This project aims to develop protocols that will lead to the establishment of a robust high-throughput pipelinefor the atomic-level structural characterization of membrane protein microcrystals grown in membrane-likeenvironment of lipidic cubic phase (LCP) using serial femtosecond nanocrystallography (SFX) at free electronX-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 arecollected prior to onset of any damage), simplify handling, as harvesting and freezing are not required, andsignificantly reduce the time from obtaining initial crystal hits to collecting full data sets. Our long term goal isthe integration of this technology into our structural determination pipeline enabling the determination of a largenumber of three-dimensional structures of G protein-coupled receptors (GPCRs)-ligand complexes addressingquestions on ligand selectivity and efficacy using structure-based drug discovery (SBDD) approaches. Ourgoal will be achieved through the following specific aims.
Aim 1 : Develop protocols for the production ofsamples of GPCR-ligand complexes and for the generation, and characterization of large number ofmicrocrystals that can be used for SFX studies.
Aim 2 : Develop protocols for SFX data collection, processingand structure solution of GPCR-ligand complexes.
Aim 3 : Integrate protocols developed in Aims 1 and 2 intothe GPCR Structure Determination Pipeline and optimize and validate the modified pipeline by determining thestructure of novel GPCRs including a number of receptor-ligand complexes. GPCRs constitute the largestfamily of membrane proteins in the human genome with approximately 800 members and are responsible fortransmitting variety of extracellular signals inside the cell, thereby controlling all major physiological responses,including vision, olfactory, immune defense, reproduction, digestion, mental behavior and others; severalGPCRs are exploited as co-receptors for entry by HIV and other pathogens. GPCR signaling through multipleeffector pathways has profound therapeutic implications, which underscores the need to understand thereceptor both biochemically and structurally in the proper context. GPCRs are the target of ~40% of currentlymarketed drugs. However, detailed understanding of their mechanism of action and ligand selectivity is limitedby a lack of structural information. The structure determination of GPCRs is hampered by the difficulty ofpreparing 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 synchrotronsources.

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 #
7R01GM108635-02
Application #
9078982
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
2015-01-01
Budget End
2015-08-31
Support Year
2
Fiscal Year
2014
Total Cost
$318,762
Indirect Cost
$125,133
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 :

Showing the most recent 10 out of 23 publications