Georgios Skiniotis - US grants

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. During cell division, chromatin remodeling (local and global structural changes in chromatin) is necessary for both transcription and chromosomal segregation. In eukaryotes, ATP-dependent chromatin remodeling is performed by large multi-protein assemblies that modulate DNA-nucleosome interactions. We are working to refine structural models of the multi-protein RSC (Remodels the Structure of Chromatin) complex in yeast (Saccharomyces cerevisiae), an ATP-dependent chromatin remodeler with homologous counterparts in humans and higher eukaryotes.

DESCRIPTION (provided by applicant): Leptin (L) and its receptor (L-R) are key players in the regulation of energy homeostasis and body weight. Complex formation between leptin and the extracellular portion of L-R results in the activation of Janus kinase 2 (JAK2) that is constitutively bound on the intracellular regions of the receptor. Leptin-instigated JAK2 signaling in hypothalamic nuclei reduces food intake and stimulates energy expenditure, while functional defects in either leptin or L-R result in morbid obesity, hyperglycemia, decreased insulin sensitivity, and hyperlipidemia. Despite the crucial impact of the leptin system on body weight and other physiological responses, little is known about the structure of the L/L-R complex and its association with JAK2. One of the reasons for this lack of insight is that both L-R and JAK2 have a relatively long and flexible multi-domain arrangement that has proved to be very challenging for both large-scale purification and implementation of X-ray crystallography. The present proposal aims to overcome these limitations in addressing the architectural prerequisites of L-R signaling by applying single- particle cryo-electron microscopy (cryo-EM) to characterize the holo-complex of full-length L/L-R and JAK2. Single-particle EM has emerged as a very powerful tool for the characterization of dynamic protein assemblies in relatively small concentrations and without the need for crystallization. We anticipate that the application of single-particle EM techniques on this system will reveal the architecture of the L/L-R assembly and JAK2 independently, and in complex. Given the underlying importance of this membrane-localized signaling complex in obesity, energy metabolism, and heart disease, the structural results obtained will be of very broad biomedical interest. Considering the current lack of structural information on any receptor/JAK complex, our studies will provide the general architectural framework for understanding how extracellular ligand binding on cytokine receptors results in intracellular JAK activation. PUBLIC HEALTH RELEVANCE: Leptin signaling is key to the regulation of mammalian energy homeostasis and body weight. Extracellular binding of leptin to its receptor results in the intracellular activation of Janus kinase 2 (JAK2) which, in turns, regulates a number of physiologically crucial signaling cascades. We propose to apply molecular electron microscopy techniques to characterize the structure of the signaling complex between leptin, its receptor, and JAK2. The elucidation of this architecture will provide a mechanistic understanding of JAK2 activation via extracellular receptor engagement by leptin. Since deficient leptin signaling is associated with severe pathologies, such as obesity and diabetes, this knowledge will be ultimately used for the design of therapeutic strategies that are based on targeting the leptin receptor complex.

? DESCRIPTION: The Center for Structural Biology at the University of Michigan has established the first cryo-electron microscopy (cryo-EM) facility on campus with a focus on molecular EM of biological assemblies. The facility aims to serve the growing needs of the scientific community within and outside UMICH towards state-of-the-art visualization and structural characterization of macromolecular complexes. The cryo-EM suite, which already includes a 100kV, a 120 kV, and a 200 kV transmission electron microscope (TEM), will soon host a high-resolution 300 kV TEM. This expansion is based on the success of the first 5 years of the facility's operation (more than 20 high-impact publications) and the need to extend its imaging capabilities in terms of applications, quality of data, and throughput. The current computational platform dedicated to EM is very limited and represents a major bottleneck for image processing of data, especially for single particle EM projects requiring many thousands of images. Given that the output of cryo-EM data will increase several fold with the combined use of the 200 and the 300 kV TEMs, both equipped with new direct electron detectors and automated image acquisition algorithms, the present proposal requests the funds for the acquisition of a powerful compute cluster that includes 96 nodes with a total of 1152 CPU cores. This high-end cluster will facilitate the processing of large cryo-EM datasets, the implementation of computationally demanding image processing algorithms, as well as the parallel processing for multiple projects. Combined with our powerful TEM platform, the addition of this advanced computer system will enable the rapid and sophisticated cryo-EM image processing for many projects addressing the structure of important biological assemblies from intermediate to high resolution. The combination of these technologies will complement numerous NIH- funded projects of well-established biological programs within and outside UMICH. For the purpose of this application, we include a number of project descriptions that would greatly benefit from the acquisition of this instrument.

