significantly increased gizzard sizes in birds dissected on day 21 and day 35. Neither dilution nor substitution of feeds with whole wheat (400 g/kg) significantly affected the level of an Eimeria acervulina infection, as measured by daily and total faecal oocyst yields.

Identification and structure of the nerve growth factor binding site on TrkA.

Nerve growth factor (NGF) is involved in the development and maintenance of the nervous system and has been implicated as a possible therapeutic target molecule in a number of neurodegenerative diseases, especially Alzheimer's disease. NGF binds with high affinity to the extracellular region of a tyrosine kinase receptor, TrkA, which comprises three leucine-rich motifs (LRMs), flanked by two cysteine-rich clusters, followed by two immunoglobulin-like (Ig-like) domains. We have expressed the second Ig-like domain as a recombinant protein in E. coli and demonstrate that NGF binds to this domain with similar affinity to the native receptor. This domain (TrkAIg(2)) has the ability to sequester NGF in vitro, preventing NGF-induced neurite outgrowth, and in vivo, inhibiting NGF-induced plasma extravasation. We also present the three-dimensional structure of the TrkAIg(2) domain in a new crystal form, refined to 2.0 A resolution.

Crystal structure of the NADP(H)-dependent ketose reductase from Bemisia argentifolii at 2.3 A resolution.

Polyhydric alcohols are widely found in nature and can be accumulated to high concentrations as a protection against a variety of environmental stresses. It is only recently, however, that these molecules have been shown to be active in protection against heat stress, specifically in the use of sorbitol by the silverleaf whitefly, Bemisia argentifolii. We have determined the structure of the enzyme responsible for production of sorbitol in Bemisia argentifolii, NADP(H)-dependent ketose reductase (BaKR), to 2.3 A resolution. The structure was solved by multiwavelength anomalous diffraction (MAD) using the anomalous scattering from two zinc atoms bound in the structure, and was refined to an R factor of 21.9 % (R(free)=25.1 %). BaKR belongs to the medium-chain dehydrogenase family and its structure is the first for the sorbitol dehydrogenase branch of this family. The enzyme is tetrameric, with the monomer having a very similar fold to the alcohol dehydrogenases (ADHs). Although the structure determined is for the apo form, a phosphate ion in the active site marks the likely position for the adenyl phosphate of NADP(H). The catalytic zinc ion is tetrahedrally coordinated to Cys41, His66, Glu67 and a water molecule, in a modification of the zinc site usually found in ADHs. This modified zinc site seems likely to be a conserved feature of the sorbitol dehydrogenase sub-family. Comparisons with other members of the ADH family have also enabled us to model a ternary complex of the enzyme, and suggest how structural differences may influence coenzyme binding and substrate specificity in the reduction of fructose to sorbitol.

Recognition of structurally diverse substrates by type II 3-hydroxyacyl-CoA dehydrogenase (HADH II)/amyloid-beta binding alcohol dehydrogenase (ABAD).

Human type II hydroxyacyl-CoA dehydrogenase/amyloid-beta binding alcohol dehydrogenase (HADH II/ABAD) is an oxidoreductase whose salient features include broad substrate specificity, encompassing 3-hydroxyacyl-CoA derivatives, hydroxysteroids, alcohols and beta-hydroxybutyrate, and the capacity to bind amyloid-beta peptide, leading to propagation of amyloid-induced cell stress. In this study, we examine the structure and enzymatic activity of the homologous rat HADH II/ABAD enzyme. We report the crystal structure of rat HADH II/ABAD as a binary complex with its NADH cofactor to 2.0 A resolution, as a ternary complex with NAD(+) and 3-ketobutyrate (acetoacetate) to 1.4 A resolution, and as a ternary complex with NADH and 17 beta-estradiol to 1.7 A resolution. This first crystal structure of an HADH II confirms these enzymes are closely related to the short-chain hydroxysteroid dehydrogenases and differ substantially from the classic, type I 3-hydroxyacyl-CoA dehydrogenases. Binding of the ketobutyrate substrate is accompanied by closure of the active site specificity loop, whereas the steroid substrate does not appear to require closure for binding. Despite the different chemical nature of the two bound substrates, the presentation of chemical groups within the active site of each complex is remarkably similar, allowing a general mechanism for catalytic activity to be proposed. There is a characteristic extension to the active site that is likely to accommodate the CoA moiety of 3-hydroxyacyl-CoA substrates. Rat HADH II/ABAD also binds amyloid-beta (1-40) peptide with a K(D) of 21 nM, which is similar to the interaction exhibited between this peptide and human HADH II/ABAD. These studies provide the first structural insights into HADH II/ABAD interaction with its substrates, and indicate the relevance of the rodent enzyme and associated rodent models for analysis of HADH II/ABAD's physiologic and pathophysiologic properties.

The structure of Antirrhinum centroradialis protein (CEN) suggests a role as a kinase regulator.

