skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Exceptionally tight membrane-binding may explain the key role of the synaptotagmin-7 C 2 A domain in asynchronous neurotransmitter release

Abstract

Synaptotagmins (Syts) act as Ca2+ sensors in neurotransmitter release by virtue of Ca2+-binding to their two C2 domains, but their mechanisms of action remain unclear. Puzzlingly, Ca2+-binding to the C2B domain appears to dominate Syt1 function in synchronous release, whereas Ca2+-binding to the C2A domain mediates Syt7 function in asynchronous release. Here we show that crystal structures of the Syt7 C2A domain and C2AB region, and analyses of intrinsic Ca2+-binding to the Syt7 C2 domains using isothermal titration calorimetry, did not reveal major differences that could explain functional differentiation between Syt7 and Syt1. However, using liposome titrations under Ca2+ saturating conditions, we show that the Syt7 C2A domain has a very high membrane affinity and dominates phospholipid binding to Syt7 in the presence or absence of L-α-phosphatidylinositol 4,5-diphosphate (PIP2). For Syt1, the two Ca2+-saturated C2 domains have similar affinities for membranes lacking PIP2, but the C2B domain dominates binding to PIP2-containing membranes. Mutagenesis revealed that the dramatic differences in membrane affinity between the Syt1 and Syt7 C2A domains arise in part from apparently conservative residue substitutions, showing how striking biochemical and functional differences can result from the cumulative effects of subtle residue substitutions. Viewed together, our results suggest that membranemore » affinity may be a key determinant of the functions of Syt C2 domains in neurotransmitter release.« less

Authors:
; ; ;
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
NIHOTHER
OSTI Identifier:
1409103
Resource Type:
Journal Article
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Volume: 114; Journal Issue: 40; Journal ID: ISSN 0027-8424
Publisher:
National Academy of Sciences, Washington, DC (United States)
Country of Publication:
United States
Language:
ENGLISH
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; 59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Voleti, Rashmi, Tomchick, Diana R., Südhof, Thomas C., and Rizo, Josep. Exceptionally tight membrane-binding may explain the key role of the synaptotagmin-7 C 2 A domain in asynchronous neurotransmitter release. United States: N. p., 2017. Web. doi:10.1073/pnas.1710708114.
Voleti, Rashmi, Tomchick, Diana R., Südhof, Thomas C., & Rizo, Josep. Exceptionally tight membrane-binding may explain the key role of the synaptotagmin-7 C 2 A domain in asynchronous neurotransmitter release. United States. doi:10.1073/pnas.1710708114.
Voleti, Rashmi, Tomchick, Diana R., Südhof, Thomas C., and Rizo, Josep. Mon . "Exceptionally tight membrane-binding may explain the key role of the synaptotagmin-7 C 2 A domain in asynchronous neurotransmitter release". United States. doi:10.1073/pnas.1710708114.
@article{osti_1409103,
title = {Exceptionally tight membrane-binding may explain the key role of the synaptotagmin-7 C 2 A domain in asynchronous neurotransmitter release},
author = {Voleti, Rashmi and Tomchick, Diana R. and Südhof, Thomas C. and Rizo, Josep},
abstractNote = {Synaptotagmins (Syts) act as Ca2+ sensors in neurotransmitter release by virtue of Ca2+-binding to their two C2 domains, but their mechanisms of action remain unclear. Puzzlingly, Ca2+-binding to the C2B domain appears to dominate Syt1 function in synchronous release, whereas Ca2+-binding to the C2A domain mediates Syt7 function in asynchronous release. Here we show that crystal structures of the Syt7 C2A domain and C2AB region, and analyses of intrinsic Ca2+-binding to the Syt7 C2 domains using isothermal titration calorimetry, did not reveal major differences that could explain functional differentiation between Syt7 and Syt1. However, using liposome titrations under Ca2+ saturating conditions, we show that the Syt7 C2A domain has a very high membrane affinity and dominates phospholipid binding to Syt7 in the presence or absence of L-α-phosphatidylinositol 4,5-diphosphate (PIP2). For Syt1, the two Ca2+-saturated C2 domains have similar affinities for membranes lacking PIP2, but the C2B domain dominates binding to PIP2-containing membranes. Mutagenesis revealed that the dramatic differences in membrane affinity between the Syt1 and Syt7 C2A domains arise in part from apparently conservative residue substitutions, showing how striking biochemical and functional differences can result from the cumulative effects of subtle residue substitutions. Viewed together, our results suggest that membrane affinity may be a key determinant of the functions of Syt C2 domains in neurotransmitter release.},
doi = {10.1073/pnas.1710708114},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
issn = {0027-8424},
number = 40,
volume = 114,
place = {United States},
year = {2017},
month = {9}
}

