Raymond C. Stevens, Ph.D. - US grants

The objective of this project is to determine the 3-dimensional structure of the human beta2-adrenergic receptor, a G-protein coupled 7 transmembrane receptor, in the presence and absence of agonist and antagonist. Two approaches will be taken to determine the three- dimensional structure. The first approach is to determine the structure of the holo-receptor. The second approach is to truncate the receptor by removing the large soluble cytoplasmic regions of the receptor. The goal of working with the truncated molecule is to aid in the stabilization of the molecule and provide an alternative molecule for crystallization trials. The technique of electron diffraction of 2-dimensional crystals will be used. Because of the similarity in structure between bacteriorhodopsin and adrenergic receptor, the procedure used to determine the structure of bacteriorhodopsin will initially be followed.

9458067 Stevens Two areas of research are being pursued that focus on the receptor molecules that are responsible for neuronal signal transduction. The first project takes a structural approach using x-ray and electron crystallography to elucidate the structure and function of the family of 7-transmembrane G- protein coupled receptors. Although there has been a great deal of information obtained about 7-transmembrane cell receptors, the information has not been expanded and consolidated so that an understanding of their structure and function is known. These molecules are key to understanding how signal transduction occurs between the cells in the brain and nervous system. The second project uses the neuronal receptors described in the first part in the design of a new molecular biosensor. The receptor biosensor harnesses the power of these selective and sensitive receptors by mimicking how cells communicate the detection of drugs, odorants, etc. The biosensor is a new material that couples the receptor biosensor to colored lipids that change color upon ligand binding induced structural changes in the receptor. By designing materials that mimic how neurons communicate, we are able to learn more about how communication occurs.

Funds are requested for an X-ray data collection system for macromolecular crystal structure determination. The instrument is the R-AXIS II imaging plate system, which includes a rotating anode X-ray generator, focusing mirrors, and a controlling computer. The new instrument will be located in Stanley Hall at the University of California, Berkeley, and will be shared by three research groups which- use X-ray crystallography as a primary research tool. Currently, the three research groups share one X-ray area detector, which is used full time, and the backlog of data collection limits progress. The three groups have a broad range of research interests. The proteins and peptides under investigation differ significantly in size and biological function. Prof. Alber asks general questions about protein folding, stability, and intra- and intermolecular protein interactions. Model systems used to test these questions include coiled-coils, T4 lysozyme, ATCase, and transcriptional accessory proteins. Prof. Nelson studies DNA binding proteins and protein-DNA interactions. Crystals of the DNA binding domain of the heat shock transcription factor diffract X-rays to at least 0.9 resolution, making this the highest resolution structure for which mutants are readily obtained. Prof. Stevens studies proteins involved in neurotransmission. Proteins under study include: two proteins involved in catecholamine biosynthesis, tyrosine hydroxylase and dopamine monoxygenase; neuroreceptors, such as human 2-adrenergic receptor, fish olfactory receptor, and yeast factor receptor; and the botulinum neurotoxin. In addition, to the individual research interests of the three groups, collaborative crystallographic projects have been initiated with other faculty at Berkeley. Clearly, an additional instrument will allow significant progress to made on a wide range of crystallographic studies. In addition to supporting the research efforts of these three groups, this instrument will have a train ing function. Between the three groups, there are 4 post-doctoral fellows, 14 graduate students, and 4 undergraduates who are pursuing crystallographic projects. The size of the joint group is expected to grow by 6 more students and fellows within the next year.

DESCRIPTION: Structure-function studies of catalytic antibodies are to focus on three systems. (1) The antibody 48G7 which was elicited to a phosphonate transition-state (TS) analogue and catalyzes the hydrolysis of the corresponding esters and carbonates. This system represents one of the simplest and most prevalent antibody-catalyzed reactions to study transition state stabilization and catalysis. Moreover, the germline genes have been cloned and expressed, permitting the study of the immunological evolution of catalysis. The functional effects of affinity maturation on TS analogue binding and catalysis can be analyzed and interpreted in terms of the three-dimensional structures of both the mature (48G7) and germline antibodies. In addition, a high level expression system in E. coli will allow a systematic mutagenesis study of both the active site and somatically mutated residues. (2) The antibody AZ-28 which was elicited against a chairlike TS analogue and catalyzes the corresponding oxy-Cope rearrangement. There are virtually no enzymes that catalyze such pericyclic rearrangements with the exception of chorismate mutase, the mechanism of which remains unclear despite extensive structural and mechanistic studies. Consequently, detailed structure-function studies of this biological catalyst may shed insight into the requirements for catalysis of the chemical transformation. Moreover, the availability of a family of antibodies that catalyze this unimolecular reaction may help to elucidate those factors essential for activity. (3) The antibody 28B4.2 which catalyzes the oxygenation reaction of a thioether to the corresponding sulfoxide. This was the first biological catalyst to use an abiological "cofactor" for activity, i.e. a periodate ion, in place of the heme and flavin cofactors used by the corresponding enzymes. The kcat values for the antibody and enzymes are comparable, suggesting that this strategy may allow the replacement of expensive cofactors with inexpensive chemical cofactors. A detailed understanding of the structure and mechanism of this antibody and the relationship of the active site structure to hapten structure may suggest generalizations and may allow engineering of antibodies with enhanced stereoselectivities or may permit modification of the antibody to other reactions such as disulfide bond formation.

