The goal of the proposed research is to characterize structures and interactions of receptors residing in plasma membranes, in particular those of immune cells. Membrane bound receptors play important roles in cells but their structures and molecular functions have been difficult to reveal. Importantly, the T-cell plasma membrane contains the T-cell receptor of antigens and the associated CD3 components crucial for signaling. Furthermore, the membrane contains the CD4/8, CCR5 and CXCR4, co-receptors of HIV. There are many other T-cell transmembrane proteins with numerous activities, such as CD2, CD45 or Ca2+ channels. For a number of these systems structures of extracellular or intracellular portions have been characterized in various groups including ours. However, interactions of transmembrane segments have been difficult to access on a structural level due to the complexity of handling membranes. Exceptions include the G-protein coupled receptors CCR5 and CXCR4, or the ? homodimer of the TCR and a model of the TCR transmembrane arrangement based on biochemical experiments and model building. Structures and functional aspects of complexes of T-cell membrane proteins with other proteins residing in other membranes are even more difficult to study but will be tackled here. We recently made a break through designing covalently circularized nanodiscs (cNDs) of variable size, which allows assembly of stable membrane protein complexes in a well defined near-native environment and dramatically facilitates structural studies of integral membrane proteins. Moreover, this approach provides access to structural studies of proteins residing in different membranes. We show preliminary data from non-circularized and circularized nanodisc since the latter was established only recently. It is now fully available and will be applied to the systems described. We will apply this technology in two specific aims:
Aim 1. Characterize the interaction of viral particles with their receptors in circularized nanodiscs. As a proof of concept we will use EM methods to reveal detail of poliovirus interaction with its receptor CD155 in cNDs. This approach will be extended for imaging the interaction of HIV pseudovirus and/or gp160 with its co-receptors and will reveal details of viral engagement and pore formation.
Aim 2 : Determine the structure of the TCR/CD3 complex in cNDs and characterize the interaction with pMHC and CD8. Receptors residing in different nanodiscs will be linked with DNA handles to allow formation of cND sandwiches that mimic interactions of receptors from different cells.

Public Health Relevance

The planned research will use a new circularized nanodisc technology to study structure and function of membrane proteins from immune cells. The approach is suitable for cryo electron microscopy. It will also image viral particle interaction with receptors on T-cells and nerve cells.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI037581-22
Application #
9386715
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Singleton, Kentner L
Project Start
1996-07-15
Project End
2021-10-31
Budget Start
2017-11-01
Budget End
2018-10-31
Support Year
22
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Harvard Medical School
Department
Biochemistry
Type
Schools of Medicine
DUNS #
047006379
City
Boston
State
MA
Country
United States
Zip Code
02115
Nasr, Mahmoud L; Baptista, Diego; Strauss, Mike et al. (2017) Covalently circularized nanodiscs for studying membrane proteins and viral entry. Nat Methods 14:49-52
Coote, Paul; Anklin, Clemens; Massefski, Walter et al. (2017) Rapid convergence of optimal control in NMR using numerically-constructed toggling frames. J Magn Reson 281:94-103
Mallis, Robert J; Reinherz, Ellis L; Wagner, Gerhard et al. (2016) Backbone resonance assignment of N15, N30 and D10 T cell receptor ? subunits. Biomol NMR Assign 10:35-9
Das, Dibyendu Kumar; Mallis, Robert J; Duke-Cohan, Jonathan S et al. (2016) Pre-T Cell Receptors (Pre-TCRs) Leverage V? Complementarity Determining Regions (CDRs) and Hydrophobic Patch in Mechanosensing Thymic Self-ligands. J Biol Chem 291:25292-25305
Takeuchi, Koh; Arthanari, Haribabu; Imai, Misaki et al. (2016) Nitrogen-detected TROSY yields comparable sensitivity to proton-detected TROSY for non-deuterated, large proteins under physiological salt conditions. J Biomol NMR 64:143-51
Das, Dibyendu Kumar; Feng, Yinnian; Mallis, Robert J et al. (2015) Force-dependent transition in the T-cell receptor ?-subunit allosterically regulates peptide discrimination and pMHC bond lifetime. Proc Natl Acad Sci U S A 112:1517-22
Mallis, Robert J; Bai, Ke; Arthanari, Haribabu et al. (2015) Pre-TCR ligand binding impacts thymocyte development before ??TCR expression. Proc Natl Acad Sci U S A 112:8373-8
Takeuchi, Koh; Sun, Zhen-Yu J; Li, Shuai et al. (2015) NMR resonance assignments of the catalytic domain of human serine/threonine phosphatase calcineurin in unligated and PVIVIT-peptide-bound states. Biomol NMR Assign 9:201-5
Takeuchi, Koh; Arthanari, Haribabu; Shimada, Ichio et al. (2015) Nitrogen detected TROSY at high field yields high resolution and sensitivity for protein NMR. J Biomol NMR 63:323-331
Gal, Maayan; Li, Shuai; Luna, Rafael E et al. (2014) The LxVP and PxIxIT NFAT motifs bind jointly to overlapping epitopes on calcineurin's catalytic domain distant to the regulatory domain. Structure 22:1016-27

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