Works referenced in this record:

How Synaptotagmin Promotes Membrane Fusion
journal, May 2007


A Complexin/Synaptotagmin 1 Switch Controls Fast Synaptic Vesicle Exocytosis
journal, September 2006


Architecture of the synaptotagmin–SNARE machinery for neuronal exocytosis
journal, August 2015

  • Zhou, Qiangjun; Lai, Ying; Bacaj, Taulant
  • Nature, Vol. 525, Issue 7567
  • DOI: 10.1038/nature14975

Augmenting neurotransmitter release by enhancing the apparent Ca2+ affinity of synaptotagmin 1
journal, December 2005

  • Rhee, J. -S.; Li, L. Y.; Shin, O. -H.
  • Proceedings of the National Academy of Sciences, Vol. 102, Issue 51
  • DOI: 10.1073/pnas.0509153102

C 2 -domains, Structure and Function of a Universal Ca 2+ -binding Domain
journal, June 1998


Structure of the first C2 domain of synaptotagmin I: A novel Ca2+/phospholipid-binding fold
journal, March 1995


Structure of Human Synaptotagmin 1 C2AB in the Absence of Ca 2+ Reveals a Novel Domain Association ,
journal, November 2007

  • Fuson, Kerry L.; Montes, Miguel; Robert, J. Justin
  • Biochemistry, Vol. 46, Issue 45
  • DOI: 10.1021/bi701651k

A Quaternary SNARE–Synaptotagmin–Ca2+–Phospholipid Complex in Neurotransmitter Release
journal, March 2007


Mechanism of Phospholipid Binding by the C 2 A-Domain of Synaptotagmin I
journal, September 1998

  • Zhang, Xiangyang; Rizo, Josep; Südhof, Thomas C.
  • Biochemistry, Vol. 37, Issue 36
  • DOI: 10.1021/bi9807512

Hydrophobic Contributions to the Membrane Docking of Synaptotagmin 7 C2A Domain: Mechanistic Contrast between Isoforms 1 and 7
journal, September 2012

  • Brandt, Devin S.; Coffman, Matthew D.; Falke, Joseph J.
  • Biochemistry, Vol. 51, Issue 39
  • DOI: 10.1021/bi3007115

Synaptotagmin I functions as a calcium regulator of release probability
journal, March 2001

  • Fernández-Chacón, Rafael; Königstorfer, Andreas; Gerber, Stefan H.
  • Nature, Vol. 410, Issue 6824
  • DOI: 10.1038/35065004

Reconstitution of the Vital Functions of Munc18 and Munc13 in Neurotransmitter Release
journal, December 2012


PIP2 increases the speed of response of synaptotagmin and steers its membrane-penetration activity toward the plasma membrane
journal, December 2003

  • Bai, Jihong; Tucker, Ward C.; Chapman, Edwin R.
  • Nature Structural & Molecular Biology, Vol. 11, Issue 1
  • DOI: 10.1038/nsmb709

A statistic for local intensity differences: robustness to anisotropy and pseudo-centering and utility for detecting twinning
journal, June 2003

  • Padilla, Jennifer E.; Yeates, Todd O.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 59, Issue 7
  • DOI: 10.1107/S0907444903007947

Membrane-Bound Orientation and Position of the Synaptotagmin C2B Domain Determined by Site-Directed Spin Labeling
journal, January 2005

