scholarly journals Cooperativity and Avidity in Membrane Binding by C2AB Tandem Domains of Synaptotagmins 1 and 7

2018 ◽  
Author(s):  
H. Tran ◽  
L. Anderson ◽  
J. Knight
Keyword(s):  
Hormone Secretion ◽  
Membrane Binding ◽  
Dissociation Rate ◽  
Dissociation Kinetics ◽  
Kinetic Measurements ◽  
C2 Domains ◽  
Synaptotagmin 1 ◽  
Fusion Pores ◽  
Fluorescence Kinetic ◽  
C2a Domain

AbstractSynaptotagmin-1 (Syt-1) and synaptotagmin-7 (Syt-7) contain analogous tandem C2 domains, C2A and C2B, which together serve as a Ca2+ sensor to bind membranes and promote the stabilization of exocytotic fusion pores. Functionally, Syt-1 triggers fast release of neurotransmitters, while Syt-7 is involved in lower-Ca2+ processes such as hormone secretion. Evidence suggests that Syt-1 C2 domains bind membranes cooperatively, penetrating farther into membranes as the C2AB tandem than as individual C2 domains. In contrast, we previously reported that the two C2 domains of Syt-7 bind membranes independently, based in part on measurements of their liposome dissociation kinetics. Here, we have investigated the effects of C2A-C2B interdomain cooperativity with Syt-1 and Syt-7 using directly comparable measurements. We report Ca2+ sensitivities, dissociation kinetics, and membrane insertion using liposomes approximating physiological lipid compositions. Equilibrium Ca2+ titrations confirm that the Syt-7 C2AB tandem has a greater Ca2+ sensitivity of membrane binding than either of its individual domains. Stopped-flow fluorescence kinetic measurements show that Syt-1 C2AB dissociates from liposome membranes much more slowly than either of its isolated C2 domains, suggesting that the two C2 domains of Syt-1 bind membranes cooperatively. In contrast, the dominant population of Syt-7 C2AB has a dissociation rate comparable to its C2A domain, indicating a lack of cooperativity, while only a small subpopulation dissociates at a slower rate. Measurements using an environment-sensitive fluorescent probe indicate that the Syt-7 C2B domain inserts more deeply into membranes as part of the C2AB tandem, similarly to Syt-1. Overall, these measurements are consistent with a model in which the structural linkage of C2A and C2B impacts the membrane-binding geometry of synaptotagmin C2B domains, but imparts little or no cooperativity to Syt-7 membrane binding and dissociation events that are dominated by its C2A domain.

scholarly journals Synaptotagmin-1 membrane binding is driven by the C2B domain and assisted cooperatively by the C2A domain

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Clémence Gruget ◽  
Oscar Bello ◽  
Jeff Coleman ◽  
Shyam S. Krishnakumar ◽  
Eric Perez ◽  
...  
Keyword(s):  
Neurotransmitter Release ◽  
Mutational Analysis ◽  
Membrane Binding ◽  
Surface Force ◽  
Membrane Interaction ◽  
C2 Domains ◽  
Binding Energetics ◽  
Synaptotagmin 1 ◽  
Site Binding ◽  
C2a Domain

Abstract Synaptotagmin interaction with anionic lipid (phosphatidylserine/phosphatidylinositol) containing membranes, both in the absence and presence of calcium ions (Ca2+), is critical to its central role in orchestrating neurotransmitter release. The molecular surfaces involved, namely the conserved polylysine motif in the C2B domain and Ca2+-binding aliphatic loops on both C2A and C2B domains, are known. Here we use surface force apparatus combined with systematic mutational analysis of the functional surfaces to directly measure Syt1-membrane interaction and fully map the site-binding energetics of Syt1 both in the absence and presence of Ca2+. By correlating energetics data with the molecular rearrangements measured during confinement, we find that both C2 domains cooperate in membrane binding, with the C2B domain functioning as the main energetic driver, and the C2A domain acting as a facilitator.

scholarly journals Synaptotagmin 1 clamps synaptic vesicle fusion in mammalian neurons independent of complexin

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Nicholas A. Courtney ◽  
Huan Bao ◽  
Joseph S. Briguglio ◽  
Edwin R. Chapman
Keyword(s):  
Synaptic Vesicle ◽  
Vesicle Fusion ◽  
Snare Proteins ◽  
Major Focus ◽  
C2 Domains ◽  
Releasable Pool ◽  
Readily Releasable Pool ◽  
Synaptotagmin 1 ◽  
Synaptic Vesicle Fusion ◽  
C2a Domain

