scholarly journals Stability, folding dynamics, and long-range conformational transition of the synaptic t-SNARE complex

2016 ◽  
Vol 113 (50) ◽  
pp. E8031-E8040 ◽  
Author(s):  
Xinming Zhang ◽  
Aleksander A. Rebane ◽  
Lu Ma ◽  
Feng Li ◽  
Junyi Jiao ◽  
...  
Keyword(s):  
Single Molecule ◽  
Plasma Membranes ◽  
Conformational Transition ◽  
Snare Complex ◽  
Structure Stability ◽  
Helix Bundle ◽  
Terminal Domain ◽  
C Terminus ◽  
Protein Receptors ◽  
Ionic Layer

Synaptic soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNAREs) couple their stepwise folding to fusion of synaptic vesicles with plasma membranes. In this process, three SNAREs assemble into a stable four-helix bundle. Arguably, the first and rate-limiting step of SNARE assembly is the formation of an activated binary target (t)-SNARE complex on the target plasma membrane, which then zippers with the vesicle (v)-SNARE on the vesicle to drive membrane fusion. However, the t-SNARE complex readily misfolds, and its structure, stability, and dynamics are elusive. Using single-molecule force spectroscopy, we modeled the synaptic t-SNARE complex as a parallel three-helix bundle with a small frayed C terminus. The helical bundle sequentially folded in an N-terminal domain (NTD) and a C-terminal domain (CTD) separated by a central ionic layer, with total unfolding energy of ∼17 kBT, where kB is the Boltzmann constant and T is 300 K. Peptide binding to the CTD activated the t-SNARE complex to initiate NTD zippering with the v-SNARE, a mechanism likely shared by the mammalian uncoordinated-18-1 protein (Munc18-1). The NTD zippering then dramatically stabilized the CTD, facilitating further SNARE zippering. The subtle bidirectional t-SNARE conformational switch was mediated by the ionic layer. Thus, the t-SNARE complex acted as a switch to enable fast and controlled SNARE zippering required for synaptic vesicle fusion and neurotransmission.

scholarly journals HOPS Proofreads the trans-SNARE Complex for Yeast Vacuole Fusion

2008 ◽  
Vol 19 (6) ◽  
pp. 2500-2508 ◽  
Author(s):  
Vincent J. Starai ◽  
Christopher M. Hickey ◽  
William Wickner
Keyword(s):  
Snare Complex ◽  
Wild Type ◽  
Fusion Event ◽  
Sm Proteins ◽  
Terminal Domain ◽  
Domain Structures ◽  
Vacuole Fusion ◽  
Protein Receptors ◽  
Optimal Fusion ◽  
Guanosine Triphosphatase

The fusion of yeast vacuoles, like other organelles, requires a Rab-family guanosine triphosphatase (Ypt7p), a Rab effector and Sec1/Munc18 (SM) complex termed HOPS (homotypic fusion and vacuole protein sorting), and soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). The central 0-layer of the four bundled vacuolar SNAREs requires the wild-type three glutaminyl (Q) and one arginyl (R) residues for optimal fusion. Alterations of this layer dramatically increase the Km value for SNAREs to assemble trans-SNARE complexes and to fuse. We now find that added purified HOPS complex strongly suppresses the fusion of vacuoles bearing 0-layer alterations, but it has little effect on the fusion of vacuoles with wild-type SNAREs. HOPS proofreads at two levels, inhibiting the formation of trans-SNARE complexes with altered 0-layers and suppressing the ability of these mismatched 0-layer trans-SNARE complexes to support membrane fusion. HOPS proofreading also extends to other parts of the SNARE complex, because it suppresses the fusion of trans-SNARE complexes formed without the N-terminal Phox homology domain of Vam7p (Qc). Unlike some other SM proteins, HOPS proofreading does not require the Vam3p (Qa) N-terminal domain. HOPS thus proofreads SNARE domain and N-terminal domain structures and regulates the fusion capacity of trans-SNARE complexes, only allowing full function for wild-type SNARE configurations. This is the most direct evidence to date that HOPS is directly involved in the fusion event.

scholarly journals Complexin induces a conformational change at the membrane-proximal C-terminal end of the SNARE complex

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Ucheor B Choi ◽  
Minglei Zhao ◽  
Yunxiang Zhang ◽  
Ying Lai ◽  
Axel T Brunger
Keyword(s):  
Conformational Change ◽  
Single Molecule ◽  
Neurotransmitter Release ◽  
Plasma Membranes ◽  
Molecular Mechanisms ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Snare Complex ◽  
Spontaneous Release ◽  
Trans Conformation

