Edward Eisenstein

Structure and control of pyridoxal phosphate dependent allosteric threonine deaminase

BACKGROUNDnFeedback inhibition of biosynthetic threonine deaminase (TD) from Escherichia coli provided one of the earliest examples of protein-based metabolic regulation. Isoleucine, the pathway end-product, and valine, the product of a parallel pathway, serve as allosteric inhibitor and activator, respectively. This enzyme is thus a useful model system for studying the structural basis of allosteric control mechanisms.nnnRESULTSnWe report the crystal structure of TD at 2.8 A resolution. The tetramer has 222 symmetry, with C-terminal regulatory domains projecting out from a core of catalytic PLP-containing N-terminal domains. The subunits, and especially the regulatory domains, associate extensively to form dimers, which associate less extensively to form the tetramer. Within the dimer, each monomer twists approximately 150 degrees around a thin neck between the domains to place its catalytic domain adjacent to the regulatory domain of the other subunit.nnnCONCLUSIONSnThe structure of TD and its comparison with related structures and other data lead to the tentative identification of the regulatory binding site and revealed several implications for the allosteric mechanism. This work prepares the way for detailed structure/function studies of the complex allosteric behaviour of this enzyme.

Biological function made crystal clear - annotation of hypothetical proteins via structural genomics.

Many of the gene products of completely sequenced organisms are hypothetical - they cannot be related to any previously characterized proteins - and so are of completely unknown function. Structural studies provide one means of obtaining functional information in these cases. A structural genomics project has been initiated aimed at determining the structures of 50 hypothetical proteins from Haemophilus influenzae to gain an understanding of their function. Each stage of the project - target selection, protein production, crystallization, structure determination, and structure analysis - makes use of recent advances to streamline procedures. Early results from this and similar projects are encouraging in that some level of functional understanding can be deduced from experimentally solved structures.

Structural reorganization of the interleukin-7 signaling complex

We report here an unliganded receptor structure in the common gamma-chain (γc) family of receptors and cytokines. The crystal structure of the unliganded form of the interleukin-7 alpha receptor (IL-7Rα) extracellular domain (ECD) at 2.15 Å resolution reveals a homodimer forming an “X” geometry looking down onto the cell surface with the C termini of the two chains separated by 110 Å and the dimer interface comprising residues critical for IL-7 binding. Further biophysical studies indicate a weak association of the IL-7Rα ECDs but a stronger association between the γc/IL-7Rα ECDs, similar to previous studies of the full-length receptors on CD4+ T cells. Based on these and previous results, we propose a molecular mechanism detailing the progression from the inactive IL-7Rα homodimer and IL-7Rα–γc heterodimer to the active IL-7–IL-7Rα–γc ternary complex whereby the two receptors undergo at least a 90° rotation away from the cell surface, moving the C termini of IL-7Rα and γc from a distance of 110 Å to less than 30 Å at the cell surface. This molecular mechanism can be used to explain recently discovered IL-7– and γc-independent gain-of-function mutations in IL-7Rα from B- and T-cell acute lymphoblastic leukemia patients. The mechanism may also be applicable to other γc receptors that form inactive homodimers and heterodimers independent of their cytokines.

Structural Basis for the Binding Specificity of Human Recepteur d'Origine Nantais (RON) Receptor Tyrosine Kinase to Macrophage-stimulating Protein

