Janet L. Smith - US grants

crystallization; aldehyde dehydrogenases; enzyme structure; protein structure function; alcoholism /alcohol abuse; ethanol; liver; cow;

This primary objective of the proposed research is to understand the control of the biochemical pathway for de novo biosynthesis of purine nucleotides. Specifically, this goal will be accomplished by determining the three-dimensional structure of glutamine PRPP amidotransferase, the enzyme that catalyzes the first and committed reaction in the pathway. Changes to the structure caused by inhibitor and activator molecules will also be determined. Most of the regulation of the purine biosynthetic pathway is directed towards this enzyme and a solid understanding of its structure and its reaction with effector molecules is critical to understanding regulation of the entire pathway. The health relatedness of the proposed research is twofold. Defects in the purine biosynthetic pathway have been associated with disorders such as hyperuricemia (gout), immunodeficiency and neurological symptoms. Secondly, components of the pathway, especially glutamine PRPP amidotransferase, are targets for the design of anticancer drugs due to the high rate of purine biosynthesis required by rapidly dividing cells. Glutamine PRPP amidotransferase contains an essential inorganic FeS center that apparently has a novel regulatory function and is not catalytic. The three-dimensional structure of the enzyme should clarify the role of this unusual metal center. The three-dimensional structure will be determined by X-ray crystallography and the structure determination should be a significant advance for the new crystallographic technology of multiwavelength anomalous dispersion. Successful application of this methodology to the glutamine PRPP amidotransferase structure will be a significant step towards proving its generality as a rapid and accurate means of structure determination for biological macromolecules.

The incorporation of nitrogen into biological molecules occurs through the action of a family of enzymes called glutamine amidotransferases. These enzymes transfer NH3 from the side chain of glutamine to an appropriate substrate. There are at least fourteen different glutamine amidotransferases, each catalyzing the transfer of NH3 to a different substrate. Sequence fingerprints have been identified that assign all known glutamine amidotransferase sequences to one of two families. Neither the mechanism of NH3 generation nor the tight coupling between NH3 generation and NH3 transport are understood for any of the enzymes. This important question in biological catalysis is being addressed by determination of three-dimensional structures for three different glutamine amidotransferases in both families by X-ray crystallography. Two of the structure determinations are being done by multiwavelength anomalous diffraction and represent important new applications of this crystallographic methodology. 1. Glutamine PRPP amidotransferase catalyzes the first and committed step in de novo synthesis of purine nucleotides. Defects in the purine pathway have been associated with disorders such as hyperuricemia (gout), immunodeficiency and neurological symptoms. Crystal structures are being determined for the enzyme from both B. subtilis and E. coli in order to elucidate the function of an unusual metal center in B. subtilis and higher eukaryotes that apparently has a novel regulatory function and is not catalytic. 2. GNP synthetase, another enzyme from the purine pathway, is a target of attempts to stop cell growth, specifically tumor cell growth and T-cell proliferation. Crystals of GMP synthetase with substrate and ATP bound and crystals of enzyme alone are an excellent system for structural studies of the coupling of NH3-generating and NH3-transfer activities. 3. Imidazole glycerol 3-phosphate synthase, an enzyme from the histidine pathway, is a heterodimeric glutamine amidotransferase.

Glutamine amidotransferases are "complex" enzymes with two different catalytic domains, each contributing to the catalysis of a single biochemical reaction. The family of sixteen enzymes catalyzes the transfer of amide nitrogen from glutamine (glutamine catalytic domain) to a variety of acceptor substrates (acceptor catalytic domain). Crystal structures have been determined for two amidotransferases of de novo purine biosynthesis. The structures of glutamine PRPP amidotransferase (GPATase) and GMP synthetase are prototypes for the two homologous families of glutamine catalytic domains, the Ntn and Triad families, respectively. The acceptor domains of both enzymes also represent homologous enzymes families, the phosphoribosyltransferases and the N-type ATP pyrophosphatases. Results from these first structures of amidotransferases have changed thinking about the enzyme family and led to new direction for future experiments. The most important new discovery of this work has been the means by which Ntn amidotransferases couple catalysis between glutamine and acceptor domains. The enzyme creates a channel for NH/3 between glutamine and acceptor active sites. Signal transduction between catalytic sites is triggered by substrate binding in the acceptor domain and involves formation of the NH/3 channel and activation of the glutamine domain. The proposed studies will investigate control and operation of the NH3 channel in GPATase and determine whether Triad amidotransferases also channel NH/3 between active sites. The product of GPATase is extremely unstable, and there is indirect evidence for channeling to GAP synthetase, the next enzyme of the purine biosynthetic pathway. This possibility will be investigated by structural studies of GAP synthetase and by mutagenesis experiments. The significant observations of the first two amidotransferases will be extended to other members of the enzyme family.