immunology; biomedical resource; biomaterials;

Botulinum neurotoxin complex serotype A is a 900 kiloDalton (kDa) protein produced as one of eight serotypes (A-G) by the anaerobic bacterium Clostridium botulinum. Among the most potent biological toxins known to man, botulinum neurotoxin causes inhibition of synaptic vesicle release at the neuromuscular junction resulting in flaccid paralysis and ultimately death. Botulinum neurotoxin type A (BoNT/A) is a potent disease agent in both food-borne botulism and Sudden Infant Death Syndrome (SIDS), an established biological weapon, and a novel therapeutic in the treatment of involuntary muscle disorders. Previously, we have determined the 3-D structure of the 150 kDNA neurotoxin component of the 900 kDa complex by x-ray crystallography. We have also completed antibody mapping experiments to determine how the 150 kDa neurotoxin is bound into the 900 kDa toxin complex. We have conducted a series of biophysical stability experiments in order to understand how the two assemblies (150 kDa toxin and 750 kDa non-toxic component) combine and stabilize the 900 kDa complex. Lastly, based on the work above, and preliminary electron microscopy work, we are designing an alternative vaccine strategy for botulism. Current vaccine programs for botulism are not very effective. The preliminary objective of this proposal is to obtain a three- dimensional structure of the 900 kDa botulinum neurotoxin complex, and understand how the neurotoxin component fits into the complex. To accomplish this goal, we will use a 2-D crystals of the 900 kDa complex to conduct 3-D image reconstruction experiments. We have already obtained 2-D crystals of the 900 kDa complex to conduct 3-D image reconstruction experiments. We have already obtained 2-D crystals of the 900 kDa complex that diffract weakly to 14 Angstroms resolution in negative strain, and a density projection map has been produced at 30 Angstroms resolution. Based on the crystal quality and the frequency with which defects were observed in the crystals used in our earlier investigation, it appears as though much higher quality crystals can be obtained. Specifically, our transfer technique is presently crude due to our new venture into this area of research, and several suggestions have been made by other program project members on how to improve our transfer techniques. We are also investigating alternative buffer conditions to help stabilize the protein further. Once optimization of the 2-D crystals has been completed, we will complete the negative stain work at the maximum resolution possible using data collection in a tilt series followed by 3-D image reconstruction. This work will be followed by attempting higher resolution studies with cryo-techniques. We will crystallize the 900 kDa complex in the presence of scFv antibody molecules that have a high affinity for exposed regions of the neurotoxin when bound to the 900 kDn complex.

Structural studies of catalytic antibodies in my laboratory focuses on six systems (i) Antibody-catalyzed ester hydrolysis, (ii) An antibody ferrochelastase, (iii) Antibody-catalyzed aminoacylation reaction, (iv) An antibody-catalyzed regio- and stereoselective ketone reduction. To date, the field of catalytic antibodies has suffered from not having high resolution crystal structures to aid in the hapten/transition state ligand design. Without improved hapten design, the field will have a difficult time in improving the catalytic rates of the abzymes and in achieving the catalytic rates that are comparable to enzymes. Thus it is critical to obtain the highest resolution data possible to carefully understand how the catalytic antibodies function.

SUBPROJECT ABSTRACT NOT AVAILABLE

The three-dimensional structure of the enzymes phenylalanine hydroxylase and tryptophan hydroxylase will be studied using crystallographic methods. The structures of the catalytic domain of human phenylalanine hydroxylase, catalytic domain plus tetramerization domain, catalytic domain plus regulatory domain, and catalytic domain with catecholamine inhibitors have previously been determined in my lab at various resolutions (1.9 Angstroms to 3.0 Angstroms). The family of aromatic amino acid hydroxylases are known to be involved in inherited metabolic disorders. The enzyme phenylalanine hydroxylase is the known cause of the disease known as PKU (phenylketonuria). The enzyme normally converts the essential amino acid phenylalanine to tyrosine. Failure of the conversion results in a buildup of phenylalanine. Excessive amounts of phenylalanine are toxic to the central nervous system and causes severe problems associated with PKU. Based on the three-dimensional structure of the modeled full-length enzyme, the mutations will be mapped and site- specific mutagenesis will be conducted to understand the structural basis of PKU. As a complement to the studies on phenylalanine hydroxylase, tryptophan hydroxylase will be studied as comparisons of it and phenylalanine hydroxylase may provide important insights into the mechanisms of PKU. A second goal of the research is to understand the structural basis of pterin-dependent hydroxylase catalysis. The three-dimensional structure of both enzymes will be determined in the presence of co-factors, substrates, and inhibitors in order to understand the mechanism of catalysis and role of inhibitors. The last goal is to crystallize and determine the three-dimensional structure of the intact holoenzyme. By combining the structures of the isolated domains with adequate overlap, a proposed full-length enzyme model can be assembled. However it is important to determine the three dimensional structure of the full-length enzyme in order to fully understand the complex regulatory, catalytic, and stability issues associated with this family of enzymes.

Abstract Not Provided.