? DESCRIPTION (provided by applicant): Metabotropic Glutamate receptors (mGluRs) belong to the Family C of G-protein coupled receptors (GPCRs) and critically regulates neuronal excitability, synaptic transmission and plasticity through recognition of the amino acid and excitatory neurotransmitter glutamate. Many disorders of the CNS have been linked to alterations in neuronal excitability via the glutamatergic system. Accordingly, mGluRs have been the subject of an enormous drug discovery effort as they represent major therapeutic targets for treating numerous physiological dysfunctions and for neurodegenerative and neuropsychiatric conditions. Apart from the prototypical seven transmembrane helix (7TM) domain, mGluRs also include a large extracellular venus fly trap' (VFT) domain that constitutes the glutamate binding site and a cysteine rich domain (CRD) that links the VFT to the 7TM. Binding of glutamate to the extracellular VFT domain triggers a large conformational change in the VFT domains from an open to a closed conformation. This clam-shell like closure of the extracellular domain results in receptor engagement and activation of G-proteins on the intracellular side of the transmembrane domain. Receptor activated G proteins then act to either enhance or repress secondary messenger signaling cascades. Despite intensive efforts, the mechanism of allosteric communication across the cell membrane by the mGluRs remains enigmatic due to the lack of structural information on full- length proteins. Here we propose to apply single-particle cryo-electron microscopy (cryo-EM) visualization in order to characterize the structure of mGluR5 and mGluR2 in activated and inactivated states and also in complex with their cognate G-proteins. The obtained structures will be used for molecular dynamics simulations aiming to unravel the molecular basis for conformational transitions coupled to signal instigation or silencing. Given that mGluRs are important drug targets for several CNS conditions including Parkinson's disease, Fragile X syndrome/autism spectrum disorders, schizophrenia, cognition, addiction, depression, anxiety and pain, the results obtained will have profound biomedical interest and will form the basis for the design of novel therapeutic strategies against neurological disorders.

? DESCRIPTION (provided by applicant): Bacterial type I polyketide synthases (PKSs) are mega-enzyme assembly lines responsible for generating the macrolactone core of a wide range of polyketide products that are biologically active natural compounds (e.g. antimicrobial, antifungal, antiviral, anticancer, and immunosuppressant compounds). Indicative of their significance, polyketide natural products currently form the basis for nearly one-third of pharmaceuticals. PKSs employ a modular multi-step mechanism to produce polyketides, and bioengineering these systems has immense potential for the creation of new chemotypes with invaluable applications in drug discovery. However, such efforts have met with limited success, reflecting our poor structural and mechanistic understanding of the modular process to generate polyketides. We recently employed cryo-electron microscopy (cryo-EM) to show the first subnanometer resolution structures of the full length PikAIII module from the pikromycin PKS biosynthetic pathway, a prototype for assembly-line PKS systems. The findings not only revealed an unexpected module architecture undergoing extensive structural rearrangements, but also showed that the type of substrate linked to a highly mobile acyl carrier protein (ACP) domain specifies its positioning in a way that facilitates assembly-line throughput. By employing recent breakthroughs in cryo-EM technologies, we now aim to obtain high resolution cryo-EM structures of PikAIII and of the terminal PikAIV module, in an effort to resolve several lingering question regarding functional interfaces and module dynamics. Our studies will include both natural and unnatural substrates, seeking to reveal the principles of substrate recognition and processing in these remarkable macromolecular factories. The findings from the proposed studies will for the basis for renewed bioengineering efforts towards the creation of PKSs that can efficiently produce novel compounds of high medicinal value.

Summary  Stanford University and the SLAC National Accelerator Laboratory (SLAC) propose to host a National Center  for Cryoelectron Microscopy ? The Stanford-­SLAC CryoEM Center (S2C2) ? to meet the emerging national  need for cryoEM as a tool for atomic-­resolution structural biology. The proposed S2C2 will (1) establish, and  keep at the forefront, state-­of-­the-­art cryo-­electron microscopes to satisfy users? needs for atomic-­resolution  image data, (2) archive image data with appropriate metadata, (3) provide computing resources to assess data  quality in real time, (4) inform and recruit potential users across the U.S. about the public accessibility of the  Center, (5) establish an open, fair, transparent and efficient process to select user proposals based on  scientific impact and specimen readiness, regardless of users? geographic locations or affiliations, (6) help  users to overcome technical hurdles and enable them to obtain high-­resolution cryoEM structures quickly with  a rigorous validation protocol, (7) form and facilitate a user network to exchange information seamlessly, (8)  train users to become independent cryoEM investigators, (9) integrate user and trainee feedback into a  continuous loop of facility enhancement, and (10) optimize operations to achieve the above tasks effectively  and efficiently. Dr. Wah Chiu, the Contact PI, has decades of experience developing high-­resolution cryoEM  technologies and directing research and training enterprises across multiple institutions. Drs. Michael Schmid,  Georgios Skiniotis , and Britt Hedman, also PIs of the Center, bring complementary expertise in cryoEM,  structural biology, and management and operation of large-­scale national user facilities. A Center Director of  Operations, three skilled cryoEM specialists, a technical assistant, and a user facility administrator will carry  out the S2C2?s day-­to-­day operations, including data collection, quality assessment, and cross-­training. This  S2C2 will reside in a new building at SLAC. It will leverage SLAC?s expertise in serving thousands of users each  year at its renowned X-­ray synchrotron and free electron laser user facilities. An Advisory Committee  comprised of external expert scientists as well as user representatives will provide advice regarding S2C2  operations and development. Our Center will build on the unique strengths and support from Stanford and  SLAC, and will create a unique environment that combines high throughput cryoEM data generation with  training the next generation of cryoEM scientists. 