Beam time was requested to perform diffraction experiments on crystals of two glutamine amidotransferase enzymes. GMP synthetase and glutamine PRPP amidotransferase. Structures have been solved for both enzymes, and high-resolution studies will be carried out to address specific questions of biological function. The enzymes have a complex organization, with two catalytic active sites that must work in a highly coordinated way to catalyze a single biochemical transformation. Diffraction experiments have been outlined with various ligand states and mutants to probe the coupling of cataltyic activities in the two active sites of each enzyme. The aim is to measure data to the highest resolution possible. A second major aim is to measure data for the high-resolution structure of the active conformation of glutamine PRPP amidotransferase, the allosteric enzyme controlling purine biosynthesis. A structure at moderate resolution has been obtained very recently for this allosteric state. However, crystals have a unit cell edge in excess of 300 [unreadable], and the diffraction limit of these crystals cannot be reached with conventional radiation. Biological significance of the projects derives from the general features of coupled catalysis by complex enzymes, and allosteric structural transitions in regulatory enzymes. All crystals can be frozen.

Glutamine amidotransferases are a family of enzymes that utilize the amide of glutamine in biosynthesis. they provide the main route for incorporation of nitrogen into amino acids, purine and pyrimidine nucleotides, amino sugars and coenzymes. There are 3 glutamine amidotransferases in the pathway for purine nucleotide synthesis. Glutamine PRPP amidotransferase catalyzes step 1 and is the key regulatory enzyme in the de novo pathway. The long term objective of this research is to determine basic mechanisms for glutamine amidotransferase catalysis and regulation as well as gene regulation in microorganisms. There are 4 specific aims. (1) Determine mechanisms for regulation of glutamine PRPP amidotransferase. The experiments focus on two regulatory events: nucleotide endproduct inhibition and processing of an NH2-terminal pro-peptide in the Bacillus subtilis enzyme. (2) Determine mechanisms for genetic regulation of Escherichia coli genes involved in the IMP to AMP and GMP branches of the pathway. (3) Determine how purine bases bind to E. coli pur regulon aporepressor and how this binding promotes the specific holorepressor-DNA interactions. (4) Determine mechanisms for transcriptional regulation of the B. subtilis pur operon which encodes the 10 step pathway to IMP and regulation of genes purA, guaA and guaB for the IMP to AMP and GMP branches. Finally, experiments are planned to determine the importance of B. subtilis pur operon gene overlaps in establishing the translational stoichiometry for enzyme synthesis. Purine metabolism has been a fertile area for development of drugs against a variety of diseases. Two inhibitors of glutamine amidotransferases in the purine pathway, acivicin and triciribine have antineoplastic activity.

This award supports improvements in the capability and reliability of the X-ray diffraction laboratory used for study of single crystals of biological macromolecules at Purdue University. The equipment to be obtained with this support will be used for in research aimed at determination of the three-dimensional crystal structures of proteins, RNA molecules, and of protein-nucleic acid complexes. Knowledge of such structures are essential to molecular level understanding of how biological macromolecules function in a variety of processes. Three types of equipment will be obtained: a new diffraction instrument that will improve throughput and, by providing a more monochromatic and intense X-ray beam, that will facilitate the study of very large macromolecules. The new instrument will allow flash-frozen crystals to be saved in their frozen state for later diffraction experiments. A new optical system for an existing diffraction instrument will improve the intensity and quality of the X-ray beam, permitting more pricise measurements and use of smaller crystals than now possible. Finally a new microspectrophotometer will be obtained to monitor enzymatic reactions and measure ligand binding of crystalline macromolecules. The award provides improved infrastructure for one of the nation's leading academic laboratories in one of the most important areas of contemporary biology.

