Mechanistic insights into the first Lygus-active ß-pore forming protein.

The cotton pests Lygus hesperus and Lygus lineolaris can be controlled by expressing Cry51Aa2.834_16 in cotton. Insecticidal activity of pore-forming proteins is generally associated with damage to the midgut epithelium due to pores, and their biological specificity results from a set of key determinants including proteolytic activation and receptor binding. We conducted mechanistic studies to gain insight into how the first Lygus-active ß-pore forming protein variant functions. Biophysical characterization revealed that the full-length Cry51Aa2.834_16 was a stable dimer in solution, and when exposed to Lygus saliva or to trypsin, the protein underwent proteolytic cleavage at the C-terminus of each of the subunits, resulting in dissociation of the dimer to two separate monomers. The monomer showed tight binding to a specific protein in Lygus brush border membranes, and also formed a membrane-associated oligomeric complex both in vitro and in vivo. Chemically cross-linking the ß-hairpin to the Cry51Aa2.834_16 body rendered the protein inactive, but still competent to compete for binding sites with the native protein in vivo. Our study suggests that disassociation of the Cry51Aa2.834_16 dimer into monomeric units with unoccupied head-region and sterically unhindered ß-hairpin is required for brush border membrane binding, oligomerization, and the subsequent steps leading to insect mortality.

Crystal structure of a Vip3B family insecticidal protein reveals a new fold and a unique tetrameric assembly.

Vegetatively expressed insecticidal proteins (VIPs) produced by Bacillus thuringiensis fall into several classes of which the third, VIP3, is known for their activity against several key Lepidopteran pests of commercial broad acre crops and because their mode of action does not overlap with that of crystalline insecticidal proteins. The details of the VIP3 structure and mode of action have remained obscure for the quarter century that has passed since their discovery. In the present article, we report the first crystal structure of a full-length VIP3 protein. Crystallization of this target required multiple rounds of construct optimization and screening-over 200 individual sequences were expressed and tested. This protein adopts a novel global fold that combines domains with hitherto unreported topology and containing elements seemingly borrowed from carbohydrate-binding domains, lectins, or from other insecticidal proteins.

Microbial HemG-type protoporphyrinogen IX oxidase enzymes for biotechnology applications in plant herbicide tolerance traits.

BACKGROUND: Protoporphyrinogen IX oxidase (PPO)-inhibiting herbicides act by inhibiting a key enzyme in the heme and chlorophyll biosynthetic pathways in plants. This enzyme, the PPO enzyme, is conserved across plant species. However, some microbes are known to utilize a unique family of PPO enzymes, the HemG family. This enzyme family carries out the same enzymatic step as the plant PPO enzymes, but does not share sequence homology with the plant PPO enzymes. RESULTS: Bioinformatic analysis was used to identify putative HemG PPO enzyme variants from microbial sources. A subset of these variants was cloned and characterized. HemG PPO variants were characterized for functionality and tolerance to PPO-inhibiting herbicides. HemG PPO variants that exhibited insensitivity to PPO-inhibiting herbicides were identified for further characterization. Expression of selected variants in maize, soybean, cotton and canola resulted in plants that displayed tolerance to applications of PPO-inhibiting herbicides. CONCLUSION: Selected microbial-sourced HemG PPO enzyme variants present an opportunity for building new herbicide tolerance biotechnology traits. These traits provide tolerance to PPO-inhibiting herbicides and, therefore, could provide additional tools for farmers to employ in their weed management systems. © 2019 Society of Chemical Industry.

Development of enzymes for robust aryloxyphenoxypropionate and synthetic auxin herbicide tolerance traits in maize and soybean crops.

BACKGROUND: Effective management of weedy species in agricultural fields is essential for maintaining favorable growing conditions and crop yields. The introduction of genetically modified crops containing herbicide tolerance traits has been a successful additional tool available to farmers to better control weeds. However, weed resistance challenges present a need for additional herbicide tolerance trait options. RESULTS: To help meet this challenge, a new trait that provides tolerance to an aryloxyphenoxypropionate (FOP) herbicide and members of the synthetic auxin herbicide family, such as 2,4-dichlorophenoxyacetic acid (2,4-D), was developed. Development of this herbicide tolerance trait employed an enzyme engineered with robust and specific enzymatic activity for these two herbicide families. This engineering effort utilized a microbial-sourced dioxygenase scaffold to generate variants with improved enzymatic parameters. Additional optimization to enhance in-plant stability of the enzyme enabled an efficacious trait that can withstand the higher temperature conditions often found in field environments. CONCLUSION: Optimized herbicide tolerance enzyme variants with enhanced enzymatic and temperature stability parameters enabled robust herbicide tolerance for two herbicide families in transgenic maize and soybeans. This herbicide tolerance trait for FOP and synthetic auxin herbicides such as 2,4-D could be useful in weed management systems, providing additional tools for farmers to control weeds. © 2019 Society of Chemical Industry.

