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 #
5R01AI037581-24
Application #
9812207
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
2019-11-01
Budget End
2020-10-31
Support Year
24
Fiscal Year
2020
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
Brazin, Kristine N; Mallis, Robert J; Boeszoermenyi, Andras et al. (2018) The T Cell Antigen Receptor ? Transmembrane Domain Coordinates Triggering through Regulation of Bilayer Immersion and CD3 Subunit Associations. Immunity 49:829-841.e6
Mallis, Robert J; Arthanari, Haribabu; Lang, Matthew J et al. (2018) NMR-directed design of pre-TCR? and pMHC molecules implies a distinct geometry for pre-TCR relative to ??TCR recognition of pMHC. J Biol Chem 293:754-766
Zhao, Zhao; Zhang, Meng; Hogle, James M et al. (2018) DNA-Corralled Nanodiscs for the Structural and Functional Characterization of Membrane Proteins and Viral Entry. J Am Chem Soc 140:10639-10643
Robson, Scott A; Takeuchi, Koh; Boeszoermenyi, Andras et al. (2018) Mixed pyruvate labeling enables backbone resonance assignment of large proteins using a single experiment. Nat Commun 9:356
Hagn, Franz; Nasr, Mahmoud L; Wagner, Gerhard (2018) Assembly of phospholipid nanodiscs of controlled size for structural studies of membrane proteins by NMR. Nat Protoc 13:79-98
Nasr, Mahmoud L; Wagner, Gerhard (2018) Covalently circularized nanodiscs; challenges and applications. Curr Opin Struct Biol 51:129-134
Coote, Paul W; Robson, Scott A; Dubey, Abhinav et al. (2018) Optimal control theory enables homonuclear decoupling without Bloch-Siegert shifts in NMR spectroscopy. Nat Commun 9:3014
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

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