Complexin regulates spontaneous and activates Ca2+-triggered neurotransmitter release, yet the molecular mechanisms are still unclear. Here we performed single molecule fluorescence resonance energy transfer experiments and uncovered two conformations of complexin-1 bound to the ternary SNARE complex. In the cis conformation, complexin-1 induces a conformational change at the membrane-proximal C-terminal end of the ternary SNARE complex that specifically depends on the N-terminal, accessory, and central domains of complexin-1. The complexin-1 induced conformation of the ternary SNARE complex may be related to a conformation that is juxtaposing the synaptic vesicle and plasma membranes. In the trans conformation, complexin-1 can simultaneously interact with a ternary SNARE complex via the central domain and a binary SNARE complex consisting of syntaxin-1A and SNAP-25A via the accessory domain. The cis conformation may be involved in activation of synchronous neurotransmitter release, whereas both conformations may be involved in regulating spontaneous release.

scholarly journals Common intermediates and kinetics, but different energetics, in the assembly of SNARE proteins

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Sylvain Zorman ◽  
Aleksander A Rebane ◽  
Lu Ma ◽  
Guangcan Yang ◽  
Matthew A Molski ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Energy Release ◽  
Optical Tweezers ◽  
Single Molecule ◽  
Folding Energy ◽  
Single Molecule Level ◽  
Terminal Domain ◽  
Evolutionarily Conserved ◽  
Protein Receptors ◽  
And Function

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are evolutionarily conserved machines that couple their folding/assembly to membrane fusion. However, it is unclear how these processes are regulated and function. To determine these mechanisms, we characterized the folding energy and kinetics of four representative SNARE complexes at a single-molecule level using high-resolution optical tweezers. We found that all SNARE complexes assemble by the same step-wise zippering mechanism: slow N-terminal domain (NTD) association, a pause in a force-dependent half-zippered intermediate, and fast C-terminal domain (CTD) zippering. The energy release from CTD zippering differs for yeast (13 kBT) and neuronal SNARE complexes (27 kBT), and is concentrated at the C-terminal part of CTD zippering. Thus, SNARE complexes share a conserved zippering pathway and polarized energy release to efficiently drive membrane fusion, but generate different amounts of zippering energy to regulate fusion kinetics.

scholarly journals Munc18-1 catalyzes neuronal SNARE assembly by templating SNARE association

2018 ◽  
Author(s):  
Junyi Jiao ◽  
Mengze He ◽  
Sarah A. Port ◽  
Richard W. Baker ◽  
Yonggang Xu ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Single Molecule ◽  
Force Spectroscopy ◽  
Snare Complex ◽  
Sm Proteins ◽  
Single Molecule Force Spectroscopy ◽  
Helix Bundle ◽  
The Stability ◽  
Sm Protein ◽  
Template Complex

AbstractSec1/Munc18-family (SM) proteins are required for SNARE-mediated membrane fusion, but their mechanism(s) of action remain controversial. Using single-molecule force spectroscopy, we found that the SM protein Munc18-1 catalyzes step-wise zippering of three synaptic SNAREs (syntaxin, VAMP2, and SNAP-25) into a four-helix bundle. Catalysis requires formation of an intermediate template complex in which Munc18-1 juxtaposes the N-terminal regions of the SNARE motifs of syntaxin and VAMP2, while keeping their C-terminal regions separated. Next, SNAP-25 binds the templated SNAREs to form a partially-zippered SNARE complex. Finally, full zippering displaces Munc18-1. Munc18-1 mutations modulate the stability of the template complex in a manner consistent with their effects on membrane fusion, indicating that chaperoned SNARE assembly is essential for exocytosis. Two other SM proteins, Munc18-3 and Vps33, similarly chaperone SNARE assembly via a template complex, suggesting that SM protein mechanism is conserved.

scholarly journals N-terminal domain of complexin independently activates calcium-triggered fusion

2016 ◽  
Vol 113 (32) ◽  
pp. E4698-E4707 ◽  
Author(s):  
Ying Lai ◽  
Ucheor B. Choi ◽  
Yunxiang Zhang ◽  
Minglei Zhao ◽  
Richard A. Pfuetzner ◽  
...  
Keyword(s):  
Membrane Binding ◽  
Snare Complex ◽  
Cooperative Interaction ◽  
Binding Property ◽  
Triggered Release ◽  
Terminal Domain ◽  
Influenza Virus Hemagglutinin ◽  
Sensitive Factor ◽  
Protein Receptors ◽  
Single Vesicle