  • Rufener, Elisabeth; Frazier, April A.; Wieser, Catherine M.
  • Biochemistry, Vol. 44, Issue 1
  • DOI: 10.1021/bi048370d

Solution Structures of the Ca 2+ -free and Ca 2+ -bound C 2 A Domain of Synaptotagmin I:  Does Ca 2+ Induce a Conformational Change?
journal, November 1998

  • Shao, Xuguang; Fernandez, Imma; Südhof, Thomas C.
  • Biochemistry, Vol. 37, Issue 46
  • DOI: 10.1021/bi981789h

Neurotransmitter Release: The Last Millisecond in the Life of a Synaptic Vesicle
journal, October 2013


Direct Interaction of a Ca 2+ -binding Loop of Synaptotagmin with Lipid Bilayers
journal, May 1998


How Does Synaptotagmin Trigger Neurotransmitter Release?
journal, June 2008


Diffraction data analysis in the presence of radiation damage
journal, March 2010

  • Borek, Dominika; Cymborowski, Marcin; Machius, Mischa
  • Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 4
  • DOI: 10.1107/S0907444909040177

Synaptic vesicle fusion
journal, July 2008

  • Rizo, Josep; Rosenmund, Christian
  • Nature Structural & Molecular Biology, Vol. 15, Issue 7
  • DOI: 10.1038/nsmb.1450

Close membrane-membrane proximity induced by Ca2+-dependent multivalent binding of synaptotagmin-1 to phospholipids
journal, February 2006

  • Araç, Demet; Chen, Xiaocheng; Khant, Htet A.
  • Nature Structural & Molecular Biology, Vol. 13, Issue 3
  • DOI: 10.1038/nsmb1056

Differential but convergent functions of Ca2+ binding to synaptotagmin-1 C2 domains mediate neurotransmitter release
journal, September 2009

  • Shin, O. -H.; Xu, J.; Rizo, J.
  • Proceedings of the National Academy of Sciences, Vol. 106, Issue 38
  • DOI: 10.1073/pnas.0908798106

Dynamic binding mode of a Synaptotagmin-1–SNARE complex in solution
journal, June 2015

  • Brewer, Kyle D.; Bacaj, Taulant; Cavalli, Andrea
  • Nature Structural & Molecular Biology, Vol. 22, Issue 7
  • DOI: 10.1038/nsmb.3035

HKL -3000: the integration of data reduction and structure solution – from diffraction images to an initial model in minutes
journal, July 2006

  • Minor, Wladek; Cymborowski, Marcin; Otwinowski, Zbyszek
  • Acta Crystallographica Section D Biological Crystallography, Vol. 62, Issue 8
  • DOI: 10.1107/S0907444906019949

Synaptotagmin-1 and Synaptotagmin-7 Trigger Synchronous and Asynchronous Phases of Neurotransmitter Release
journal, November 2013


Synaptotagmin-1 and -7 are functionally overlapping Ca2+ sensors for exocytosis in adrenal chromaffin cells
journal, February 2008

  • Schonn, J. -S.; Maximov, A.; Lao, Y.
  • Proceedings of the National Academy of Sciences, Vol. 105, Issue 10
  • DOI: 10.1073/pnas.0712373105

The Ca 2+ Affinity of Synaptotagmin 1 Is Markedly Increased by a Specific Interaction of Its C2B Domain with Phosphatidylinositol 4,5-Bisphosphate
journal, July 2009

  • Radhakrishnan, Anand; Stein, Alexander; Jahn, Reinhard
  • Journal of Biological Chemistry, Vol. 284, Issue 38
  • DOI: 10.1074/jbc.M109.042499

Surprises and pitfalls arising from (pseudo)symmetry
journal, December 2007

  • Zwart, Peter H.; Grosse-Kunstleve, Ralf W.; Lebedev, Andrey A.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 64, Issue 1
  • DOI: 10.1107/S090744490705531X

NMR View: A computer program for the visualization and analysis of NMR data
journal, September 1994