Disabled insecticidal proteins: A novel tool to understand differences in insect receptor utilization.

The development of insect resistance to pesticides via natural selection is an acknowledged agricultural issue. Likewise, resistance development in target insect populations is a significant challenge to the durability of crop traits conferring insect protection and has driven the need for novel insecticidal proteins (IPs) with alternative mechanism of action (MOA) mediated by different insect receptors. The combination or "stacking" of transgenes encoding different insecticidal proteins in a single crop plant can greatly delay the development of insect resistance, but requires sufficient knowledge of MOA to identify proteins with different receptor preferences. Accordingly, a rapid technique for differentiating the receptor binding preferences of insecticidal proteins is a critical need. This article introduces the Disabled Insecticidal Protein (DIP) method as applied to the well-known family of three-domain insecticidal proteins from Bacillus thuringiensis and related bacteria. These DIP's contain amino acid substitutions in domain 1 that render the proteins non-toxic but still capable of competing with active proteins in insect feeding assays, resulting in a suppression of the expected insecticidal activity. A set of insecticidal proteins with known differences in receptor binding (Cry1Ab3, Cry1Ac.107, Cry2Ab2, Cry1Ca, Cry1A.105, and Cry1A.1088) has been studied using the DIP method, yielding results that are consistent with previous MOA studies. When a native IP and an excess of DIP are co-administered to insects in a feeding assay, the outcome depends on the overlap between their MOAs: if receptors are shared, then the DIP saturates the receptors to which the native protein would ordinarily bind, and acts as an antidote whereas, if there is no shared receptor, the toxicity of the native insecticidal protein is not inhibited. These results suggest that the DIP methodology, employing standard insect feeding assays, is a robust and effective method for rapid MOA differentiation among insecticidal proteins.

Rational protein engineering in action: the first crystal structure of a phenylalanine tRNA synthetase from Staphylococcus haemolyticus.

In this article, we describe for the first time the high-resolution crystal structure of a phenylalanine tRNA synthetase from the pathogenic bacterium Staphylococcus haemolyticus. We demonstrate the subtle yet important structural differences between this enzyme and the previously described Thermus thermophilus ortholog. We also explain the structure-activity relationship of several recently reported inhibitors. The native enzyme crystals were of poor quality--they only diffracted X-rays to 3-5A resolution. Therefore, we have executed a rational surface mutagenesis strategy that has yielded crystals of this 2300-amino acid multidomain protein, diffracting to 2A or better. This methodology is discussed and contrasted with the more traditional domain truncation approach.

Serendipitous discovery of novel bacterial methionine aminopeptidase inhibitors.

In this article we describe the application of structural biology methods to the discovery of novel potent inhibitors of methionine aminopeptidases. These enzymes are employed by the cells to cleave the N-terminal methionine from nascent peptides and proteins. As this is one of the critical steps in protein maturation, it is very likely that inhibitors of these enzymes may prove useful as novel antibacterial agents. Involvement of crystallography at the very early stages of the inhibitor design process resulted in serendipitous discovery of a new inhibitor class, the pyrazole-diamines. Atomic-resolution structures of several inhibitors bound to the enzyme illuminate a new mode of inhibitor binding.

The structure of Yersinia pestis V-antigen, an essential virulence factor and mediator of immunity against plague.

The LcrV protein (V-antigen) is a multifunctional virulence factor in Yersinia pestis, the causative agent of plague. LcrV regulates the translocation of cytotoxic effector proteins from the bacterium into the cytosol of mammalian cells via a type III secretion system, possesses antihost activities of its own, and is also an active and passive mediator of resistance to disease. Although a crystal structure of this protein has been actively sought for better understanding of its role in pathogenesis, the wild-type LcrV was found to be recalcitrant to crystallization. We employed a surface entropy reduction mutagenesis strategy to obtain crystals of LcrV that diffract to 2.2 A and determined its structure. The refined model reveals a dumbbell-like molecule with a novel fold that includes an unexpected coiled-coil motif, and provides a detailed three-dimensional roadmap for exploring structure-function relationships in this essential virulence determinant.