[unreadable] DESCRIPTION (provided by applicant): The goal of the JCSG Center for Innovative Membrane Protein Technologies (JCIMPT) is to develop and disseminate methods and technologies for structure-grade production of membrane proteins. JCIMPT is focusing on integral membrane proteins expressed in cell-free, baculovirus and mammalian cell systems. Close integration and collaboration with the Joint Center for Structural Genomics will provide access to databases, database tracking, and biophysical characterization technologies. In addition, the JCSG and the JCIMPT will collaborate on bacterial expression technologies. Miniaturization and automation are major components in our systematic investigation of membrane protein expression and the development of novel genetically engineered expression systems and sample preparation technologies. JCIMPT will synthesize and test a matrix of new molecules to stabilize integral membrane proteins. To develop the necessary methods and technologies, we have assembled a consortium of investigators that have extensive experience in both technology development and technology transfer for structural biology as well as for membrane protein production and structure determination by electron microscopy, NMR spectroscopy, and X-ray crystallography. A clear path for technology dissemination of the expression and sample preparation developments are in place through key collaborations with companies that already supply many structural biologists with the necessary tools to conduct their research. [unreadable] [unreadable] [unreadable]

ABSTRACT NOT PROVIDED

The mammalian fatty acid amides (FAAs) have been directly linked to the regulation of pain thresholds, body temperature, sleep cycles, appetite, and higher-level cognitive processes such as memory and learning. Nonetheless, how FAAs influence nervous system function is poorly understood. While some of these molecules trigger the central cannabinoid receptor CB1, other members of this class lack described molecular targets. The enzyme fatty acid amide hydrolase (FAAH) controls the levels of fatty acid amides in vivo, setting the baseline function of their various corresponding physiologies. We have recently determined the three dimensional structure of this integral membrane protein, and we are now prepared to begin second-generation structure determination efforts to extend our knowledge of the mechanisms of action of this important enzyme. The studies described in this application (Project II of the Program Project) aim to determine higher resolution FAAH structures, as well as structures of human FAAH, apo-FAAH, FAAH-inhibitor/product complexes, and key FAAH mutants, including the natural P129T variant associated with problem drug use. Information accrued from our studies will not only enlighten our understanding of FAAH but will also serve as a guide for the development of agents designed to intersect the cannabinoid and other FAA systems in vivo, possibly to therapeutic benefit.

[unreadable] DESCRIPTION (provided by applicant): The goal of the JCSG Center for Innovative Membrane Protein Technologies (JCIMPT) is to develop and disseminate methods and technologies for structure-grade production of membrane proteins. JCIMPT is focusing on integral membrane proteins expressed in cell-free, baculovirus and mammalian cell systems. Close integration and collaboration with the Joint Center for Structural Genomics will provide access to databases, database tracking, and biophysical characterization technologies. In addition, the JCSG and the JCIMPT will collaborate on bacterial expression technologies. Miniaturization and automation are major components in our systematic investigation of membrane protein expression and the development of novel genetically engineered expression systems and sample preparation technologies. JCIMPT will synthesize and test a matrix of new molecules to stabilize integral membrane proteins. To develop the necessary methods and technologies, we have assembled a consortium of investigators that have extensive experience in both technology development and technology transfer for structural biology as well as for membrane protein production and structure determination by electron microscopy, NMR spectroscopy, and X-ray crystallography. A clear path for technology dissemination of the expression and sample preparation developments are in place through key collaborations with companies that already supply many structural biologists with the necessary tools to conduct their research. [unreadable] [unreadable] [unreadable]

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Immunization with transition state analogue results in a germline-encoded antibody that catalyses the rearrangement of hexadiene to aldehyde with a rate approaching that of a related pericyclic reaction catalysed by the enzyme chorismate mutase. Affinity maturation gives antibody AZ-28, which has six amino acid substitutions, one of which results in a decrease in catalytic rate. To understand the relationship between binding and catalytic rate in this system we characterized a series of active-site mutants and determined the 3-D crystal structure of the complex of AZ-28 with the transition state analogue. This analysis indicates that the activation energy depends on a complex balance of several stereoelectronic effects which are controlled by an extensive network of binding interactions in the active site.

ABSTRACT NOT PROVIDED

DESCRIPTION (provided by applicant): Synchrotron beam time has always been a needed and limited resource for macromolecular crystallographers, even with the advancements seen in higher brilliance facilities. Improvements in protein expression and crystallization have expanded the number and range of biologically interesting targets, the number of researchers capable of conducting this type of research, and the high value of the information obtained from such studies. Recently, with NIH and DOE support, the macromolecular crystallography beamlines in the United States are becoming more automated with the installation of robotic crystal sample changers that have impressive robustness and utility. With the improvement in beam line efficiency, in-house pre-screening of protein crystals becomes limiting leading to extensive screening of samples at the synchrotron beam lines (screening time estimated at 70% by several different laboratories), particularly for membrane proteins, protein-protein complexes, and structural genomics efforts. Because of limited synchrotron beam time and the improvements in the other aspects of protein crystallographic research, one (1) can no longer screen all of the crystalline samples available and therefore there is a critical need for robotics equipment on in-house x-ray sources, particularly for institutions with very large crystallographic communities. The opportunity to substantially improve the quantity and quality of the crystal screening process in the home laboratory will result in much more efficient usage of synchrotron beamtime as well as enabling new scientific knowledge gained from the subsequent structural studies. We are thus requesting funds to purchase an automated crystal sample changing and characterization system capable of automatically mounting, aligning, imaging (visual & diffraction) and dismounting batches of 288 cryogenically-cooled crystals on an in-house high brilliance (FR-D) x-ray source. The robotics system is identical to that at SSRL, using the 96 pin cassette system that can also handle pucks from the MSC/ALS robotics system. 15 NIH funded research laboratories at TSRI and the Burnham Institute (~180 researchers) plan to utilize the automated screening facility.