structural biology; replicase; virus replication; virus protein; protein structure function; biomedical resource;

[unreadable] DESCRIPTION (provided by applicant): The long-term goal is to relate molecular structure to specificity and reactivity in selected enzymes, with an emphasis on enzymes that require vitamin-based cofactors or essential metals. Three of the enzymes to be studied, B12-dependent methionine synthase (MetH), methylenetetrahydrofolate reductase (MTHFR), and betaine-homocysteine methyltransferase (BHMT), are key catalysts in the metabolism of one-carbon units and control the cellular and plasma levels of homocysteine and methionine. These metabolites are believed to be important indicators of risk for cardiovascular disease and neural tube defects. High-resolution x-ray analysis will be the primary tool to examine local and global conformation changes that are fundamental features of catalysis and control in these enzymes. Mutagenesis and biophysical techniques will also be employed to determine how these enzymes exploit conformation changes and how mutation leads to dysfunction. [unreadable] [unreadable] Descriptions of domain rearrangements will be obtained for B12-dependent methionine synthase (MetH), a prototype for complex proteins that undergo large domain movements. In MetH from E. coli, a large enzyme with four ligand binding modules, the B12-binding domain is required to move long distances to interact in turn with homocysteine, methyltetrahydrofolate, and S-adenosylmethionine. Structures of each module were determined earlier; the current goal is to understand how the modules interact and what drives their movements. Impairment of human methionine synthase, an ortholog of the E. coli enzyme, is responsible for many of the manifestations of B12 deficiency. Comparisons will be made with B12-independent methionine synthase, which catalyzes methylation of homocysteine without requiring an intermediate methyl carrier. [unreadable] [unreadable] Structure-function studies of human BHMT, an important enzyme in homocysteine homeostasis in liver, are aimed at understanding the conformation changes that produce ordered binding of substrates. [unreadable] [unreadable] The structure of methylenetetrahydrofolate reductase (MTHFR) from E. coli has been determined as a model for the catalytic module of human MTHFR, an enzyme that lies at a critical branch point in one-carbon metabolism and is allosterically controlled by S-adenosylmethionine. The bacterial model has been used to show how folates protect the activity of MTHFR, thereby lowering homocysteine levels. The larger human enzyme, containing both catalytic and regulatory modules, has now been expressed by our collaborators; structural studies are aimed at understanding the regulatory mechanisms. [unreadable] [unreadable]

Project Summary Polyketide natural products are the basis for a substantial number of pharmaceuticals, including some with newly discovered indications for inflammatory diseases. Development of polyketide lead compounds is challenging due to their chemical complexity and the inability to obtain most of them from natural sources. Thus the biosynthetic pathways are attractive targets for expanding chemical space through chemo-enzymatic synthesis and for engineering pathways to new function. The type I polyketide synthases (PKS) are assembly- line megasynthases where several distinct modules sequentially extend and modify a polyketide intermediate. This project examines fundamental aspects of PKS module function and of PKS enzyme domains that catalyze unusual reactions. In Aim 1, a robust assay system using authentic, late-stage polyketide intermediates as substrates will be employed to determine the selectivity of PKS catalytic domains for their natural acyl carrier protein (ACP) partners and the limits of enzyme tolerance for foreign ACPs. Details of ACP-enzyme interaction will be visualized in crystal structures of crosslinked ACP-enzyme complexes and will aid future efforts to design of a broadly tolerated generic ACP. In Aim 2, dehydratase domains will be investigated to determine the structural basis for two unusual activities: a rare dual dehydratase-isomerase activity and the unprecedented dehydration of ?-hydroxyacyl substrates. In Aim 3, the structural basis for C-methyltransferase activity by domains embedded in PKS modules will be investigated and the substrate range will be explored. Macrolide antibiotics are among the most interesting polyketides, and a final aim will address the determinants of macrocycle formation by the thioesterases (TE) that offload polyketide products. Using four macrocycle- forming TEs of known structure and a panel of natural and non-natural substrates, Aim 4 will test the hypothesis that the active site of each TE is adapted to bind its natural substrate in conformations that are most conducive to cyclization and not hydrolysis to a linear product. Details of trapped acyl-enzyme intermediates will be visualized in crystal structures. These fundamental studies will have direct relevance to efforts to tap the amazing diversity of polyketide natural products by exploiting the biosynthetic pathways in development of new compounds. The enzymes that catalyze reactions of dehydration, isomerization, methyl transfer and macrocycle formation can be deployed as tools to expand the chemical potential of polyketide bio- activity.