  • Johnson, Bruce A.; Blevins, Richard A.
  • Journal of Biomolecular NMR, Vol. 4, Issue 5
  • DOI: 10.1007/BF00404272

MolProbity : all-atom structure validation for macromolecular crystallography
journal, December 2009

  • Chen, Vincent B.; Arendall, W. Bryan; Headd, Jeffrey J.
  • Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 1
  • DOI: 10.1107/S0907444909042073

Coot model-building tools for molecular graphics
journal, November 2004

  • Emsley, Paul; Cowtan, Kevin
  • Acta Crystallographica Section D Biological Crystallography, Vol. 60, Issue 12, p. 2126-2132
  • DOI: 10.1107/S0907444904019158

Postsynaptic synaptotagmins mediate AMPA receptor exocytosis during LTP
journal, March 2017

  • Wu, Dick; Bacaj, Taulant; Morishita, Wade
  • Nature, Vol. 544, Issue 7650
  • DOI: 10.1038/nature21720

The Synaptic Vesicle Release Machinery
journal, June 2015


PHENIX: a comprehensive Python-based system for macromolecular structure solution
journal, January 2010

  • Adams, Paul D.; Afonine, Pavel V.; Bunkóczi, Gábor
  • Acta Crystallographica Section D Biological Crystallography, Vol. 66, Issue 2, p. 213-221
  • DOI: 10.1107/S0907444909052925

Identification of patterns in diffraction intensities affected by radiation exposure
journal, December 2012

  • Borek, Dominika; Dauter, Zbigniew; Otwinowski, Zbyszek
  • Journal of Synchrotron Radiation, Vol. 20, Issue 1
  • DOI: 10.1107/S0909049512048807

Measurement errors and their consequences in protein crystallography
journal, October 2003

  • Borek, Dominika; Minor, Wladek; Otwinowski, Zbyszek
  • Acta Crystallographica Section D Biological Crystallography, Vol. 59, Issue 11
  • DOI: 10.1107/S0907444903020924

NMRPipe: A multidimensional spectral processing system based on UNIX pipes
journal, November 1995

  • Delaglio, Frank; Grzesiek, Stephan; Vuister, GeertenW.
  • Journal of Biomolecular NMR, Vol. 6, Issue 3
  • DOI: 10.1007/BF00197809

Membrane Fusion: Grappling with SNARE and SM Proteins
journal, January 2009


Genetic analysis of synaptotagmin-7 function in synaptic vesicle exocytosis
journal, February 2008

  • Maximov, A.; Lao, Y.; Li, H.
  • Proceedings of the National Academy of Sciences, Vol. 105, Issue 10
  • DOI: 10.1073/pnas.0712372105

Crystal Structure of the Cytosolic C2a-C2b Domains of Synaptotagmin III: Implications for Ca
journal, November 1999

  • Sutton, R. Bryan; Ernst, James A.; Brunger, Axel T.
  • The Journal of Cell Biology, Vol. 147, Issue 3
  • DOI: 10.1083/jcb.147.3.589

The Janus-faced nature of the C2B domain is fundamental for synaptotagmin-1 function
journal, October 2008

  • Xue, Mingshan; Ma, Cong; Craig, Timothy K.
  • Nature Structural & Molecular Biology, Vol. 15, Issue 11
  • DOI: 10.1038/nsmb.1508

Multiparametric scaling of diffraction intensities
journal, April 2003

  • Otwinowski, Zbyszek; Borek, Dominika; Majewski, Wladyslaw
  • Acta Crystallographica Section A Foundations of Crystallography, Vol. 59, Issue 3
  • DOI: 10.1107/S0108767303005488

Three-Dimensional Structure of the Synaptotagmin 1 C2B-Domain
journal, December 2001


Phaser crystallographic software
journal, July 2007

  • McCoy, Airlie J.; Grosse-Kunstleve, Ralf W.; Adams, Paul D.
  • Journal of Applied Crystallography, Vol. 40, Issue 4
  • DOI: 10.1107/S0021889807021206