The botulinum neurotoxins (BoNTs), produced by Clostridium botulinum are among the most potent toxins known to man. The Centers for Disease Control and Prevention (CDC) has classified it as a potential bioweapon, Category A, because of its extreme potency and lethality, its ease of production and transport, and the need for prolonged intensive care among affected persons. Recently, it has been observed that the majority of therapeutic and diagnostic BoNT programs are creating narrow spectrum reagents (molecules specific for only one very specific subtype of BoNT) and that the number of subtypes is much larger than originally anticipated. This project focuses on determining the three-dimensional structure of prioritized serotypes and subtypes of BoNTs. Serotype/subtype information is critical in the development of broad spectrum therapeutics and diagnostics that are needed. The overall goal is structurally characterizing these variants at the protein level and developing effective countermeasures through the creation of new antibody reagents and small molecule inhibitors via collaborations. This study will conduct a series of X-ray crystallographic studies to determine the three dimensional structure of the prioritized holotoxin serotypes/subtypes, prioritized catalytic and binding domain serotype/subrypes, and complexes with antibodies and small molecule inhibitors. In addition to the structures, the project will also produce highly purified protein samples that will be made available to its collaborators and other workers in the biodefense consortium for assay and antibody development.

[unreadable] DESCRIPTION (provided by applicant): The goal of the JCSG Center for Innovative Membrane Protein Technologies (JCIMPT) is to develop and disseminate methods and technologies for structure-grade production of membrane proteins. JCIMPT is focusing on integral membrane proteins expressed in cell-free, baculovirus and mammalian cell systems. Close integration and collaboration with the Joint Center for Structural Genomics will provide access to databases, database tracking, and biophysical characterization technologies. In addition, the JCSG and the JCIMPT will collaborate on bacterial expression technologies. Miniaturization and automation are major components in our systematic investigation of membrane protein expression and the development of novel genetically engineered expression systems and sample preparation technologies. JCIMPT will synthesize and test a matrix of new molecules to stabilize integral membrane proteins. To develop the necessary methods and technologies, we have assembled a consortium of investigators that have extensive experience in both technology development and technology transfer for structural biology as well as for membrane protein production and structure determination by electron microscopy, NMR spectroscopy, and X-ray crystallography. A clear path for technology dissemination of the expression and sample preparation developments are in place through key collaborations with companies that already supply many structural biologists with the necessary tools to conduct their research. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Phenylketonuria (PKU) is a metabolic disorder that results from impaired activity of hepatic phenylalanine hydroxylase (PAH), the enzyme responsible for disposal of the majority of phenylalanine intake. Currently, the only known treatment for classical PKU is a very difficult to maintain diet for life. Development of a therapeutic treatment would assist the current dietary treatment and prevent the neurological damages inflicted on those individuals with PKU, particularly for those patients with the most severe forms of the disease. This proposal combines the basic PKU research efforts of the Stevens laboratory at The Scripps Research Institute with the clinical PKU research efforts of the Scriver laboratory at the McGill University and Montreal Children's Hospital. Based on our recent success in developing a less immunogenic injectable PKU enzyme substitute using pegylated phenylalanine ammonia-lyase (PEG-PAL; reported by Gamez, et al. (2005) Mo/. 77?er.,11;986-989 and Wang, et al. (2005) Mol. Genet. Metab. 86:134-140), this proposal details the development of a structure-based PKU enzyme substitution therapy using modified PAL or pegylated variations of modified PAL (for stability, protease resistance, and reduced immunogenicity) to create an oral therapeutic. The specific objectives of this study are to use structure- based molecular engineering to optimize a form of PAL with improved characteristics for potential use in PKU oral therapy (Aim 1). Further optimization of PAL will incorporate pegylation procedures to develop a form of the enzyme for PKU oral therapy use (Aim 2). Animal studies will be conducted starting in the first year on the most promising modified PAL candidates, and PK/PD/ADME/toxicity studies will be completed by the end of the funding period, making a favorable orally available therapeutic PAL molecule available for human trials (Aim 3). [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Phenylketonuria (PKU) is a metabolic disorder that results from impaired activity of hepatic phenylalanine hydroxylase (PAH), the enzyme responsible for disposal of the majority of phenylalanine intake. Currently, the only known treatment for classical PKU is a very difficult to maintain diet for life. Development of a therapeutic treatment would assist the current dietary treatment and prevent the neurological damages inflicted on those individuals with PKU, particularly for those patients with the most severe forms of the disease. This proposal combines the basic PKU research efforts of the Stevens laboratory at The Scripps Research Institute with the clinical PKU research efforts of the Scriver laboratory at the McGill University and Montreal Children's Hospital. Based on our recent success in developing a less immunogenic injectable PKU enzyme substitute using pegylated phenylalanine ammonia-lyase (PEG-PAL; reported by Gamez, et al. (2005) Mo/. 77?er.,11;986-989 and Wang, et al. (2005) Mol. Genet. Metab. 86:134-140), this proposal details the development of a structure-based PKU enzyme substitution therapy using modified PAL or pegylated variations of modified PAL (for stability, protease resistance, and reduced immunogenicity) to create an oral therapeutic. The specific objectives of this study are to use structure- based molecular engineering to optimize a form of PAL with improved characteristics for potential use in PKU oral therapy (Aim 1). Further optimization of PAL will incorporate pegylation procedures to develop a form of the enzyme for PKU oral therapy use (Aim 2). Animal studies will be conducted starting in the first year on the most promising modified PAL candidates, and PK/PD/ADME/toxicity studies will be completed by the end of the funding period, making a favorable orally available therapeutic PAL molecule available for human trials (Aim 3). [unreadable] [unreadable] [unreadable]