DESCRIPTION (provided by applicant): Acquired resistance to antibacterial drugs is a growing public health problem due to the spread of multidrug-resistant bacteria. The long-term objective of this project is to build the knowledge to engineer new combinations of polyketide synthase (PKS) catalytic domains with reliable predictions of the chemical and stereochemical outcome. Modular PKSs are among the most desirable target pathways for "combinatorial biosynthesis" because of the rich chemical diversity of their products. They are also among the most amenable pathways to this approach because of their modular organization. Recombination of PKS domains to build synthetic pathways for novel compounds requires an understanding of the basis for substrate specificity and stereochemical outcome for each catalytic domain, which at present is lacking. Crystal structures of some domains are available, but, apart from the preliminary studies for this application, the structures lack bound substrates or analogs. Affinity labels have been demonstrated to be an effective means to establish the mechanism of pikromycin thioesterase (Pik TE). The central hypothesis of the proposed research is that polyketide-based affinity labels are powerful tools to probe the structure and mechanism of isolated PKS didomains. The specific aims of this application are: (1) to determine the structural basis for substrate specificity and stereochemical outcome of several NADP-dependent ketoreductase (KR) domains, (2) to explore the substrate range of KR domains for non-natural substrates, and (3) to understand the mechanisms of macrolactone formation by terminal thioesterase (TE) domains. The research design and methods will utilize the acyl carrier protein (ACP) of didomain constructs, KR-ACP and ACP-TE, to deliver substrate and product mimics to the KR and TE active sites. Natural KR-ACP and ACP-TE didomains will be covalently modified by vinyl ketone affinity label mimics, and their crystal structures determined. Didomains from the PKS systems for pikromycin, erythromycin, and tylosin will be examined since these molecules are established lead compounds for antibiotic drug discovery. The proposed research is significant because the rational engineering of PKS systems is expected to provide novel macrolide antibiotics to combat the rising tide of multidrug-resistant bacteria. PUBLIC HEALTH RELEVANCE: Complex mega-enzyme machines known as polyketide synthases (PKS) produce many antibiotics and other bio-active molecules. With appropriate engineering, these proteins are potentially rich sources of new pharmaceuticals. This project will examine the details of substrate interactions with several PKS domains. Substrate mimics will be synthesized that covalently attach to the enzyme, and crystal structures of substrate mimic trapped in the enzyme will be solved. The goal is to understand how the substrate specificity and range for each reaction, and thereby allow design of new enzymes to make new bio-active molecules.