Crystal structure of the Yersinia pestis GTPase activator YopE.

Yersinia pestis, the causative agent of bubonic plague, evades the immune response of the infected organism by using a type III (contact-dependent) secretion system to deliver effector proteins into the cytosol of mammalian cells, where they interfere with signaling pathways that regulate inflammation and cytoskeleton dynamics. The cytotoxic effector YopE functions as a potent GTPase-activating protein (GAP) for Rho family GTP-binding proteins, including RhoA, Rac1, and Cdc42. Down-regulation of these molecular switches results in the loss of cell motility and inhibition of phagocytosis, enabling Y. pestis to thrive on the surface of macrophages. We have determined the crystal structure of the GAP domain of YopE (YopE(GAP); residues 90-219) at 2.2-A resolution. Apart from the fact that it is composed almost entirely of alpha-helices, YopE(GAP) shows no obvious structural similarity with eukaryotic RhoGAP domains. Moreover, unlike the catalytically equivalent arginine fingers of the eukaryotic GAPs, which are invariably contained within flexible loops, the critical arginine in YopE(GAP) (Arg144) is part of an alpha-helix. The structure of YopE(GAP) is strikingly similar to the GAP domains from Pseudomonas aeruginosa (ExoS(GAP)) and Salmonella enterica (SptP(GAP)), despite the fact that the three amino acid sequences are not highly conserved. A comparison of the YopE(GAP) structure with those of the Rac1-ExoS(GAP) and Rac1-SptP complexes indicates that few, if any, significant conformational changes occur in YopE(GAP) when it interacts with its G protein targets. The structure of YopE(GAP) may provide an avenue for the development of novel therapeutic agents to combat plague.

Structure of the full-length insecticidal protein Cry1Ac reveals intriguing details of toxin packaging into in vivo formed crystals.

For almost half a century, the structure of the full-length Bacillus thuringiensis (Bt) insecticidal protein Cry1Ac has eluded researchers, since Bt-derived crystals were first characterized in 1965. Having finally solved this structure we report intriguing details of the lattice-based interactions between the toxic core of the protein and the protoxin domains. The structure provides concrete evidence for the function of the protoxin as an enhancer of native crystal packing and stability.

Structural biology approaches to antibacterial drug discovery.

Antibacterial drug discovery has undertaken a major experiment in the 12 years since the first bacterial genomes were sequenced. Genome mining has identified hundreds of potential targets that have been distilled to a relatively small number of broad-spectrum targets ('low-hanging fruit') using the genetics tools of modern microbiology. Prosecuting these targets with high-throughput screens has led to a disappointingly small number of lead series that have mostly evaporated under closer scrutiny. In the meantime, multi-drug resistant pathogens are becoming a serious challenge in the clinic and the community and the number of pharmaceutical firms pursuing antibacterial discovery has declined. Filling the antibacterial development pipeline with novel chemical series is a significant challenge that will require the collaboration of scientists from many disciplines. Fortunately, advancements in the tools of structural biology and of in silico modeling are opening up new avenues of research that may help deal with the problems associated with discovering novel antibiotics.

Design and synthesis of a series of novel pyrazolopyridines as HIF-1alpha prolyl hydroxylase inhibitors.

Recently resolved X-ray crystal structure of HIF-1alpha prolyl hydroxylase was used to design and develop a novel series of pyrazolopyridines as potent HIF-1alpha prolyl hydroxylase inhibitors. The activity of these compounds was determined in a human EGLN-1 assay. Structure-based design aided in optimizing the potency of the initial lead (2, IC(50) of 11 microM) to a potent (11l, 190 nM) EGLN-1 inhibitor. Several of these analogs were potent VEGF inducers in a cell-based assay. These pyrazolopyridines were also effective in stabilizing HIF-1alpha.

A novel series of imidazo[1,2-a]pyridine derivatives as HIF-1alpha prolyl hydroxylase inhibitors.

Utilizing modeling information from a recently resolved structure of human HIF-1alpha prolyl hydroxylase (EGLN1) and structure-based design, a novel series of imidazo[1,2-a]pyridine derivatives was prepared. The activity of these compounds was determined in a human EGLN1 assay and a limited SAR was developed.