DESCRIPTION: The NIH Roadmap Initiative was created to focus on critically important and challenging areas of biomedical importance that were not being adequately addressed, due in part to a lack of non-hypothesis driven funding and a lack of suitable methods and new technologies. Since membrane proteins play such a critical role in so many cellular and physiological processes and membrane proteins are so difficult to express, solubilize and purify, the production of membrane proteins was identified as a serious roadblock to understanding their structure and function and chosen as one of the top Roadmap Initiatives. The first phase of the Structural Biology Roadmap Initiative focused on creating Centers for Innovation in Membrane Protein Production. Two Centers one at University of California at San Francisco (UCSF) and the other at The Scripps Research Institute (TSRI) were funded and established in 2004. Because innovation was not the exclusive domain of these centers, a second and third initiative requested P01, R21 &R01s and more recently, additional R01 applications, focused on novel approaches to obtaining intact and active membrane proteins for structural studies. To date, 20 grants have been funded through this mechanism. Because of the potentially valuable information that could be generated and shared, NIH staff decided that there should be an annual Inter-Center meeting between the Centers. The first Inter-Center meeting, hosted by Bob Stroud at UCSF in May, 2006, was very successful and the consensus among the center directors and NIH program directors was that all investigators funded through the Structural Biology Roadmap Initiative should be included and invited to the next annual meeting. This proposal requests funds to establish an expanded NIH Roadmap meeting to include all funded Roadmap investigators, with the first extended meeting to be held at TSRI November 1-2, 2007. The meeting will provide a unique opportunity for members of the two Roadmap membrane protein centers as well as those from projects funded through the RFA Membrane Protein Production and Structure Determination (RFA-RM-04-026 and RFA-RM-07-003) to discuss the current status of their projects (Table 1). Presentation and discussion will deal with progress as well as barriers to progress that could transform membrane protein structure determination processes to produce results comparable to that now seen in studies of soluble proteins. The organizers view the meeting as a possible catalyst for the start of fruitful collaborations among conference participants and will encourage their formation. Three to four leading experts in the field will also be invited to help further catalyze the discussions and disseminate the technologies. Plans also include the publication of the proceedings of the meeting in a high-impact structural biology journal thus providing a wide distribution of the discussions and results presented in the meeting. This proposal requests funds to establish an expanded NIH Roadmap meeting to include all funded Roadmap investigators, with the first extended meeting to be held at TSRI November 1-2, 2007. The meeting will provide a unique opportunity for members of the two Roadmap membrane protein centers as well as those from projects funded through the RFA Membrane Protein Production and Structure Determination (RFA-RM-04-026 and RFA-RM-07-003) to discuss the current status of their projects (Table 1). Presentation and discussion will deal with progress as well as barriers to progress that could transform membrane protein structure determination processes to produce results comparable to that now seen in studies of soluble proteins.

DESCRIPTION (provided by applicant): The Joint Center for Integral Membrane Protein Technologies-Complexes (JCIMPT-Complexes) is a research network comprising seven integrated projects working in three focused areas of membrane protein expression, stabilization, and biophysical characterization. The primary mission is to develop and disseminate novel and enabling methods and technologies to the scientific community that lead to the structure determination of human membrane proteins and their complexes. Miniaturization and automation are the major themes in the systematic development of new technologies to study membrane proteins and membrane protein complexes. Our extensive experience with human G protein-coupled receptors and their complexes make them the ideal target protein family for the planned focused technology development. In several active collaborations with others, we will work in parallel on targets including transporters and ion channels. Focus area A will work on production of eukaryotic (particularly human) monomeric, homomeric, and heteromeric membrane proteins and their complexes. A major effort will be in completing and reducing to a robust method the JCIMPT protocol of using parallel microexpression, characterization, and purification of human membrane protein constructs for selecting the best construct for producing structure grade protein;thus reducing cost and effort, and increasing success rates. Single molecule spectroscopic studies will be used to characterize and understand the nature of multi-protein complexes with the goal of developing technology to evaluate and produce functional and stable assemblies. Focus area B will work on design and validation of new compounds (e.g. lipids, detergents, lipidic cubic phase) for stabilizing membrane proteins. New compounds that form lipid cubic phase, as well as sponge phase, will be designed and tested in stabilization and crystallization studies. Focus area C will work on characterization and structure solution of membrane proteins and complexes using NMR spectroscopy. X-ray diffraction, small angle scattering, and electron microscopy. Finally, a new outreach program will disseminate knowledge (e.g. methods and protocols) as well as reagents. Over the five-year funding period, we will conduct workshops and host meetings, as well as maintain a comprehensive and up-to-date website that will become a powerful portal for those scientists interested in membrane protein structural biology. PUBLIC HEALTH RELEVANCE: Membrane proteins are the most difficult family of biological macromolecules to characterize at any level given their complex interaction with both the lipid and water soluble environments. These proteins (e.g. human G-protein coupled receptors) are the target of the majority of therapeutic drug targets. There is a critical need to develop breakthrough technologies to enable better characterization of this protein family.