DESCRIPTION (provided by applicant): Marine cyanobacteria are extraordinarily rich in their production of biologically active and structurally unique natural products. A number of these secondary metabolites or their derivatives are lead compounds in drug development programs aimed at providing new therapies to treat cancer, bacterial infections, inflammatory responses and in crop protection to kill harmful microbial pathogens and insects. Isolation and structural analysis of marine and terrestrial cyanobacterial natural products has provided access to an unusually large number of mixed non-ribosomal peptide synthetase/polyketide synthase (NRPS/PKS) systems. The corresponding metabolic systems are comprised of an intriguing set of complex multifunctional proteins that along with allied enzymes generate structurally complex molecules via a modular multi-step process. Over the past several years the Sherman, Gerwick and Smith laboratories have developed a complementary program to clone and characterize the biosynthetic pathways of novel cyanobacterial secondary metabolites that possess significant potential for biotechnological applications. Despite considerable progress, a full understanding of the molecular mechanisms, catalytic activities, kinetic properties, and substrate specificities within cyanobacterial biosynthetic pathways is just beginning to unfold. The proposed research will build upon our accomplishments on the curacin, jamaicamide and cryptophycin/ arenastatin metabolic systems, three robust pathways that have been a rich source of new information. The expected metabolic, biochemical and structural understanding will facilitate the design of new biosynthetic systems that harness the growing potential of cyanobacterial natural product pathways. The full promise of cyanobacterial natural products to yield new lead compounds for development as useful pharmaceuticals will only be realized by closing a series of key gaps in knowledge and technology. Solving these challenges will require development and optimization of genetic and biochemical methods that allow us to 1) manipulate cyanobacterial natural product metabolic systems to produce analog structures, 2) utilize unique secondary metabolite enzymes for creation of novel bioactive molecules and, 3) screen new compounds and analogs to identify promising new anticancer compounds for further development. The specific aims are: 1. To harness the inherent versatility of cyanobacterial natural product systems to create new anticancer lead compounds. Sub-aims include: a. Investigate ability of cyanobacterial biosynthetic pathways to generate novel analogs using unique laboratory culture and mutasynthesis methodologies. b. Investigate the unique enzymatic capabilities of marine cyanobacterial pathways to engineer new metabolic systems and tailoring processes to generate new bioactive compounds. c. Employ structural biology and site-directed mutagenesis approaches to understand the precise biochemical mechanisms of unique biosynthetic enzymes. d. Develop new chemoenzymatic, in vivo, and in vitro pathways to create new anticancer agents with enhanced medicinal properties 2. Perform bioassays on new compounds resulting from Specific Aim 1. a. New compounds derived from the proposed research will be transferred to Eisai Research Institute and University of Michigan Center for Chemical Genomics for analysis of biological activity using a series of biochemical and cell based assays relevant to cancer.

DESCRIPTION (provided by applicant): A bacterial type I polyketide synthase (PKS) is comprised of an intriguing set of complex multifunctional proteins that along with allied enzymes generate structurally complex and clinically important natural products via a modular multi-step process. Numerous systems of this type have been discovered over the past decade, paving the way to engineered PKSs that generate novel natural products. Access to affordable high throughput genome sequencing of diverse microbial systems is revealing new PKS, non- ribosomal peptide synthetase (NRPS) and mixed PKS-NRPS systems at an ever-increasing rate. Moreover, bioinformatic tools to predict the structural outcome of these metabolic systems are providing rapid access to new natural products. Despite increasing access to new information, obtaining a detailed biochemical understanding of PKS-NRPS systems is necessary to test functional predictions and demands the application of rigorous experimental approaches. Understanding these details will not only expand our basic knowledge of PKS-NRPS molecular machines, but also provide new strategies to manipulate them to expand chemical diversity. Such systems are attractive due to their potential to create new chemotypes with valuable applications in drug discovery and development. Despite remarkable progress, an understanding of the molecular mechanisms, catalytic activities, kinetic properties, substrate specificity and protein-protein recognition in both natural and hybrid PKSs remains limited. This competing renewal application proposes to employ the versatile and well-characterized Streptomyces venezuelae pikromycin PKS, as well as a series of additional pathways whose detailed analysis has been initiated during the previous cycle of support. These systems each bear fascinating biochemical attributes that will expand our understanding of the specificity and structural features that lead to functional activity within and between native and hybrid PKS modules. Our objectives and approach will focus on assessing the molecular details of polyketide chain initiation, elongation, 2-branching and termination that lead to the remarkable chemical diversity of polyketide natural products. This detailed biochemical analysis, and the integration of structural biology to probe substrate specificity and synthetic chemistry to develop chemoenzymatic approaches will allow pursuit of our long term objective of engineering PKS systems that efficiently generate novel structures with significant potential as therapeutic agents. Specific aims include: I. Molecular Analysis of Modular Polyketide Synthases. Design and employ synthetic substrates and Pik, DEBS, and Tyl terminal modules to explore selectivity and tolerance in chain loading, elongation and processing. II. Molecular recognition as the basis for protein-protein interactions in modular PKSs. Explore molecular parameters of docking selectivity by designing and constructing effective pathways using native, and heterologous docking domain combinations. III. Analysis of the molecular basis for termination in modular systems. Explore the determinants of macrolactone formation vs. hydrolysis by the terminating thioesterases in the PKSs for pikromycin, erythromycin, tylosin, tautomycetin, curacin, and carmabin. IV. Analysis of new catalytic domains and molecular interactions in modular PKSs that synthesize 2-branched products. Pursue analysis of the bryostatin biosynthetic system (Bry) including HMG synthase and 2-branching leading to the modified pyrone ring system. Explore the basis for acyl-ACP cognate enzyme interactions in Bry including ACPD::HMGS, ACPD::KS, and KS::HMGS). PUBLIC HEALTH RELEVANCE: The proposed research will focus on elucidating the detailed function of complex biosynthetic machines that create chemically diverse, biologically active natural products. The ability to understand and subsequently engineer these remarkable biochemical systems will create new opportunities to discover and develop effective drugs for the treatment of human diseases, including cancer, infectious diseases, and Alzheimer's.

