Two modes of interaction between the membrane-embedded TARP stargazin's C-terminal domain and the bilayer visualized by electron crystallography

Matthew F. Roberts; David W. Taylor; Vinzenz M. Unger

doi:10.1016/j.jsb.2011.03.012

Two modes of interaction between the membrane-embedded TARP stargazin's C-terminal domain and the bilayer visualized by electron crystallography

Matthew F. Roberts, David W. Taylor, Vinzenz M. Unger^*

^*Corresponding author for this work

Molecular Biosciences

Research output: Contribution to journal › Article › peer-review

7 Scopus citations

Abstract

Glutamate-mediated neurotransmission through ligand-gated, ionotropic glutamate receptors is the main form of excitatory neurotransmission in the vertebrate central nervous system where it plays central roles in learning, memory and a variety of disorders. Acting as auxiliary subunits, transmembrane α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) regulatory proteins (TARPs) are essential regulators for glutamate-mediated neurotransmission in the central nervous system. Here, we report the first electron crystallographic reconstructions of full-length mouse stargazin (γ-2) at ∼20. Å resolution in a membrane bilayer environment. Formation of ordered arrays required anionic lipids and was modulated by cholesterol and monovalent cations. Projection structures revealed that the C-termini of stargazin monomers closely interacted with the bilayer surface in an extended conformation that placed the C-terminal PDZ-binding motif ∼100. Å away from the transmembrane domain and in close proximity to a membrane re-entrant region. The C-termini interaction with the bilayer was modulated by the ionic strength of the solution and overall protein secondary structure increased when membrane-bound. Our data suggest that stargazin interactions with and within the membrane play significant roles in TARP structure and directly visualize TARP functional mechanisms essential for AMPAR trafficking and clustering.

Original language	English (US)
Pages (from-to)	542-551
Number of pages	10
Journal	Journal of Structural Biology
Volume	174
Issue number	3
DOIs	https://doi.org/10.1016/j.jsb.2011.03.012
State	Published - Jun 2011

Funding

We thank Susumu Tomita for the gift of the full-length mouse stargazin pGEMHE construct and much constructive criticism. His evaluation of this work was essential for its completion and we are extremely grateful for his help. We thank Chen Xu of Brandeis University for use of cryo-EM facility equipment and his help in generating vitrified specimens. We also acknowledge the generous support of the Yale School of Medicine towards support of the local cryo-EM facility and especially thank Hongwei Wang for his help with single particle analysis. This work was supported by National Institutes of Health Grants T32 GM007223 (Predoctoral Program in Cellular and Molecular Biology; to M.F.R.), F31 NS064748 (to M.F.R.), and DA024101 (to V.M.U.); D.W.T. is a National Science Foundation Graduate Research Fellow.

Keywords

Electron crystallography
Stargazin
Three dimensional electron microscopy
Transmembrane AMPA receptor regulatory protein
Two-dimensional crystallization

ASJC Scopus subject areas

Structural Biology

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10.1016/j.jsb.2011.03.012

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@article{25635cd12eb84f57acfb9ef73339c1fb,

title = "Two modes of interaction between the membrane-embedded TARP stargazin's C-terminal domain and the bilayer visualized by electron crystallography",

abstract = "Glutamate-mediated neurotransmission through ligand-gated, ionotropic glutamate receptors is the main form of excitatory neurotransmission in the vertebrate central nervous system where it plays central roles in learning, memory and a variety of disorders. Acting as auxiliary subunits, transmembrane α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) regulatory proteins (TARPs) are essential regulators for glutamate-mediated neurotransmission in the central nervous system. Here, we report the first electron crystallographic reconstructions of full-length mouse stargazin (γ-2) at ∼20. {\AA} resolution in a membrane bilayer environment. Formation of ordered arrays required anionic lipids and was modulated by cholesterol and monovalent cations. Projection structures revealed that the C-termini of stargazin monomers closely interacted with the bilayer surface in an extended conformation that placed the C-terminal PDZ-binding motif ∼100. {\AA} away from the transmembrane domain and in close proximity to a membrane re-entrant region. The C-termini interaction with the bilayer was modulated by the ionic strength of the solution and overall protein secondary structure increased when membrane-bound. Our data suggest that stargazin interactions with and within the membrane play significant roles in TARP structure and directly visualize TARP functional mechanisms essential for AMPAR trafficking and clustering.",

keywords = "Electron crystallography, Stargazin, Three dimensional electron microscopy, Transmembrane AMPA receptor regulatory protein, Two-dimensional crystallization",

