Qinghai Zhang, Ph.D. - US grants

DESCRIPTION (provided by applicant): Experimentally determining the 3-D structure of the G protein-coupled receptor (GPCR), CXCR4 and its complexes, the overall goal of the proposed study, is critical in developing a deeper understanding of its role in HIV viral entry and toxicity;availability of these data is expected to facilitate the development of new therapeutic strategies. Although CCR5 is the primary co-receptor responsible for HIV transmission, emergence of CXCR4-tropic (X4-tropic) viruses later in infection, correlates with a more rapid CD4 decline and a faster progression to AIDS. CXCR4 is also implicated in neuroaids, triggering neuronal cell death and possibly causing onset of dementia. Known conformational flexibility of GPCRs as well as results from preliminary studies have shown that stabilization of the CXCR4 receptor is a significant bottleneck in attempts to crystallize and solve its structures. The two PI's of this research proposal, one a chemist, and the other a crystallographer, propose to develop new chemical tools optimized for structural studies of the HIV co-receptor CXCR4, attempting crystallization and structural determination of the receptors and its complexes. Chemical tools will be designed and optimized for stabilizing, purifying, and detecting the receptor and for measuring its functional behavior. R21 studies will have two aims: (1) Develop tool compounds optimized for structural studies of CXCR4 and (2) Validate new ligands, detergents, and lipids by assessing their impact on protein sample production processes and by producing diffracting crystals of CXCR4. Results from R21 studies will be used to achieve the following in R33 phase of the work: (3) Determine the high-resolution X-ray structure of CXCR4, and (4) Assess the utility of new molecular tools and technologies in stabilizing and crystallizing CXCR4 in complex with gp120. Success of the R21 studies will be achieved by the production of diffracting CXCR4 crystals along with the production of new compound tools that significantly improves receptor sample behavior (e.g. improved stability over time, increased Tm). PUBLIC HEALTH RELEVANCE: Availability of a 3D structure of CXCR4 along with its complexes will lead to developing a deeper understanding of how the HIV virus enters the cell and will help in the design of new therapeutics. CXCR4 is also involved in important physiological process and have been implicated in a number of diseases such as cancer and availability of its structure will likewise have important biomedical implications.

DESCRIPTION (provided by applicant): Compared to soluble proteins, it is much more challenging to obtain well diffracting membrane protein crystals suitable for X-ray analysis. Membrane protein crystals are characterized by small hydrophilic protein- protein interactions that are crucial for formation of a 3D crystal lattice. Approaches to expand or modify the polar surface of membrane proteins are effective for crystal growth but still represent significant challenges. The overarching goal of our proposal is to develop an innovative approach of using intelligently designed amphiphiles or lipids, the essential component required to stabilize membrane proteins, to mediate ordered protein surface interactions so as to increase the crystallization propensity and improve crystal diffraction. This approach is orthogonal and complementary to available protein engineering techniques and applicable to both detergent micelle and lipid bilayer based crystallization protocols. To achieve our goal, we will develop new design principles for the creation of novel amphiphiles. We will use biochemical assays and various biophysical techniques to study the thermodynamic interaction and binding between the amphiphiles and membrane proteins, as these properties govern protein stability and function. Crystallization experiments will be performed to identify molecules that mediate ordered membrane protein crystal contacts and reveal molecular details of the amphiphile-protein interaction. Through this work, structurally novel stabilization reagents will be developed to overcome the crystallization bottleneck that cannot be fully addressed by currently available detergents, lipids or other novel amphiphiles that have been tested in the last two decades. We aim to identify a robust set of reagents that can be generally applicable to the structural solution of different families of membrane proteins, not ones limited to a single protein or a single class. By improving the resolution of previously solved structures and facilitating the structural determination of new membrane proteins, our study will have a direct impact on biology.