HIV; RNA;

DESCRIPTION (provided by applicant): The 27th Annual Symposium of The Protein Society aims to act as a nexus for scientists from all disciplines, and at all stages of career development, who share a common research interest in protein structure, function, dynamics, design and their implications with regard to human health and disease. A gathering of biologists, chemists, physicists, mathematicians and others, the Symposium is designed to facilitate the dissemination of protein-related knowledge outside the silos of discipline and sector, and has long served to accelerate the advancement of health- related discovery. The central role of proteins in human biology, combined with the ever- expanding array of methodologies, tools, and investigative strategies used to understand them, make opportunities for coactive exploration more critical than ever before. The program will consist of 10 scientific sessions that focus on leading-edge topics including protein interactions in the cellular milieu, protein folding proteins and human disease, big machines, chemical biology, post-translational modifications, protein design and directed evolution, functional dynamics, protein science for sustainability, and nanotechnology. It will feature presentations by nearly 50 researchers (including 20 Young Investigator talks, selected from submitted abstracts of students and postdocs), as well as 2 plenary sessions. Recognizing the enduring nature of the struggle against human disease, the Protein Society Symposium places a special emphasis upon fostering the growth and development of young scientists by providing unique and plentiful opportunities for mentorship and interaction with established researchers, career guidance through educational workshops led by established academics, as well as numerous prospects for recognition of their work. And, true to the mission and character of the Society, a focus on the participation of traditionally underrepresented groups means that attendees benefit from varied perspectives, enriching the quality and effectiveness of the overall scientific program. A special edition of the Society journal, Protein Science, containing the submitted abstracts of conference participants, will be published in conjunction with the Symposium and made available to participants and interested investigators in print and online. Held in Boston (July 20- 23, 2013), the Symposium is expected to include more than 1,000 attendees, and will offer complimentary attendance to interested undergraduate students, with a concerted effort to encourage participation by representatives of graduate programs at leading academic institutions, and to act as a forum for connection of the two.

Core 2: Crystallography - Summary The CRNA Crystallography Core will collaborate with CRNA investigators to solve crystal structures of RNA-protein complexes, RNAs and proteins relevant to HIV RNA- mediated processes. The Core also provides essential expertise and materials that extend far beyond the crystallography mission and support many macromolecule preparation and analysis aspects of CRNA research. The Core team, led by Smith and Stuckey, offers collaborative expertise in molecular biology tailored for RNAs and RNA- protein complexes, including construct design, expression testing, and development of purification protocols. The resulting vectors, expression plasmids and protocols as well as purified RNAs, RNA-protein complexes and proteins are provided to collaborating investigators in the CRNA, with the Core lab at the University of Michigan acting as a repository for materials and protocols. Two new members of the Crystallography Core team, CRNA investigator Pornillos (U Virginia) and Significant Contributor Zhang (NIDDK, NIH), bring exceptional expertise in the design, production and validation of RNAs and RNA-protein complexes, particularly for crystallization. In CRNA 2.0, the Core will develop novel methods for expression of RNAs, formation of RNA-protein complexes by co-expression, and design of chaperones to aid RNA crystallization. The Core will also establish an in vitro RNA synthesis capability at Michigan. In support of its crystallography mission, the Core will develop cutting-edge methods to aid RNA crystallization, to improve the diffraction quality of crystals, to solve the phase problem for RNA-containing crystals, to improve crystallographic refinement for RNA-containing structures, and to facilitate the combination of crystallography and electron microscopy. !