Background: RON and MET receptors bind their ligands MSP and HGF selectively and activate different signaling pathways. Results: Crystallographic and analytical ultracentrifugation studies provide important information about RON-MSP interaction. Conclusion: RON-MSP and MET-HGF exhibit 2:2 complex stoichiometry, but differences within the respective interfaces explain the strict ligand-receptor specificity. Significance: Signaling pathways must be exquisitely regulated with no cross-reactivity between related systems. Recepteur dorigine nantais (RON) receptor tyrosine kinase and its ligand, serum macrophage-stimulating protein (MSP), play important roles in inflammation, cell growth, migration, and epithelial to mesenchymal transition during tumor development. The binding of mature MSPαβ (disulfide-linked α- and β-chains) to RON ectodomain modulates receptor dimerization, followed by autophosphorylation of tyrosines in the cytoplasmic receptor kinase domains. Receptor recognition is mediated by binding of MSP β-chain (MSPβ) to the RON Sema. Here we report the structure of RON Sema-PSI-IPT1 (SPI1) domains in complex with MSPβ at 3.0 Å resolution. The MSPβ serine protease-like β-barrel uses the degenerate serine protease active site to recognize blades 2, 3, and 4 of the β-propeller fold of RON Sema. Despite the sequence homology between RON and MET receptor tyrosine kinase and between MSP and hepatocyte growth factor, it is well established that there is no cross-reactivity between the two receptor-ligand systems. Comparison of the structure of RON SPI1 in complex with MSPβ and that of MET receptor tyrosine kinase Sema-PSI in complex with hepatocyte growth factor β-chain reveals the receptor-ligand selectivity determinants. Analytical ultracentrifugation studies of the SPI1-MSPβ interaction confirm the formation of a 1:1 complex. SPI1 and MSPαβ also associate primarily as a 1:1 complex with a binding affinity similar to that of SPI1-MSPβ. In addition, the SPI1-MSPαβ ultracentrifuge studies reveal a low abundance 2:2 complex with ∼10-fold lower binding affinity compared with the 1:1 species. These results support the hypothesis that the α-chain of MSPαβ mediates RON dimerization.

Gene identification in black cohosh (Actaea racemosa L.): expressed sequence tag profiling and genetic screening yields candidate genes for production of bioactive secondary metabolites

Black cohosh (Actaea racemosa L., syn. Cimicifuga racemosa, Nutt., Ranunculaceae) is a popular herb used for relieving menopausal discomforts. A variety of secondary metabolites, including triterpenoids, phenolic dimers, and serotonin derivatives have been associated with its biological activity, but the genes and metabolic pathways as well as the tissue distribution of their production in this plant are unknown. A gene discovery effort was initiated in A. racemosa by partial sequencing of cDNA libraries constructed from young leaf, rhizome, and root tissues. In total, 2,066 expressed sequence tags (ESTs) were assembled into 1,590 unique genes (unigenes). Most of the unigenes were predicted to encode primary metabolism genes, but about 70 were identified as putative secondary metabolism genes. Several of these candidates were analyzed further and full-length cDNA and genomic sequences for a putative 2,3 oxidosqualene cyclase (CAS1) and two BAHD-type acyltransferases (ACT1 and HCT1) were obtained. Homology-based PCR screening for the central gene in plant serotonin biosynthesis, tryptophan decarboxylase (TDC), identified two TDC-related sequences in A. racemosa. CAS1, ACT1, and HCT1 were expressed in most plant tissues, whereas expression of TDC genes was detected only sporadically in immature flower heads and some very young leaf tissues. The cDNA libraries described and assorted genes identified provide initial insight into gene content and diversity in black cohosh, and provide tools and resources for detailed investigations of secondary metabolite genes and enzymes in this important medicinal plant.

Structural Insights into the Inhibitory Mechanism of an Antibody against B7-H6, a Stress-Induced Cellular Ligand for the Natural Killer Cell Receptor NKp30

Antibodies have been shown to block signaling through cell surface receptors using several mechanisms. The two most common are binding to the ligand-binding site of the receptor and, conversely, binding to the receptor-binding site of the ligand. Here, we investigated the inhibitory mechanism of an antibody (17B1.3) against human B7-H6, a stress-induced cellular ligand for the natural killer (NK) cell receptor NKp30. Binding of this antibody to B7-H6, a transmembrane protein expressed on tumor and other stressed cells, but not on normal cells, prevents NK cell activation via NKp30. We determined the crystal structure of antibody 17B1.3 in complex with the ectodomain of B7-H6 to 2.5Å resolution. Surprisingly, 17B1.3 binds to a site on B7-H6 that is completely distinct from the binding site for NKp30, such that 17B1.3 does not block the NKp30-B7-H6 interaction. We then asked whether 17B1.3 prevents signaling by binding to a putative site for B7-H6 dimerization. However, structure-based mutations designed to disrupt potential B7-H6 dimerization through this site did not diminish NKp30-mediated cell activation. We conclude that the bulky 17B1.3 antibody most likely acts by sterically interfering with close cell-cell contacts at the NK cell-target cell interface that are required for NK cell activation. A similar inhibitory mechanism may apply to other antibodies, including therapeutic antibodies that block signaling through cell surface receptors whose ligands are also cell surface proteins.

