Investigating the Structural Basis of Transmembrane Signaling by GPCRs
(from Du et al., Cell 2019, 177, 1232-1242. PMCID PMC6763313; DOI: 10.1016/j.cell.2019.04.022)
The activation of G proteins by G protein-coupled receptors (GPCRs) underlies the majority of transmembrane signaling by hormones and neurotransmitters, and represents an attractive pharmaceutical target. Recent structures of GPCR-G protein complexes from crystallography and cryo-EM reveal similar interactions between GPCRs and the alpha subunit of different G protein isoforms, with no clear structural explanation for G protein subtype-selectivity. Moreover, all of these complexes are stabilized in the nucleotide-free state, a condition that does not exist in living cells. During the scientific commissioning of XFP, the CSB and colleagues at CWRU initiated a collaboration with Brian Kobilka’s lab at Stanford to use time-resolved x-ray footprinting methods to better understand the structural basis of coupling specificity in assembly of the β2 adrenergic receptor GPCR with target G proteins. A bespoke time-resolved capillary flow x-ray footprinting device was assembled for mixing experiments allowing us to probe timescales over 4 orders of magnitude (10 ms to >10s). This data, in concert with results from HDX-MS and fluorescence methods, allowed the team to demonstrate the existence of one or more transient intermediate states that serve as selectivity filters and precede the formation of the stable nucleotide-free GPCR-G protein complexes observed in crystal and cryo-EM structures. This work demonstrates the power of time-resolved synchrotron x-ray footprinting methods to provide answers to challenging biophysical questions not obtainable by other structural probes.
Multimodal Approaches to Structure Determination of Intrinsically Disordered Proteins
(from Y. Peng et al., Structure 2019, 27, 229-240.E4. PMCID PMC6365180; DOI: 10.1016/j.str.2018.10.026)
The N-terminal transactivation domain (NTD) of estrogen receptor alpha, a well-known member of the family of intrinsically disordered proteins, mediates the receptor's transactivation function. However, an accurate molecular dissection of NTD's structure-function relationships remains elusive. A recent report from Prof. Sichun Yang (CWRU Center for Proteomics & Bioinformatics) and colleagues at CWRU and NSLS-II shows that the NTD adopts a mostly disordered, unexpectedly compact conformation that undergoes structural expansion on chemical denaturation. They employed a multimodal approach that combined small-angle X-ray scattering, hydroxyl radical protein footprinting, and computational modeling to derive the ensemble-structures of the NTD and determine its ensemble-contact map revealing metastable long-range contacts. Furthermore, mutation of one of these contacts, a known phosphorylation site, promotes conformational changes and increases coactivator binding. 19F nuclear magnetic resonance was used to validate the identity of the long-range contact postulated from XF and side-directed mutagenesis. These findings extend our understanding of how specific contact metastability mediates critical functions of disordered proteins, and amply demonstrates the power of multiple synchrotron techniques to address challenging structural biology problems.
Searching for the Structural Basis of Prion-based Disease
(from Q. Li et al., J. Biol. Chem. 2018, 293, 18494–18503. PMCID PMC6290147; DOI: 10.1074/jbc.RA118.005622).
Prion diseases are neurodegenerative disorders that affect many mammalian species, and are caused by prion proteins (PrPs) that can misfold into many different aggregates. However, only a small subpopulation of these structures is infectious, and a major question in prion research involves identifying the specific structural features of the misfolded protein aggregates that are important for prion infectivity in vivo. In this work, Witold Surewicz’s group at CWRU used synchrotron x-ray footprinting at the NSLS-II XFP beamline, along with other mass-spectrometry based probes, to examine the structures of two mouse PrP aggregates, one of which is highly infectious while the second is non-infectious. They demonstrated structural differences between the two forms in both the polypeptide backbone structure and quaternary packing arrangement. Notably, infectious prions show a region with a well-ordered, stable conformation that is not seen in non-infectious prions. The structural constraints provided by this analysis should facilitate the development of experimentally based high-resolution structural models of infectious mammalian prions, and demonstrate the ability of synchrotron x-ray footprinting to probe the structure of disordered amyloidogenic proteins having biomedical importance.
2014-2016 NSLS to NSLS-II Transition at ALS
Two representative publications made possible by our contribution to / collaboration with a team developing a new X-ray footprinting resource at the ALS during the CSB’s transition to NSLS-II:
Probing the structure of ribosome assembly intermediates in vivo using DMS and hydroxyl radical footprinting
(from RM Hulscher et al., Methods. 2016, 103, 49-56. PMCID PMC4921310; DOI: 10.1016/j.ymeth.2016.03.012)
Local and Global Structural Drivers for the Photoactivation of the Orange Carotenoid Protein
(from S. Gupta et al., Proc. Natl. Acad. Sci. USA. 2015, 112, E5567-74. PMCID PMC4611662; DOI: 10.1073/pnas.1512240112)