Abstract Synaptic vesicle (SV) exocytosis is mediated by SNARE proteins. Reconstituted SNAREs are constitutively active, so a major focus has been to identify fusion clamps that regulate their activity in synapses: the primary candidates are synaptotagmin (syt) 1 and complexin I/II. Syt1 is a Ca2+ sensor for SV release that binds Ca2+ via tandem C2-domains, C2A and C2B. Here, we first determined whether these C2-domains execute distinct functions. Remarkably, the C2B domain profoundly clamped all forms of SV fusion, despite synchronizing residual evoked release and rescuing the readily-releasable pool. Release was strongly enhanced by an adjacent C2A domain, and by the concurrent binding of complexin to trans-SNARE complexes. Knockdown of complexin had no impact on C2B-mediated clamping of fusion. We postulate that the C2B domain of syt1, independent of complexin, is the molecular clamp that arrests SVs prior to Ca2+-triggered fusion.

scholarly journals Synaptotagmin-1 C2B domains cooperatively stabilize the fusion stalk via a master-servant mechanism

2021 ◽  
Author(s):  
Ary Lautaro Di Bartolo ◽  
Diego Masone
Keyword(s):  
Free Energy ◽  
Membrane Fusion ◽  
Membrane Binding ◽  
Vesicle Fusion ◽  
Fusion Process ◽  
Total Work ◽  
C2 Domains ◽  
Domain Interactions ◽  
Synaptotagmin 1 ◽  
Data Point

Synaptotagmin-1 is a low-affinity Ca2+ sensor that triggers synchronous vesicle fusion. It contains two similar C2 domains (C2A and C2B) that cooperate in membrane binding, being the C2B domain the main responsible for the membrane fusion process due to its polybasic patch KRLKKKKTTIKK (321-332). In this work, a master-servant mechanism between two identical C2B domains is shown to control the formation of the fusion stalk. Two regions in C2B are essential for the process, the well-known polybasic patch and a recently described pair of arginines (398,399). The master domain shows strong PIP2 interactions with its polybasic patch and its pair of arginines. At the same time, the servant analogously cooperates with the master to reduce the total work to form the fusion stalk. The strategic mutation (T328E,T329E) in both master and servant domains disrupts the cooperative mechanism, drastically increasing the free energy needed to induce the fusion stalk, however with negligible effects on the master domain interactions with PIP2. These data point to a difference in the behavior of the servant domain, which is unable to sustain its PIP2 interactions neither through its polybasic patch nor through its pair of arginines, in the end losing its ability to assist the master in the formation of the fusion stalk.

scholarly journals A Polybasic Patch on Synaptotagmin-1 C2A Domain is Essential for Evoked Release and Dilation of Fusion Pores

2020 ◽  
Vol 118 (3) ◽  
pp. 400a
Author(s):  
Zhenyong Wu ◽  
Lu Ma ◽  
Jie Zhu ◽  
Nicholas Courtney ◽  
Yongli Zhang ◽  
...  
Keyword(s):  
Synaptotagmin 1 ◽  
Fusion Pores ◽  
C2a Domain

scholarly journals Mutations that disrupt Ca2+-binding activity endow Doc2β with novel functional properties during synaptic transmission

2014 ◽  
Vol 25 (4) ◽  
pp. 481-494 ◽  
Author(s):  
Jon D. Gaffaney ◽  
Renhao Xue ◽  
Edwin R. Chapman
Keyword(s):  
Plasma Membrane ◽  
Synaptic Transmission ◽  
Functional Properties ◽  
Binding Activity ◽  
Mutant Form ◽  
C2 Domains ◽  
Synaptotagmin 1 ◽  
Asynchronous Release ◽  
Vesicle Pool ◽  
C2a Domain

Double C2-domain protein (Doc2) is a Ca2+-binding protein implicated in asynchronous and spontaneous neurotransmitter release. Here we demonstrate that each of its C2 domains senses Ca2+; moreover, the tethered tandem C2 domains display properties distinct from the isolated domains. We confirm that overexpression of a mutant form of Doc2β, in which two acidic Ca2+ ligands in the C2A domain and two in the C2B domain have been neutralized, results in markedly enhanced asynchronous release in synaptotagmin 1–knockout neurons. Unlike wild-type (wt) Doc2β, which translocates to the plasma membrane in response to increases in [Ca2+]i, the quadruple Ca2+-ligand mutant does not bind Ca2+ but is constitutively associated with the plasma membrane; this effect is due to substitution of Ca2+ ligands in the C2A domain. When overexpressed in wt neurons, Doc2β affects only asynchronous release; in contrast, Doc2β Ca2+-ligand mutants that constitutively localize to the plasma membrane enhance both the fast and slow components of synaptic transmission by increasing the readily releasable vesicle pool size; these mutants also increase the frequency of spontaneous release events. Thus, mutations in the C2A domain of Doc2β that were intended to disrupt Ca2+ binding result in an anomalous enhancement of constitutive membrane-binding activity and endow Doc2β with novel functional properties.

scholarly journals Differences in Membrane Binding Cooperativity between the Tandem C2 Domains of Synaptotagmin 1 and Synaptotagmin 7