The botulinum neurotoxins, BoNTs, produced by Clostridium botulinum are among the most potent toxins known to man. The Centers for Disease Control and Prevention (CDC) has classified it as a potential bioweapon, Category A, because of its extreme potency and lethality, its ease of production and transport, and the need for prolonged intensive care among affected persons. This project participates on a collaboration focusing on the various serotypes and subtypes of the toxin with the overall goal of characterizing these variants at the DMA and protein level and developing a deeper understanding of neurotoxin action. We will focus on three major research areas: (1) continuing to deepen our understanding of the cell receptor recognition and binding processes as well as the translocation process by determining the structures of the toxin in complex with cell recognition receptor molecules, (2) developing a detailed understanding on how the recently developed antibodies (Abs), work and how we can then improve them, and (3) using structurebased approaches engineer the next generation broad spectrum diagnostic antibodies (working towards one antibody to recognize multiple toxin subtypes and serotypes). The study will characterize a number of BoNT variants to assess their impact on the development of diagnostics and therapeutics and to understand differences in biological action. In addition to the structures, the project will also produce highly purified protein samples that will be made available to its collaborators and other workers in the biodefense consortium.

2-arachidonylglycerol; Active Sites; Address; analog; anandamide; Apoenzymes; Binding (Molecular Function); Brain; Carbamates; Chemistry; Cognition; Complex; Data; Desire for food; Drug or chemical Tissue Distribution; Emotions; Employee Strikes; Endocannabinoids; enzyme structure; Enzymes; Esthesia; Ethanolamines; Exhibits; fatty acid amide hydrolase; Goals; Human; Hydrolase; in vivo; inhibitor/antagonist; insight; Integral Membrane Protein; interest; Isoenzymes; Knowledge; Lead; Mammals; member; Membrane; Metabolic; Metabolism; Monoacylglycerol Lipases; Mus; mutant; Neurologic; Pain; Pathway interactions; Physiological; Physiology; protein complex; Proteins; Proteomics; Reaction; Reporting; Research; Research Personnel; Rodent; Role; Serine Hydrolase; structural biology; Structure; Substrate Specificity; System; tool; Triad Acrylic Resin; Tropism; Upper arm; Urea

Antibodies; beamline; California; Chronic Myeloproliferative Disorder; Collaborations; college; Crystallization; Data Collection; design and construction; Diffusion; experience; Florida; Foundations; G Protein-Coupled Receptor Genes; gel electrophoresis; Genes; Human; Illinois; Laboratories; Letters; Membrane Proteins; Methods; Molecular Sieve Chromatography; Process; Publications; Recording of previous events; Research; Resources; Sampling; Senior Scientist; Structure; Time; Universities

Project 1 is responsible for achieving the Center‟s Aim 1 goals; the immediate goal of Aim 1 studies is the structure solution of GPCRs and their co-crystals with ligands. The long range goal is to develop a better understanding of the structure-function relationship in GPCRs. Ultimately enough structures will be determined to provide a fine sampling of the various GPCR sequence families and, thus, enable reliable modeling and predictive docking studies of other close family members. Structures of receptors in complex with different ligands will be essential for developing a better understanding of their binding properties. Ultimately, this newly acquired knowledge can then be used to validate and to generate hypothesis regarding GPCRs mechanisms of action (e.g., in signal processing pathways). These new structures will have an enormous impact on furthering our understanding of a number of diseases and most probably will help accelerate success rates in drug discovery and therapeutics development studies.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This project focuses on the development and use of transmission electron microscopy in characterizing membrane protein-protein complexes. We will develop an automated assay to image, select, and classify membrane protein complexes in terms of their aggregation state, size, conformational state and domain arrangement. One goal is to develop a more systematic understanding of the effect of detergents and lipids on the stability and conformation of membrane proteins and their complexes in order to help optimize crystallization efforts. A more ambitious goal is to map out individual protein domains in order to better understand the interactions between proteins in a complex that might be conformationally or structurally heterogeneous. This low resolution information can be combined with high-resolution models of individual subunits or subcomplexes, derived from X-ray and NMR studies, in order to develop a more detailed understanding of the protein-protein interactions within complexes.

Biology; Chronic Myeloproliferative Disorder; Collaborations; Commit; Communities; Data; Decision Making; Deposition; Development; Ensure; experience; G Protein-Coupled Receptor Genes; Goals; Institution; knowledge base; Principal Investigator; programs; Protein Structure Initiative; Research; Research Personnel; research study; response; structural genomics; Technology; U-Series Cooperative Agreements; Work

