This project aims to develop protocols that will lead to the establishment of a robust high-throughput pipeline for the atomic-level structural characterization of membrane protein microcrystals grown in membrane-like environment of lipidic cubic phase (LCP) using serial femtosecond nanocrystallography (SFX) at free electron X-ray laser sources (XFELs). With the use of SFX, we will obviate the need for obtaining large crystals, effectively eliminate radiation damage issues through diffraction before destruction (i.e., diffraction data are collected prior to onset of any damage), simplify handling, as harvesting and freezing are not required, and significantly reduce the time from obtaining initial crystal hits to collecting full data sets. Our long term goal is the integration of this technology into our structural determination pipeline enabling the determination of a large number of three-dimensional structures of G protein-coupled receptors (GPCRs)-ligand complexes addressing questions on ligand selectivity and efficacy using structure-based drug discovery (SBDD) approaches. Our goal will be achieved through the following specific aims.
Aim 1 : Develop protocols for the production of samples of GPCR-ligand complexes and for the generation, and characterization of large number of microcrystals that can be used for SFX studies.
Aim 2 : Develop protocols for SFX data collection, processing and structure solution of GPCR-ligand complexes.
Aim 3 : Integrate protocols developed in Aims 1 and 2 into the GPCR Structure Determination Pipeline and optimize and validate the modified pipeline by determining the structure of novel GPCRs including a number of receptor-ligand complexes. GPCRs constitute the largest family of membrane proteins in the human genome with approximately 800 members and are responsible for transmitting variety of extracellular signals inside the cell, thereby controlling all major physiological responses, including vision, olfactory, immune defense, reproduction, digestion, mental behavior and others; several GPCRs are exploited as co-receptors for entry by HIV and other pathogens. GPCR signaling through multiple effector pathways has profound therapeutic implications, which underscores the need to understand the receptor both biochemically and structurally in the proper context. GPCRs are the target of ~40% of currently marketed drugs. However, detailed understanding of their mechanism of action and ligand selectivity is limited by a lack of structural information. The structure determination of GPCRs is hampered by the difficulty of preparing large amounts of homogenous and stable samples and growing sufficiently large crystals for high- resolution structure determination even when using state-of-the art microfocus beamlines at synchrotron sources.

Public Health Relevance

The proposed studies will develop protocols that will enable high-throughput determination of high-resolution structures of G protein-coupled receptors and other membrane proteins, which are an important class of drug targets for diverse pathophysiological conditions in vision, olfactory, immune defense, reproduction, digestion, mental behavior, and others including cancers.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM108635-03
Application #
8925910
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Edmonds, Charles G
Project Start
2014-09-15
Project End
2018-08-31
Budget Start
2015-09-01
Budget End
2016-08-31
Support Year
3
Fiscal Year
2015
Total Cost
$363,000
Indirect Cost
$143,000
Name
University of Southern California
Department
Type
DUNS #
072933393
City
Los Angeles
State
CA
Country
United States
Zip Code
90089
Stauch, Benjamin; Cherezov, Vadim (2018) Serial Femtosecond Crystallography of G Protein-Coupled Receptors. Annu Rev Biophys 47:377-397
Ishchenko, Andrii; Gati, Cornelius; Cherezov, Vadim (2018) Structural biology of G protein-coupled receptors: new opportunities from XFELs and cryoEM. Curr Opin Struct Biol 51:44-52
Zhang, Xianjun; Zhao, Fei; Wu, Yiran et al. (2017) Crystal structure of a multi-domain human smoothened receptor in complex with a super stabilizing ligand. Nat Commun 8:15383
Martin-Garcia, Jose M; Conrad, Chelsie E; Nelson, Garrett et al. (2017) Serial millisecond crystallography of membrane and soluble protein microcrystals using synchrotron radiation. IUCrJ 4:439-454
Johansson, Linda C; Stauch, Benjamin; Ishchenko, Andrii et al. (2017) A Bright Future for Serial Femtosecond Crystallography with XFELs. Trends Biochem Sci 42:749-762
Zhang, Haonan; Qiao, Anna; Yang, Dehua et al. (2017) Structure of the full-length glucagon class B G-protein-coupled receptor. Nature 546:259-264
Zhang, Haitao; Han, Gye Won; Batyuk, Alexander et al. (2017) Structural basis for selectivity and diversity in angiotensin II receptors. Nature 544:327-332
Ishchenko, Andrii; Wacker, Daniel; Kapoor, Mili et al. (2017) Structural insights into the extracellular recognition of the human serotonin 2B receptor by an antibody. Proc Natl Acad Sci U S A 114:8223-8228
Zhou, X Edward; He, Yuanzheng; de Waal, Parker W et al. (2017) Identification of Phosphorylation Codes for Arrestin Recruitment by G Protein-Coupled Receptors. Cell 170:457-469.e13
Ishchenko, Andrii; Cherezov, Vadim; Liu, Wei (2016) Preparation and Delivery of Protein Microcrystals in Lipidic Cubic Phase for Serial Femtosecond Crystallography. J Vis Exp :

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