Project Summary G protein-coupled receptors are important conduits to relay extracellular signals to downstream intracellular signal transduction pathways. Their central role in intercellular communication together with the shear magnitude of the gene family (>800 genes) have therefore made GPCRs superb therapeutic targets. Understanding the mechanism of hormone action on GPCRs and understanding how drugs modulate their behavior is an important fundamental endeavor but also an important mission for health scientists. The primary goal of this ongoing research program is to study the mechanism of GPCR regulation of their primary signaling partners, G proteins. In this renewal we will use biochemical and biophysical approaches to delineate the mechanism of GPCR·G protein (R·G) interactions to try to resolve the extraordinary selectivity of G protein isoforms for specific members of the GPCR superfamily. We will focus on a narrow but representative collection of GPCRs (b2AR, M2 & M3AChR, µOR and NTSR1) and their coupling to different G protein isoforms (Gs, Gi/o, Gq/11 and G12/13). Our major goal is to gain insight into the structural and dynamic bases underlying R·G specificity by determining how these family members couple to and activate specific G protein isoforms. In the previous funding cycle we made several breakthroughs by solving the structures of 6 different R·G complexes: µ opioid receptor (µOR)·Gi1, neurotensin receptor subtype 1 (NTSR1)·Gi1, cannabinoid receptor subtype 1 (CB1)·Gi1, muscarinic M2AChR·GoA, muscarinic M1AChR·G11, and glucagon receptor (GCGR)·Gs. These structures reveal key regions on the receptors and G proteins that we suspect confers receptor and G protein isoform selectivity. In this renewal we propose to apply a spectrum of biochemical and biophysical approaches to interrogate the interaction sites revealed in the R·G structures. In addition, our recent studies suggests various conformational states of the R·G complex, strongly suggesting the existence of intermediate states. In this renewal we propose to examine these intermediate states and probe their potential to contribute toward R·G specificity, and toward receptor-catalyzed nucleotide exchange. We will utilize cutting-edge approaches including cryo-electron microscopy (CryoEM), double electron-electron resonance (DEER) spectroscopy, fluorescence resonance energy transfer (FRET), single molecule spectroscopy (SMS) and interferometry to study these R-G interactions. We will study the nature of the R-G specificity, whether the underlying mechanism may be at the pre- association (perhaps through an intermediate state), or at the coupling stage. We feel that with our expertise, the generation of innovative reagents, the incorporation of cutting edge biophysical approaches and the generation of strong preliminary data together make this proposal tractable.

Abstract The ?-aminobutyric acid B receptor (GABABR) and the metabotropic glutamate receptors (mGluRs) belong to the Family C of G protein coupled receptors (GPCRs) and critically regulate neuronal excitability, synaptic transmission and plasticity. Many disorders of the CNS have been linked to alterations in neuronal excitability via the glutamatergic and GABAergic system. Accordingly, mGluRs and GABABR have been the subject of an enormous drug discovery effort as they represent major therapeutic targets for treating numerous physiological dysfunctions and for neurodegenerative and neuropsychiatric conditions. Apart from the prototypical seven transmembrane helix (7TM) domain, Family C GPCRs also include a large extracellular ?venus fly trap? (VFT) domain that constitutes the orthosteric ligand binding site. Binding of ligand to the extracellular VFT domain triggers a large conformational change in the VFT domains from an open to a closed conformation. This clam- shell like closure of the extracellular domain results in receptor engagement and activation of G proteins on the intracellular side of the transmembrane domain with a mechanism that remains unclear. Receptor activated G proteins then act to either enhance or repress secondary messenger signaling cascades. We recently showed cryoEM structures of near-full length mGluR5 and GABABR in inactive and active conformations, revealing extensive transitions in the organization of the 7TM dimer upon ligand binding to the VFT. Notwithstanding this progress, several key questions remain regarding the allosteric communication across the cell membrane by Family C GPCRs, and particularly the mechanism of G protein coupling and activation. To address these questions, we propose to obtain the structures of mGluR2, mGluR5 and GABABR in complex with their cognate G proteins and probe the structural insights using molecular dynamics simulations and mutagenesis coupled to functional assays. The similarities and differences amongst these receptor-G protein complexes will allow us to contrast and compare our findings and examine aspects of G protein coupling and selectivity. Collectively, these studies will enable us to create a detailed mechanistic framework to understand Family C GPCR signaling and will form the basis for the design of novel therapeutic strategies targeting these receptors.