Structure-based design, synthesis, and SAR evaluation of a new series of 8-hydroxyquinolines as HIF-1alpha prolyl hydroxylase inhibitors.

A new series of potent 8-hydroxyquinolines was designed based on the newly resolved X-ray crystal structure of EGLN-1. Both alkyl and aryl 8-hydroxyquinoline-7-carboxyamides were good HIF-1alpha prolyl hydroxylase (EGLN) inhibitors. In subsequent VEGF induction assays, these exhibited potent VEGF activity. In addition, this class of compounds did show the ability to stabilize HIF-1alpha.

Engineering the catalytic domain of human protein tyrosine phosphatase beta for structure-based drug discovery.

Protein tyrosine phosphatases (PTPs) play roles in many biological processes and are considered to be important targets for drug discovery. As inhibitor development has proven challenging, crystal structure-based design will be very helpful to advance inhibitor potency and selectivity. Successful application of protein crystallography to drug discovery heavily relies on high-quality crystal structures of the protein of interest complexed with pharmaceutically interesting ligands. It is very important to be able to produce protein-ligand crystals rapidly and reproducibly for as many ligands as necessary. This study details our efforts to engineer the catalytic domain of human protein tyrosine phosphatase beta (HPTPbeta-CD) with properties suitable for rapid-turnaround crystallography. Structures of apo HPTPbeta-CD and its complexes with several novel small-molecule inhibitors are presented here for the first time.

Design and synthesis of substituted pyridine derivatives as HIF-1alpha prolyl hydroxylase inhibitors.

Structure-guided de novo drug design led to the identification of a novel series of substituted pyridine derivatives as HIF-1alpha prolyl hydroxylase inhibitors. Pyridine carboxyamide derivatives bearing a substituted aryl group at the 5-position of the pyridine ring show appreciable activity, while constraining the side chain by placing a pyrazole carboxylic acid generated a potent lead series with consistent activity against EGLN-1.

Structural basis for the fast maturation of Arthropoda green fluorescent protein.

Since the cloning of Aequorea victoria green fluorescent protein (GFP) in 1992, a family of known GFP-like proteins has been growing rapidly. Today, it includes more than a hundred proteins with different spectral characteristics cloned from Cnidaria species. For some of these proteins, crystal structures have been solved, showing diversity in chromophore modifications and conformational states. However, we are still far from a complete understanding of the origin, functions and evolution of the GFP family. Novel proteins of the family were recently cloned from evolutionarily distant marine Copepoda species, phylum Arthropoda, demonstrating an extremely rapid generation of fluorescent signal. Here, we have generated a non-aggregating mutant of Copepoda fluorescent protein and solved its high-resolution crystal structure. It was found that the protein beta-barrel contains a pore, leading to the chromophore. Using site-directed mutagenesis, we showed that this feature is critical for the fast maturation of the chromophore.

1,2,3,4-Tetrahydroisoquinolinyl sulfamic acids as phosphatase PTP1B inhibitors.

High-throughput screening of the P&GP corporate repository against several protein tyrosine phosphatases identified the sulfamic acid moiety as potential phosphotyrosine mimetic. Incorporation of the sulfamic acid onto a 1,2,3,4-tetrahydroisoquinoline scaffold provided a promising starting point for PTP1B inhibitor design.

Design and synthesis of potent, non-peptidic inhibitors of HPTPbeta.

The sulfamic acid phosphotyrosine mimetic was coupled with a previously known malonate template to obtain highly selective and potent inhibitors of HPTPbeta. Potentially hydrolyzable malonate ester functionalities were replaced with 1,2,4-oxadiazoles without a significant effect on HPTPbeta potency.

Differential effects of short affinity tags on the crystallization of Pyrococcus furiosus maltodextrin-binding protein.

Pyrococcus furiosus maltodextrin-binding protein readily forms large orthorhombic crystals that diffract to high resolution. This protein was used as a model system to investigate the influence of five short affinity tags (His(6), Arg(5), Strep tag II, FLAG tag and the biotin acceptor peptide) on the formation of protein crystals and their ability to diffract X-rays. The results indicate that the amino-acid sequence of the tag can have a profound effect on both of these parameters. Consequently, the ability to obtain diffracting crystals of a particular protein may depend as much on which affinity tag is selected as it does on whether an affinity tag is used at all.