DESCRIPTION (provided by applicant): G-protein coupled receptors (GPCRs) constitute the largest family of membrane proteins in the human genome with approximately 800 members. GPCR signaling through multiple effector pathways has profound therapeutic implications, making these receptors the targets of ~40% of currently marketed drugs. Modulation of GPCR activity by small molecule ligands is emerging as a new strategy for development of anticancer therapeutics. However, design of new drugs with higher efficacy and lower side effects, and detailed understanding of GPCR mechanism of action are hampered by the limited structural information. Structural studies of many GPCRs are in turn limited by a scarcity of high-affinity ligands that stabilize GPCRs and enable their crystallization. The project attempts to develop a new platform using the in situ click chemistry approach to discover novel ligands that bind GPCRs to serve as tool compounds for structural and functional studies or as starting point for drug discovery. Target-guided synthesis approaches including in situ click chemistry, by which reactive fragments are joined in the binding sites of a biological target, have shown great promise in drug discovery but have not been applied to GPCRs. The challenges lie in the GPCRs' membrane disposition, their high conformational dynamics, low stability and low expression levels. The proposed platform is being developed targeting all GPCRs, with initial focus primarily on those known to be cancer targets. In the two years of this study, we will focus on smoothened (SMO) receptor which is an orphan receptor and a key signal transducer in the Hedgehog (Hh) signaling pathway activated during development, and thus is a target of a number of antitumor drugs in clinical trials. Our goal will be attained by achieving the following two aims: (1) Establish an in situ click chemistry platform to generate high-affinity GPCR ligands, (2) Characterize the interaction between selected ligands from Aim 1 and SMO by crystallography. The study will be supported by a team that had developed the GPCR Structure Determination Pipeline as well as its underlying technologies which have led to the structure determination of 12 GPCR structures since 2007.

DESCRIPTION (provided by applicant): The energy-linked inner mitochondrial membrane enzyme nicotinamide nucleotide transhydrogenase (TH) couples the proton motive force (pmf) generated by respiration to formation of NADPH. In the absence of NADPH reactive oxygen species (ROS) lead to mitochondrial dysfunction which is strongly correlated with neurodegenerative diseases. In pancreatic beta cells, oxidative stress arising from TH mutations is correlated with type 2 diabetes due to loss of the redox signaling that controls insulin secretion. To understand the relationship between ROS, mitochondrial dysfunction and disease, it is necessary to understand the mechanism by which TH couples the pmf with formation of NADPH. Extensive biochemical and genetic data are available, but to define a mechanism crystal structures of the entire enzyme and its constituent subunits are required. Further, knowledge of the dispositions of the subunits during hydride transfer and proton pumping are required. A viable strategy employs TH from Thermus thermophilus for crystallization of the enzyme and its individual components. Diffraction quality crystals of the membrane intercalated domains have been obtained in the lipidic cubic phase (LCP); structures of the membrane domains in complex with the soluble, NADP(H) binding domain (domain III) will also be obtained via construct design and co- crystallization. High resolution structures of the soluble NAD(H) subunit alone and in complex with domain III have been solved. Large prismatic crystals of intact TH in multiple morphologies are available for diffraction experiments. The component crystal structures will be positioned within the TH electron density envelop using lower resolution X-ray data and EM methods (single particle averaging and electron diffraction), revealing for the first time a complete structure of TH. SAXS experiments in the presence of NAD(H) and NADP(H), together with the crystal structures, will identify motions of domain III within the complex. Structure guided site- directed mutagenesis and biochemical assays (cyclic and reverse hydride transfer, vectorial proton pumping) will define residue functions. Knowledge of structure, conformational states, and function will enable a mechanism for coupling proton translocation and NADPH formation to be deduced.