G protein-coupled receptors sense an astonishing variety of extracellular molecular signals and trigger complex intracellular and physiological responses. They share a common architecture of seven transmembrane helices connected by a broad range of intra- and extra-cellular loops and terminal domains. Structure determination feasibility of this protein family was demonstrated recently with the first high resolution studies on the human Beta2 adrenergic, turkey Beta1 adrenergic, and human adenosine A2A receptors. The Center for Membrane Protein Structure Determination (CMPD) has been created to use a protein family specific platform to determine the high resolution structures of 15-20 representative GPCRs distributed across the phylogenetic tree. Receptor structures are needed at a biologically relevant granularity, for small molecule ligand receptors, peptide and protein receptors, lipid receptors, class B-F receptors, and of receptors in the active and inactive functional states. Each receptor structure will be determined with a set of different pharmacological ligands to define the receptor binding site(s). Solution studies will be conducted with purified receptors bound to different ligands to understand receptor dynamics using hydrogen-deuterium exchange and NMR spectroscopy. In collaboration with the NIH screening center, a library of small molecule probes will be used to analyze each receptor and discover allosteric binding sites using a high throughput thermal stability screen. Through a biologically informed selection of representative receptors, we will maximize the CMPD's impact through computational modeling of close homolog's and functional studies by external collaborators thereby establishing The PSI GPCR Network. The generated data will be provided to the community in a time frame consistent with the guidelines of the Protein Structure Initiative. Technology access will be achieved through on-site training, workshops, meetings, and publications. Processing access to the CMPD core facility will be provided through a 30% pipeline capacity commitment for the PSI: Biology Network nominated targets. Based on the experience of the CMPD investigators, preference will be for human or eukaryotic membrane proteins to maximally leverage the CMPD capabilities.

Production of large quantities of hiighly purified receptors is an underlying requirement for all the projects in ttie Program Project -Structure-Function of Opioid Receptors for Drug Discovery. The production of these samples will be carried out using well established protocols that have now been optimized for use with GPCRs as part of GPCR Network's GPCR Structure Determination Pipeline. These production protocols now include the use of newly developed GPCR fusion partner toolchest for stabilization and crystallization that has increased the quantity and quality of structures that we are able to determine. Most notably, with the use of a new fusion partner, apo-cytochrome b562 (RIL) mutant, BRIL, we were able to determine the structure of the A2A adenosine receptor- ZM241385 to 1.8 A resolution, the structure of NOP, and more recently 2 agonist bound serotonin receptors in an activated state. Structural studies will demand the use of large volumes of highly purified protein for crystallization studies while others doing functional studies will need much less. Studies that led to the structure solution of the K-opioid receptor and the nociception/oprhanin FQ peptide receptor required a large number of constructs and an average of 25 mgs of highly purified protein or about 100 liters of biomass. For receptor-ligand complex structural studies we estimate that 5-10 mgs will be required per structure.

Substances addressing the opioid system are widely used pharmaceuticals. Agonists (narcotic analgesics) are used for the treatment of chronic pain, whereas antagonists are generally effective in the treatment of addictive disorders, including substance abuse (opiate, alcohol, amphetamine) and non-substance, i.e. behavioral addictions such as gaming, gambling and over-eating. The proposed program project will use structural, biophysical, biochemical, and pharmacological approaches to understand ligand binding properties of all opioid receptors (p-, 6, K (MOR, DOR, KOR), and the nociceptin/orphanin FQ peptide receptor (NOP)) and to define the structural basis for molecular recognition and activation. The three overall aims are (1): Understand opioid receptor molecular recognition and functional selectivity using X-ray crystallography to determine structures of ligand-receptor complexes and NMR to understand dynamic behavior. (2) Understand the molecular mechanisms for functional and pharmacological selectivity for the four opioid receptors using computational approaches, and (3) Understand ligand-directed signaling (G- protein-, arrestin-, JNK-dependent) and its relevance in mediating opioid receptor action. Management and administration of the project will be carried out by the Management and Administration Core. Its main responsibilities include (1) Overall management of all project to ensure progress, (2) Prioritization of studies, (3) Assembly of a Scientific Advisory Board, (4) Preparation of required progress reports, (5) Establishment and facilitation of communication routes within the POI as well as with external stakeholders (e.g. SAB, NIH), and (6) Assurance ofthe high quality of studies and results.

DESCRIPTION (provided by applicant): Substances addressing the opioid system are widely used pharmaceuticals; agonists (narcotic analgesics) for the treatment of chronic pain; antagonists generally for the treatment of addictive disorders, including substance abuse (opiate, alcohol, amphetamine) and non-substance, i.e. behavioral addictions. Given the difficulties associated with opioid agonist therapy, high risk of death due to overdose (more victims were reported in 2008, than overdose of heroin and cocaine combined) and the development of tolerance and addiction, there is a need for safer narcotic analgesics. The proposed research program uses structural, computational, biophysical, biochemical, and pharmacological approaches: (1) to develop a new level of understanding of the action the opioid receptors, | j , 6, K, and the nociceptin receptor, defining the structural basis for molecuar recognition and activation, and (2) to design new binding ligands. Aims are to (1) understand opioid receptor molecular recognition and functional selectivity using X-ray crystallography and NMR; (2) understand the molecular mechanisms for functional and pharmacological selectivity for the four receptors using computational approaches; and (3) understand ligand-directed signaling (G-protein-, arrestin-, JNK-dependent) and its relevance in mediating opioid receptor action. Three projects supported by four core groups via inter-project collaborations will address important but difficult questions at a scale not imagined prior to this year. Project 1 will carry ut structural determination on ligand bound complexes. Project 2 will carry out computational studies to define the binding pockets of opioid receptors and perform computer-assisted SBDD to design a new series of optimized compounds including allosteric, functionally selective and bitopic ligands. Project 3, will carry out in vitro and in vivo studies to characterize pharmacological properties of ligands and generate new receptor biased mutants to test hypotheses related to functional selectivity and opioid receptor actions. Core groups support the aims of all 3 projects and will be responsible for (1) large-scale production of purified opioid receptors; (2) synthesis of opiate ligands including newly designed tool compounds for structural and functional studies; (3) molecular profiling and screening of new small molecules, and (4) management and administration of the overall program. Facility resources will be provided by the NIGMS GPCR Networks' Structural Determination Pipeline for large-scale protein production and structure solution and the NIMH Psychoactive Drug Screening Program resources for selectivity profiling of novel small molecules.