Project Summary Flaviviruses are insect-transmitted human pathogens that most notably cause Zika-associated microcephaly, dengue fever and West Nile fever. This study aims to elucidate the molecular mechanisms of the flaviviral nonstructural protein 1 (NS1), a multi-functional virulence protein. Intracellular NS1 is essential to replication of the viral RNA genome, whereas secreted NS1 interacts with innate immunity proteins and, in some cases, induces disease phenotypes. Our crystal structures of full-length, glycosylated NS1 from Zika virus (ZIKV), dengue virus serotype 2 (DENV2) and West Nile virus (WNV) will guide experiments to identify which of the distinct domains of NS1 are responsible for which of its several functions. Despite their overall similarity (~50% identical amino acid sequences), several properties specific to individual flaviviruses are attributed to the NS1 proteins, making comparative analysis especially powerful in dissecting the molecular functions of NS1. An extensive panel of mutants based on comparative mutagenesis of DENV2 and WNV NS1 will be expanded to include ZIKV NS1. Based on observations in the initial comparative study that NS1 affects virus particle assembly, we will test the hypothesis that NS1 acts as an infectivity factor by aiding viral structural protein folding or virus particle transit through the secretory system. Intracellular NS1 is localized to the ER lumen as a membrane-associated dimer with a critical role in replication through association with viral transmembrane proteins. We will probe the interaction with viral protein NS4B through mutagenesis and biophysical experiments. Electron cryo-microscopy or crystallography will be used to investigate the structure of secreted NS1, a hexameric lipo-protein particle. An initial observation that NS1 remodels membranes will be followed by detailed experiments using light microscopy with fluorescently tagged lipids to determine any lipid preference in this key association. Binding experiments will identify which domains of NS1 interact with which domains of two proteins of the complement system and the innate immunity Toll-like receptor 4. The results will provide a foundation for development of antiviral drugs and/or effective vaccines, which are not available or of limited use for Zika, dengue or West Nile viruses.

PROJECT SUMMARY/ABSTRACT (STRUCTURE AND DRUG SCREENING) In the previous CCSG renewal, Structural Biology (SB) and High-Throughput Screening (HTS) were both approved and funded as new Shared Resources (SRs). During the project period, the independent SRs frequently coordinated services for University of Michigan Comprehensive Cancer Center (UMCCC) members to increase the efficiency and impact of cancer-related projects. This collaboration demonstrated the benefit of working together from project initiation. The SRs and UMCCC leaders viewed the amalgamation of the SR as a natural progression and a way for members to receive maximum benefit from these vital resources. With support from UMCCC Senior Leadership, Janet Smith, PhD was joined by Co-Director Vince Groppi, PhD in 2015 when they formally merged the SRs into one unit. During this process, Medicinal Chemistry (MC) was added to the SR to meet existing needs of cancer researchers. In 2016, UMCCC approved the establishment of a comprehensive unified facility, the Structure and Drug Screening (SDS) SR. The SDS SR enables UMCCC members to use advanced structure-based drug design and/or nonbiased HTS strategies to identify chemical matter that can be advanced through a milestone-driven research plan resulting in efficient and effective discovery and development of precision oncology medicines; a strategic goal of UMCCC. This is accomplished by use of three service disciplines, each of which is guided by an experienced faculty leader: SB, under the direction of Dr. Smith; HTS, under the direction of Dr. Groppi, and MC under the direction of Andrew White, PhD. The SR's cohesive activities, services and processes yield four key benefits to UMCCC members: I) projects are advanced along the translational pipeline, from basic science discoveries into early cancer drug discovery; II) a project manager guides researchers in the effective and efficient use of appropriate services matched to their needs; III) an integrated team approach provides researchers a complete, `one-stop' set of expertise for the duration of their projects; and IV) researchers accrue cost savings from a streamlined administrative support structure.