? DESCRIPTION (provided by applicant): P-glycoprotein (Pgp) is a highly dynamic ATP-binding cassette (ABC) membrane transporter that effluxes a diversity of molecules out of cells. It is a major determinant of drug absorption, distribution, and excretion in intestines, liver, kidney and brain, and also an impediment to successful chemotherapy in some cancers, HIV and central nervous system (CNS) diseases. About half of marketed drugs are estimated to be transport substrates or inhibitors of Pgp, and the evaluation of Pgp susceptibility of drug candidates has become an important step in the development of new therapeutics in the pharmaceutical industry. However, Pgp binding molecules exhibit complex activities, acting as ATPase stimulators, inhibitors, transport substrates, or non- transportable ligands. Despite extensive studies, we still have very limited understanding of the mechanisms for the complex and polyspecific Pgp-drug interactions pertaining to Pgp transport, inhibition and evasion. This study aims to (1) define how structural and chemical properties of a ligand affect its interactions with Pgp, (2) characterize how Pgp reacts upon the binding of different classes of ligands, and (3) rationalize chemical synthesis to modify existing drugs to evade Pgp transport. We will use an integrative chemistry, structural and functional approach to tackle these aims, which is based on several major advances in the literature and that we have most recently made on Pgp structural determination and ligand interactions. First, we propose to conduct a thorough structure-activity relationship study within a focused library of ligands bearing common scaffolds. We will use a battery of functional assays, including ATPase activity, detailed drug binding and competition, cell-based transport, and drug resistance assays, as well as structural characterizations to evaluate these compounds. Second, we will use several complementary biophysical techniques, including X- ray crystallography, single particle electron microscopy (EM), and luminance resonance energy transfer (LRET), for the determination of Pgp/ligand complex structures, conformational distributions, and the kinetics of conformational changes. The characterization of Pgp conformations by EM and LRET, together with the screen of novel ligands, detergents and lipids, as well as new constructs to stabilize Pgp (and certain conformations), will facilitate higher resolution (< 3.0 Å) structural studies, a critical barrier that has eluded Pgp thus far. Third, we will modify several Pgp drug substrates on positions that have been identified without diminution of drug potency, thus with a focus on resulting changes in Pgp interactions. By the related three aim studies we will achieve a detailed and fundamental understanding of polyspecific Pgp-drug interactions, which will have a far- reaching impact on drug discovery given the pharmacological and clinical significance of Pgp and that many current and investigational drugs are susceptible to Pgp efflux.

Membrane proteins (MPs) are important but arguably the most challenging biological molecules to study in solution. One major concern with studies of MPs is the requirement for extraction from their native environment, the membrane. Over several decades the chemistry and MP communities have contributed diverse membrane mimetics that help to solubilize and stabilize MPs. Despite their broad applications, these membrane mimetics do not fully replicate the native bilayer setting of MPs which contains hundreds of diverse lipids. This is a fundamental issue because lipid bilayers and specific lipid interactions are crucial to the structural assembly and function of many MPs. Notwithstanding, key challenges remain as to how to isolate and stabilize fragile MPs and MP complexes with little disturbance to their conformation, oligomer assembly, and function, tasks that are nevertheless crucial to structural, functional and drug interaction studies. To overcome these important challenges in MP research, we propose to develop a strategy to directly enrich or purify MPs within small patches of cell membranes. Our proposal exploits the favorable properties of two types of popular membrane reagents, i.e. small molecule detergents and amphiphilic polymers, and meanwhile avoid their shortcomings in that 1) detergent is effective and inevitably used to disperse cell membranes in most MP pruritions, but the detergent- solubilized membrane patches are highly dynamic and unstable structures; and 2) an amphiphilic polymer scaffold may entrap and stabilize a central bilayer patch, but most polymers used to stabilize MPs are ineffective to disperse cell membranes. To resolve the dilemma, we propose an ex novo polymer design to encircle small membrane domains by crosslinking detergents in situ, upon the dispersion of cell membranes. To establish the feasibility of this approach, we will explore chemistry for detergent crosslinking to solubilize and stabilize small patches of cell membranes (Aim 1) and validate chemical tools in the purification of diverse MPs (Aim 2). If successful, our development will provide a completely new and versatile approach to preparing MPs in nearly native environment. Achieving this goal together with new tool development will have a far-reaching impact on many areas of MP research in which the isolation of MPs is needed.