Systems Approaches to Unraveling Plant Metabolism: Identifying Biosynthetic Genes of Secondary Metabolic Pathways

The diversity of useful compounds produced by plant secondary metabolism has stimulated broad systems biology approaches to identify the genes involved in their biosynthesis. Systems biology studies in non-model plants pose interesting but addressable challenges, and have been greatly facilitated by the ability to grow and maintain plants, develop laboratory culture systems, and profile key metabolites in order to identify critical genes involved their biosynthesis. In this chapter we describe a suite of approaches that have been useful in Actaea racemosa (L.; syn. Cimicifuga racemosa, Nutt., black coshosh), a non-model medicinal plant with no genome sequence and little horticultural information available, that have led to the development of initial gene-metabolite relationships for the production of several bioactive metabolites in this multicomponent botanical therapeutic, and that can be readily applied to a wide variety of under-characterized medicinal plants.

ABRF–MIRG Benchmark Study: Molecular Interactions in a Three-Component System

Protein-protein interactions identified through high-throughput proteomics efforts continue to advance our understanding of the protein interactome. In addition to highly specific protein-protein interactions, it is becoming increasingly more common for yeast two-hybrid, pull-down assays, and other proteomics techniques to identify multiple protein ligands that bind to the same target protein. A resulting challenge is to accurately characterize the assembly of these multiprotein complexes and the competition among multiple protein ligands for a given target. The Association of Biomolecular Resource Facilities-Molecular Interactions Research Group recently conducted a benchmark study to assess participants ability to correctly describe the interactions between two protein ligands and their target protein using primarily biosensor technologies, such as surface plasmon resonance. Participants were provided with microgram quantities of three proteins (A, B, and C) and asked to determine if a ternary A-B-C complex can form or if protein-B and protein-C bind competitively to protein-A. This article will summarize the experimental approaches taken by participants to characterize the molecular interactions, the interpretation of the data, and the results obtained using different biosensor instruments.

Biosystems Design. Report from the July 2011 Workshop

Executive Summary G rowing energy demand cannot be met by nonrenew-able fossil fuels. Consequently, alternative energy sources are being intensively pursued. Cellulose, a very stable carbohydrate that makes up plant cell walls, is a potential source of renewable fuels. Transforming cellulose into liquid fuels, however, requires substantial chemical and biological processing, first to extract the sugars that comprise the cellulose and then to convert those sugars into fuels. These requirements pose major hurdles to sustainable biofuels production , but overcoming them may be possible by engineering new plants that facilitate the extraction and conversion of their cell walls into liquid fuels. Similarly, newly designed microbes capable of metabolizing plant cell wall components can simplify biomass processing for fuel conversion. Other microorganisms such as microalgae and cyanobacteria can be redesigned to incorporate new functionalities. For example, these photosynthetic organisms, which capture light and convert it into chemical energy, can be re-engineered to produce biofuels, including biodiesel, directly from sunlight. Technological advances enabling the design of new biological systems are already moving biofuels closer to becoming viable, alternative renewable energy resources. Further advances are necessary for developing useful bioenergy crops that not only allow facile conversion of biomass into biofuels, but also do not compete with food crops for arable land. In other words, crops must be rationally redesigned to produce high biomass yields on marginal agricultural lands and under changing weather conditions. To explore the current state of the art in the field of bio-systems design, discuss new biodesign technologies and approaches, and identify key scientific challenges and knowledge gaps, the Department of Energys Office of Biological and Environmental Research organized the Biosystems Design Workshop in July 2011 in Bethesda, Maryland. The workshops goal was to bring together scientific leaders in microbiology, plant biology, metabolic engineering, systems biology, bioinformatics, computational modeling, and other relevant disciplines to examine fundamental aspects of biosystems design from molecules to organisms to communities. The focus was on the fundamental biological principles that must be harnessed to make biological design possible and the tools and computer-aided testbeds needed to design, prototype, and functionally validate multiscale natural and hybrid biological systems. Building on the outcomes of the genomic revolution that altered the course of biological research in the last decades, biosystems design research will identify modular components that can be modified, enhanced, and exchanged among different organisms, enabling the manipulation of biological systems. With the help of computational …