2016 ◽  
Vol 110 (3) ◽  
pp. 253a
Author(s):  
Hai T. Tran ◽  
Daniel T. Giardina ◽  
Kan Chantranuvatana ◽  
Matthew D. Coffman ◽  
Joseph K. Vasquez ◽  
...  
Keyword(s):  
Membrane Binding ◽  
C2 Domains ◽  
Synaptotagmin 1

scholarly journals Phosphatidylinositol Phosphates as Co-activators of Ca2+ Binding to C2 Domains of Synaptotagmin 1

2006 ◽  
Vol 281 (23) ◽  
pp. 15845-15852 ◽  
Author(s):  
LiYi Li ◽  
Ok-Ho Shin ◽  
Jeong-Seop Rhee ◽  
Demet Araç ◽  
Jong-Cheol Rah ◽  
...  
Keyword(s):  
C2 Domains ◽  
Synaptotagmin 1 ◽  
Phosphatidylinositol Phosphates

scholarly journals The structure and flexibility analysis of the Arabidopsis Synaptotagmin 1 reveal the basis of its regulation at membrane contact sites

2021 ◽  
Author(s):  
Juan Luis Benavente ◽  
Dritan Siliqi ◽  
Lourdes Infantes ◽  
Laura Lagartera ◽  
Alberto Mills ◽  
...  
Keyword(s):  
Cell Function ◽  
Lipid Extraction ◽  
Membrane Lipid ◽  
Lipid Binding ◽  
Lipid Transfer ◽  
C2 Domains ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Cellular Environment ◽  
Synaptotagmin 1

Cell function requires the maintenance of membrane lipid homeostasis as changes in cellular environment unbalance this equilibrium. The non-vesicular lipid transfer at endoplasmic reticulum (ER) and plasma membrane (PM) contact sites (CS) is central to restore it. Extended synaptotagmins (E-Syts) are ER proteins that play a central role in this process as they act as molecular tethers with PM and as lipid transfer proteins between these organelles. E-Syts are constitutively anchored to the ER through an N-terminal hydrophobic segment and bind to the PM via C-terminal C2 domains. In plants, synaptotagmins (SYTs) are orthologous of E-Syts and regulate the ER-PM communication by the activity of their two C2 domains in response to abiotic stresses. We have combined macromolecular crystallography, small-angle X-ray scattering, structural bioinformatics and biochemical data to analyze the regulation of plant synaptotagmin 1 (SYT1). Our data show that the binding of SYT1 to the PM is regulated by the interaction of the first C2 domain through a Ca2+-dependent lipid binding site and by a site for phosphorylated forms of phosphatidylinositol in such a way that two different molecular signals are integrated in response to stress. In addition, our data show that SYT1 is highly flexible by virtue of up to three hinge points, including one that connects the two C2 domains. This feature provides conformational freedom to SYT1 to define a large and complementary interaction surface with the PM. This structural plasticity, in turn, may facilitate lipid extraction, protein loading and subsequent transfer between PM and ER.

scholarly journals Binding Dynamics of Hepatitis C Virus' NS5A Amphipathic Peptide to Cell and Model Membranes

2007 ◽  
Vol 81 (12) ◽  
pp. 6682-6689 ◽  
Author(s):  
Nam-Joon Cho ◽  
Kwang Ho Cheong ◽  
ChoongHo Lee ◽  
Curtis W. Frank ◽  
Jeffrey S. Glenn
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Critical Role ◽  
Membrane Binding ◽  
Lipid Binding ◽  
Model Membranes ◽  
Cellular Membranes ◽  
Kinetic Measurements ◽  
Amphipathic Peptide ◽  
Target Membrane

ABSTRACT Membrane association of the hepatitis C virus NS5A protein is required for viral replication. This association is dependent on an N-terminal amphipathic helix (AH) within NS5A and is restricted to a subset of host cell intracellular membranes. The mechanism underlying this specificity is not known, but it may suggest a novel strategy for developing specific antiviral therapy. Here we have probed the mechanistic details of NS5A AH-mediated binding to both cell-derived and model membranes by use of biochemical membrane flotation and quartz crystal microbalance (QCM) with dissipation. With both assays, we observed AH-mediated binding to model lipid bilayers. When cell-derived membranes were coated on the quartz nanosensor, however, significantly more binding was detected, and the QCM-derived kinetic measurements suggested the existence of an interacting receptor in the target membranes. Biochemical flotation assays performed with trypsin-treated cell-derived membranes exhibited reduced AH-mediated membrane binding, while membrane binding of control cytochrome b5 remained unaffected. Similarly, trypsin treatment of the nanosensor coated with cellular membranes abolished AH peptide binding to the cellular membranes but did not affect the binding of a control lipid-binding peptide. These results therefore suggest that a protein plays a critical role in mediating and stabilizing the binding of NS5A's AH to its target membrane. These results also demonstrate the successful development of a new nanosensor technology ideal both for studying the interaction between a protein and its target membrane and for developing inhibitors of that interaction.

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