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Title: Exceptionally tight membrane-binding may explain the key role of the synaptotagmin-7 C2A 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 membrane affinitymore » may be a key determinant of the functions of Syt C2 domains in neurotransmitter release.« less

Authors:
 [1];  [1];  [2];  [1]
  1. Univ. of Texas Southwestern Medical Center, Dallas, TX (United States)
  2. Stanford Univ. Medical School, Stanford, CA (United States)
Publication Date:
Research Org.:
Argonne National Laboratory (ANL), Argonne, IL (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER); National Institutes of Health (NIH); Welch Foundation
OSTI Identifier:
1409103
Grant/Contract Number:  
AC02-06CH11357; S10OD018027; S10RR026461; I-1304; R35 NS097333; NS037200; NS049044
Resource Type:
Journal Article: Accepted Manuscript
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
Country of Publication:
United States
Language:
ENGLISH
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; 59 BASIC BIOLOGICAL SCIENCES; synaptotagmin-7; synaptotagmin-1; membrane binding; X-ray crystallography; neurotransmitter release

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 C2A 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 C2A domain in asynchronous neurotransmitter release. United States. https://doi.org/10.1073/pnas.1710708114
Voleti, Rashmi, Tomchick, Diana R., Südhof, Thomas C., and Rizo, Josep. 2017. "Exceptionally tight membrane-binding may explain the key role of the synaptotagmin-7 C2A domain in asynchronous neurotransmitter release". United States. https://doi.org/10.1073/pnas.1710708114. https://www.osti.gov/servlets/purl/1409103.
@article{osti_1409103,
title = {Exceptionally tight membrane-binding may explain the key role of the synaptotagmin-7 C2A 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},
url = {https://www.osti.gov/biblio/1409103}, 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 = {Mon Sep 18 00:00:00 EDT 2017},
month = {Mon Sep 18 00:00:00 EDT 2017}
}

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Cited by: 25 works
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Works referenced in this record:

Three distinct kinetic groupings of the synaptotagmin family: Candidate sensors for rapid and delayed exocytosis
journal, March 2005


How Synaptotagmin Promotes Membrane Fusion
journal, May 2007


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


Bipartite Ca2+-Binding Motif in C2 Domains of Synaptotagmin and Protein Kinase C
journal, July 1996


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


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


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


Genetic analysis of synaptotagmin 2 in spontaneous and Ca2+-triggered neurotransmitter release
journal, April 2006


The C2B Ca2+-binding motif of synaptotagmin is required for synaptic transmission in vivo
journal, July 2002


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


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


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


Phosphatidylinositol 4,5-Bisphosphate Increases Ca 2+ Affinity of Synaptotagmin-1 by 40-fold
journal, March 2012


Towards reconstitution of membrane fusion mediated by SNAREs and other synaptic proteins
journal, November 2014


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


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


Ca2+ binding to synaptotagmin: how many Ca2+ ions bind to the tip of a C2-domain?
journal, July 1998


Synaptotagmins I and IV promote transmitter release independently of Ca2+ binding in the C2A domain
journal, July 2002


Analysis of SNARE Complex/Synaptotagmin-1 Interactions by One-Dimensional NMR Spectroscopy
journal, May 2013


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


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


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


Prevalent mechanism of membrane bridging by synaptotagmin-1
journal, August 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


Synaptic vesicle fusion
journal, July 2008


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


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


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


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


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


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


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


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


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


Structural basis for the evolutionary inactivation of Ca2+ binding to synaptotagmin 4
journal, August 2004


Phosphatidylinositol Phosphates as Co-activators of Ca 2+ Binding to C 2 Domains of Synaptotagmin 1
journal, April 2006


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


Detection and Use of Pseudo-Translation in Determination of Protein Structures
journal, September 1998


The Synaptic Vesicle Release Machinery
journal, June 2015


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


Structural and Mutational Analysis of Functional Differentiation between Synaptotagmins-1 and -7
journal, September 2010


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


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


Membrane-Bound Orientation and Position of the Synaptotagmin I C2A Domain by Site-Directed Spin Labeling
journal, January 2003


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


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


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


Synaptotagmins: Why So Many?
journal, December 2001


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


Multiparametric scaling of diffraction intensities
journal, April 2003


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


Phaser crystallographic software
journal, July 2007


The C 2 B Domain of Synaptotagmin I Is a Ca 2+ -Binding Module
journal, May 2001


Synaptotagmin function in dense core vesicle exocytosis studied in cracked PC12 cells
journal, June 2002


Works referencing / citing this record:

Hypothesis - buttressed rings assemble, clamp, and release SNAREpins for synaptic transmission
journal, October 2017


The high-affinity calcium sensor synaptotagmin-7 serves multiple roles in regulated exocytosis
journal, May 2018


Synaptotagmin Ca2+ Sensors and Their Spatial Coupling to Presynaptic Cav Channels in Central Cortical Synapses
journal, January 2019


Presynaptic Calcium Channels
journal, May 2019


Presynaptic calcium channels
journal, February 2018


The high-affinity calcium sensor synaptotagmin-7 serves multiple roles in regulated exocytosis
journal, May 2018