DESCRIPTION (provided by applicant): The project Platform for Structure-Function Studies of Adhesion GPCRs implicated in Cancer focuses on establishing the essential basis for studying adhesion GPCRs, which are the second largest family of G-protein coupled receptors (GPCRs). These receptors regulate the migration and development of cells by controlling cell-cell interactions, as well as the intracellular signaling, which makes them of utmost importance because of their roles in tumorigenesis and cancer progression. Their importance, as novel cancer therapeutic targets, is well documented and they are considered biomarkers for specific types of cancers. However, adhesion GPCRs are also the least understood GPCR family. Structural and functional/biochemical studies have been limited or almost non-existent due to difficulties in producing purified samples. Structural information of proteins is a powerful resource for understanding their molecular activities and mechanisms of function, as well as for designing specific therapeutics making them key tools for drug development. Here, we propose to initiate the road to structural determination for members of adhesion GPCRs, by focusing on one member, CD97 receptor. CD97 is a member of the epidermal growth factor (EGF) receptor family involved in progression of thyroid, breast, gastric, esophageal, pancreatic, colorectal cancers, etc. and thus has been suggested as a direct tumor suppressor target. We aim to establish the basis for large-scale production of stabilized CD97 constructs for crystallization and biochemical characterization, which include the use of antibodies. The outcome of the proposed study will: 1) provide quantities of CD97 for functional studies, 2) enable the determination of the structure of the first adhesion GPCR, and 3) provide a platform for further studies of the entire adhesion GPCR family. Thus, this study will set the essentials for more comprehensive studies of adhesion GPCRs towards understanding how they regulate cancer development and progression and provide a basis for designing new drugs.

? DESCRIPTION (provided by applicant): Structure-function characterization of a key protein component of the endocannabinoid system, the human cannabinoid receptor 1 (CB1), is the central focus of this research proposal. It aims to develop a fundamental understanding of the structural basis of CB1 function, with the ultimate translational goal of establishing a robust structure-based drug design (SBDD) program based on experimentally determined 3-dimensional structures. The endocannabinoid system is a complex network of lipid ligands, receptors, and metabolic enzymes involved in a wide range of important physiological processes, including nociception, inflammation, sleep, and drug addiction. As with other G protein coupled receptors, CB1 can exhibit preferential signaling events in response to different ligands. This functional selectivity offers the opportunity to discover new medications with improved pharmacological profiles, enhanced therapeutic properties and reduced side effects. The study will provide the structural basis for the design and development of functionally distinct CB1 selective compounds as useful pharmacological tools and/or leads for the future development of therapeutics. Several crystal structures will be solved to better understand molecular recognition, signaling, and to assist in the design of novel compounds that could then serve as prototypes for later generation leads and drug candidates. The study has three specific aims: (1) Design and synthesize covalent ligands representing key classes of cannabinergic ligands that have been shown to have distinct functional profiles, (2) Develop a better understanding of the CB1 orthosteric binding site by solving the 3D structure of several receptor-ligand complexes, and (3) Develop a better understanding of the CB1 active state by solving the structure of the CB1 signaling complex.

PROJECT SUMMARY The central focus of this Multi-PI R01 research proposal is the structure-function characterization of the human cannabinoid receptor 2 (CB2), a key protein component of the endocannabinoid system. We aim to develop a fundamental understanding of the structural basis of CB2 function, with the ultimate translational goal of establishing a robust structure-based drug design (SBDD) program. The ECS is a complex network of lipid ligands, receptors, and metabolic enzymes involved in a wide range of important physiological processes. There have been important implications that targeting CB2 may be useful as a means for treating inflammation, pain, neurological disorders and addiction. As with other G protein-coupled receptors (GPCRs), CB2 can exhibit preferential signaling events in response to different ligands. This functional selectivity offers the opportunity to refine therapeutic approaches, to improve beneficial properties, and reduce side effect liability. The study will provide the structural basis for the design and development of pharmacologically distinct CB2-selective compounds as useful biological probes and/or leads for the future development of therapeutics. To enhance our effort in obtaining high quality crystal structures, we shall use carefully designed ligands with high affinities and selectivities for CB2, and which are also capable of tight attachment at or near the receptor?s binding domain(s) coupled with their abilities to form crystallizable ligand-receptor complexes. The study has three specific aims: (1) Design and synthesize novel irreversible ligands representing key classes of CB2 selective compounds with distinct functional profiles. (2) Extensive characterization of the newly synthesized ligands in order to identify compounds with pharmacologically diverse profiles, including the partial agonists, inverse agonists, neutral antagonists and allosteric modulators. The crystallization candidates and their chemical derivatives will also be characterized for their reversible binding nature using functional assays. (3) Develop a clear understanding of CB2 ligand binding sites by determining the 3-D structures of the several receptor-ligand complexes. Towards these goals, several crystal structures will be solved to better understand molecular recognition, signaling, and to assist in the design of novel compounds that could then serve as prototypes for later generation leads and drug candidates.