author = "Roberts, {Matthew F.} and Taylor, {David W.} and Unger, {Vinzenz M.}",

note = "Funding Information: We thank Susumu Tomita for the gift of the full-length mouse stargazin pGEMHE construct and much constructive criticism. His evaluation of this work was essential for its completion and we are extremely grateful for his help. We thank Chen Xu of Brandeis University for use of cryo-EM facility equipment and his help in generating vitrified specimens. We also acknowledge the generous support of the Yale School of Medicine towards support of the local cryo-EM facility and especially thank Hongwei Wang for his help with single particle analysis. This work was supported by National Institutes of Health Grants T32 GM007223 (Predoctoral Program in Cellular and Molecular Biology; to M.F.R.), F31 NS064748 (to M.F.R.), and DA024101 (to V.M.U.); D.W.T. is a National Science Foundation Graduate Research Fellow. ",

year = "2011",

month = jun,

doi = "10.1016/j.jsb.2011.03.012",

language = "English (US)",

volume = "174",

pages = "542--551",

journal = "Journal of Structural Biology",

issn = "1047-8477",

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T1 - Two modes of interaction between the membrane-embedded TARP stargazin's C-terminal domain and the bilayer visualized by electron crystallography

AU - Roberts, Matthew F.

AU - Taylor, David W.

AU - Unger, Vinzenz M.

N1 - Funding Information: We thank Susumu Tomita for the gift of the full-length mouse stargazin pGEMHE construct and much constructive criticism. His evaluation of this work was essential for its completion and we are extremely grateful for his help. We thank Chen Xu of Brandeis University for use of cryo-EM facility equipment and his help in generating vitrified specimens. We also acknowledge the generous support of the Yale School of Medicine towards support of the local cryo-EM facility and especially thank Hongwei Wang for his help with single particle analysis. This work was supported by National Institutes of Health Grants T32 GM007223 (Predoctoral Program in Cellular and Molecular Biology; to M.F.R.), F31 NS064748 (to M.F.R.), and DA024101 (to V.M.U.); D.W.T. is a National Science Foundation Graduate Research Fellow.

PY - 2011/6

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N2 - Glutamate-mediated neurotransmission through ligand-gated, ionotropic glutamate receptors is the main form of excitatory neurotransmission in the vertebrate central nervous system where it plays central roles in learning, memory and a variety of disorders. Acting as auxiliary subunits, transmembrane α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) regulatory proteins (TARPs) are essential regulators for glutamate-mediated neurotransmission in the central nervous system. Here, we report the first electron crystallographic reconstructions of full-length mouse stargazin (γ-2) at ∼20. Å resolution in a membrane bilayer environment. Formation of ordered arrays required anionic lipids and was modulated by cholesterol and monovalent cations. Projection structures revealed that the C-termini of stargazin monomers closely interacted with the bilayer surface in an extended conformation that placed the C-terminal PDZ-binding motif ∼100. Å away from the transmembrane domain and in close proximity to a membrane re-entrant region. The C-termini interaction with the bilayer was modulated by the ionic strength of the solution and overall protein secondary structure increased when membrane-bound. Our data suggest that stargazin interactions with and within the membrane play significant roles in TARP structure and directly visualize TARP functional mechanisms essential for AMPAR trafficking and clustering.

AB - Glutamate-mediated neurotransmission through ligand-gated, ionotropic glutamate receptors is the main form of excitatory neurotransmission in the vertebrate central nervous system where it plays central roles in learning, memory and a variety of disorders. Acting as auxiliary subunits, transmembrane α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) regulatory proteins (TARPs) are essential regulators for glutamate-mediated neurotransmission in the central nervous system. Here, we report the first electron crystallographic reconstructions of full-length mouse stargazin (γ-2) at ∼20. Å resolution in a membrane bilayer environment. Formation of ordered arrays required anionic lipids and was modulated by cholesterol and monovalent cations. Projection structures revealed that the C-termini of stargazin monomers closely interacted with the bilayer surface in an extended conformation that placed the C-terminal PDZ-binding motif ∼100. Å away from the transmembrane domain and in close proximity to a membrane re-entrant region. The C-termini interaction with the bilayer was modulated by the ionic strength of the solution and overall protein secondary structure increased when membrane-bound. Our data suggest that stargazin interactions with and within the membrane play significant roles in TARP structure and directly visualize TARP functional mechanisms essential for AMPAR trafficking and clustering.

KW - Electron crystallography

KW - Stargazin

KW - Three dimensional electron microscopy

KW - Transmembrane AMPA receptor regulatory protein

KW - Two-dimensional crystallization

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DO - 10.1016/j.jsb.2011.03.012

M3 - Article

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AN - SCOPUS:79955675073

SN - 1047-8477

VL - 174

SP - 542

EP - 551

JO - Journal of Structural Biology

JF - Journal of Structural Biology

IS - 3

ER -

Two modes of interaction between the membrane-embedded TARP stargazin's C-terminal domain and the bilayer visualized by electron crystallography

Abstract

Funding

Keywords

ASJC Scopus subject areas

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