Expression of the plant protein centroradialis (CEN) leads to a morphological switch between shoot growth and the development of flower structures (inflorescence). We have determined the crystal structure of Antirrhinum CEN to 1.9 A resolution. This structure confirms the CEN proteins as a subset of the family of phosphatidylethanolamine-binding proteins (PEBP), as predicted from sequence homology. Mammalian forms of PEBP have been found to act as inhibitors of MAP kinase signalling, a central signalling cascade regulating cell differentiation. CEN and PEBP proteins share a similar topology dominated by a large central beta-sheet. The strong conservation of a binding pocket at one end of this sheet which is capable of binding phosphoryl ligands, suggests the biological effects of CEN, like PEBP, arise from the ability of this region to form complexes with phosphorylated ligands, hence interfering with kinases and their effectors.

Function from structure? The crystal structure of human phosphatidylethanolamine-binding protein suggests a role in membrane signal transduction.

BACKGROUND: Proteins belonging to the phosphatidylethanolamine-binding protein (PEBP) family are highly conserved throughout nature and have no significant sequence homology with other proteins of known structure or function. A variety of biological roles have previously been described for members of this family, including lipid binding, roles as odorant effector molecules or opioids, interaction with the cell-signalling machinery, regulation of flowering plant stem architecture, and a function as a precursor protein of a bioactive brain neuropeptide. To date, no experimentally derived structural information has been available for this protein family. In this study we have used X-ray crystallography to determine the three-dimensional structure of human PEBP (hPEBP), in an attempt to clarify the biological role of this unique protein family. RESULTS: The crystal structures of two forms of hPEBP have been determined: one in the native state (at 2.05 A resolution) and one in complex with cacodylate (at 1.75 A resolution). The crystal structures reveal that hPEBP adopts a novel protein topology, dominated by the presence of a large central beta sheet, and is expected to represent the archaetypal fold for this family of proteins. Two potential functional sites have been identified from the structure: a putative ligand-binding site and a coupled cleavage site. hPEBP forms a dimer in the crystal with a distinctive dipole moment that may orient the oligomer for membrane binding. CONCLUSIONS: The crystal structure of hPEBP suggests that the ligand-binding site could accommodate the phosphate head groups of membrane lipids, therefore allowing the protein to adhere to the inner leaf of bilipid membranes where it would be ideally positioned to relay signals from the membrane to the cytoplasm. The structure also suggests that ligand binding may lead to coordinated release of the N-terminal region of the protein to form the hippocampal neurostimulatory peptide, which is known to be active in the development of the hippocampus. These studies are consistent with a primary biological role for hPEBP as a transducer of signals from the interior membrane surface.

VL:VH domain rotations in engineered antibodies: crystal structures of the Fab fragments from two murine antitumor antibodies and their engineered human constructs.

The crystal structures of two pairs of Fab fragments have been determined. The pairs comprise both a murine and an engineered human form, each derived from the antitumor antibodies A5B7 and CTM01. Although antigen specificity is maintained within the pairs, antigen affinity varies. A comparison of the hypervariable loops for each pair of antibodies shows their structure has been well maintained in grafting, supporting the canonical loop model. Detailed structural analysis of the binding sites and domain arrangements for these antibodies suggests the differences in antigen affinity observed are likely to be due to inherent flexibility of the hypervariable loops and movements at the VL:VH domain interface. The four structures provide the first opportunity to study in detail the effects of protein engineering on specific antibodies.

Structure of the Fab fragment of a monoclonal antibody specific for carcinoembryonic antigen.

The three-dimensional structure of the Fab fragment of the murine monoclonal antibody A5B7, which is specific for carcinoembryonic antigen (CEA) a protein expressed on carcinoma cell surfaces, has been determined. The structure was solved by molecular replacement and has been refined to an R factor for 21.2% (all data 8-2.1 A). The conformation of the hypervariable loops, which form the antigen binding site, are consistent with canonical loop predictions. Hypervariable loop H3 is unusual in surface exposing many hydrophobic groups at the expense of burying an aspartic acid in the protein core. Other regions of the structure that influence the conformation of the binding site are identified. This structure forms a basis for analysing the effects of amino-acid substitutions in both hypervariable and framework regions in engineering studies of the A5B7 antibody.

Direct observation of the iron binding sites in a ferritin.

X-Ray analysis of the ferritin of Escherichia coli (Ec-FTN) and of Ec-FTN crystals soaked in (NH4)2Fe(SO4)2 has revealed the presence of three iron-binding sites per subunit. Two of these form a di-iron site in the centre of the subunit as has been proposed for the 'ferroxidase centres' of human ferritin H chains. This di-iron site, lying within the 4-alpha-helix bundle, resemble those of ribonucleotide reductase, methane monoxygenase and haemerythrin. The third iron is bound by ligands unique to Ec-FTN on the inner surface of the protein shell. It is speculated that this state may represent the nucleation centre of a novel type of Fe(III) cluster, recently observed in Ec-FTN.