G protein-coupled receptors (GPCRs) are involved in most cellular processes and are the target of over 50% of the pharmaceuticals currently on the market. Despite their importance, the only known high-resolution structure of a GPCR is the crystal structure of the inactive state of the visual receptor rhodopsin. The goal of the proposed research is to establish the structure of metarhodopsin II, the activated state of rhodopsin. The experimental approach is to incorporate 13C labels into both the vitamin A (retinal) chromophore and the rhodopsin protein for measurements of internuclear 13C...13C distances using solid- state magic angle spinning NMR spectroscopy. The first three specific aims target different regions of the rhodopsin protein to address specific questions involving the activation mechanism. Distance measurements are planned of 1) retinal-protein contacts to establish the location of the retinal relative to transmembrane helices H5, H6, and H7, 2) helix-helix contacts to establish how transmembrane helices H5-H7 move relative to the H1-H4 core of rhodopsin, and 3) the extracellular and intracellular loops to establish their location relative to the retinal chromophore and the C- terminal peptide of the Galpha subunit of transducin. Together these studies are intended to establish how isomerization of the retinal chromophore is coupled to motion of the transmembrane helices and cytoplasmic loops.
A final aim i s to extend our structural studies to CCR5, the chemokine receptor in T-cells that serves as the co-receptor for HIV. The expression levels of CCR5 are now sufficient for solid-state NMR spectroscopy. We plan to establish the structure and location of inhibitors that bind to CCR5 and block HIV entry into T-cells. These studies will facilitate the rational design of inhibitors for the prevention of AIDS. Selective NMR measurements are also proposed that will allow comparisons to be made of the activated state structure of CCR5 with that of metarhodopsin II.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM041412-18
Application #
7687371
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Chin, Jean
Project Start
1988-12-01
Project End
2011-08-31
Budget Start
2009-09-01
Budget End
2011-08-31
Support Year
18
Fiscal Year
2009
Total Cost
$379,107
Indirect Cost
Name
State University New York Stony Brook
Department
Biochemistry
Type
Schools of Medicine
DUNS #
804878247
City
Stony Brook
State
NY
Country
United States
Zip Code
11794
Sanchez-Reyes, Omar B; Cooke, Aidan L G; Tranter, Dale B et al. (2017) G Protein-Coupled Receptors Contain Two Conserved Packing Clusters. Biophys J 112:2315-2326
Kimata, Naoki; Pope, Andreyah; Eilers, Markus et al. (2016) Retinal orientation and interactions in rhodopsin reveal a two-stage trigger mechanism for activation. Nat Commun 7:12683
Kimata, Naoki; Pope, Andreyah; Sanchez-Reyes, Omar B et al. (2016) Free backbone carbonyls mediate rhodopsin activation. Nat Struct Mol Biol 23:738-43
Opefi, Chikwado A; Tranter, Dale; Smith, Steven O et al. (2015) Construction of stable mammalian cell lines for inducible expression of G protein-coupled receptors. Methods Enzymol 556:283-305
Kimata, Naoki; Reeves, Philip J; Smith, Steven O (2015) Uncovering the triggers for GPCR activation using solid-state NMR spectroscopy. J Magn Reson 253:111-8
Kimata, Naoki; Pope, Andreyah; Rashid, Dawood et al. (2015) Sequential structural changes in rhodopsin occurring upon photoactivation. Methods Mol Biol 1271:159-71
Pope, Andreyah; Eilers, Markus; Reeves, Philip J et al. (2014) Amino acid conservation and interactions in rhodopsin: probing receptor activation by NMR spectroscopy. Biochim Biophys Acta 1837:683-93
Goncalves, Joseph; Eilers, Markus; South, Kieron et al. (2013) Magic angle spinning nuclear magnetic resonance spectroscopy of G protein-coupled receptors. Methods Enzymol 522:365-89
Xu, Bing; Chakraborty, Raja; Eilers, Markus et al. (2013) High-level expression, purification and characterization of a constitutively active thromboxane A2 receptor polymorphic variant. PLoS One 8:e76481
Opefi, Chikwado A; South, Kieron; Reynolds, Christopher A et al. (2013) Retinitis pigmentosa mutants provide insight into the role of the N-terminal cap in rhodopsin folding, structure, and function. J Biol Chem 288:33912-26

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