Complexin activates Ca2+-triggered neurotransmitter release and regulates spontaneous release in the presynaptic terminal by cooperating with the neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and the Ca2+-sensor synaptotagmin. The N-terminal domain of complexin is important for activation, but its molecular mechanism is still poorly understood. Here, we observed that a split pair of N-terminal and central domain fragments of complexin is sufficient to activate Ca2+-triggered release using a reconstituted single-vesicle fusion assay, suggesting that the N-terminal domain acts as an independent module within the synaptic fusion machinery. The N-terminal domain can also interact independently with membranes, which is enhanced by a cooperative interaction with the neuronal SNARE complex. We show by mutagenesis that membrane binding of the N-terminal domain is essential for activation of Ca2+-triggered fusion. Consistent with the membrane-binding property, the N-terminal domain can be substituted by the influenza virus hemagglutinin fusion peptide, and this chimera also activates Ca2+-triggered fusion. Membrane binding of the N-terminal domain of complexin therefore cooperates with the other fusogenic elements of the synaptic fusion machinery during Ca2+-triggered release.

The role of the C-terminus and Kpn loop in the quaternary structure stability of nucleoside diphosphate kinase from Leishmania parasites

2015 ◽  
Vol 192 (3) ◽  
pp. 336-341 ◽  
Author(s):  
Plínio Salmazo Vieira ◽  
Priscila Oliveira de Giuseppe ◽  
Arthur Henrique Cavalcante de Oliveira ◽  
Mario Tyago Murakami
Keyword(s):  
Quaternary Structure ◽  
Nucleoside Diphosphate Kinase ◽  
Structure Stability ◽  
C Terminus ◽  
Leishmania Parasites

scholarly journals Apparent lack of physical or functional interaction between CaV1.1 and its distal C terminus

2015 ◽  
Vol 145 (4) ◽  
pp. 303-314 ◽  
Author(s):  
Joshua D. Ohrtman ◽  
Christin F. Romberg ◽  
Ong Moua ◽  
Roger A. Bannister ◽  
S. Rock Levinson ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Channel Activity ◽  
Functional Interaction ◽  
Adult Skeletal Muscle ◽  
Apparent Lack ◽  
Terminal Domain ◽  
C Terminus ◽  
Decay Times

CaV1.1 acts as both the voltage sensor that triggers excitation–contraction coupling in skeletal muscle and as an L-type Ca2+ channel. It has been proposed that, after its posttranslational cleavage, the distal C terminus of CaV1.1 remains noncovalently associated with proximal CaV1.1, and that tethering of protein kinase A to the distal C terminus is required for depolarization-induced potentiation of L-type Ca2+ current in skeletal muscle. Here, we report that association of the distal C terminus with proximal CaV1.1 cannot be detected by either immunoprecipitation of mouse skeletal muscle or by colocalized fluorescence after expression in adult skeletal muscle fibers of a CaV1.1 construct labeled with yellow fluorescent protein (YFP) and cyan fluorescent protein on the N and C termini, respectively. We found that L-type Ca2+ channel activity was similar after expression of constructs that either did (YFP-CaV1.11860) or did not (YFP-CaV1.11666) contain coding sequence for the distal C-terminal domain in dysgenic myotubes null for endogenous CaV1.1. Furthermore, in response to strong (up to 90 mV) or long-lasting prepulses (up to 200 ms), tail current amplitudes and decay times were equally increased in dysgenic myotubes expressing either YFP-CaV1.11860 or YFP-CaV1.11666, suggesting that the distal C-terminal domain was not required for depolarization-induced potentiation. Thus, our experiments do not support the existence of either biochemical or functional interactions between proximal CaV1.1 and the distal C terminus.

scholarly journals Studying Binding, Conformational Transition and Assembly of E. Coli Cytolysin a Pore Forming Toxin by Single Molecule Fluorescence

2017 ◽  
Vol 112 (3) ◽  
pp. 524a
Author(s):  
Pradeep Sathyanarayana ◽  
Satyaghosh Maurya ◽  
Ganapathy Ayappa ◽  
Sandhya S. Visweswariah ◽  
Rahul Roy
Keyword(s):  
Single Molecule ◽  
Conformational Transition ◽  
Single Molecule Fluorescence ◽  
E Coli

scholarly journals Crystal structure of the N-terminal domain of VqsR fromPseudomonas aeruginosaat 2.1 Å resolution

2017 ◽  
Vol 73 (7) ◽  
pp. 431-436
Author(s):  
Qing He ◽  
Kang Wang ◽  
Tiantian Su ◽  
Feng Wang ◽  
Lichuan Gu ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Sequence Similarity ◽  
Transcriptional Regulator ◽  
Binding Domain ◽  
Structural Comparison ◽  
Homoserine Lactones ◽  
Terminal Domain ◽  
C Terminus ◽  
Common Motif ◽  
The Common

VqsR is a quorum-sensing (QS) transcriptional regulator which controls QS systems (las,rhlandpqs) by directly downregulating the expression ofqscRinPseudomonas aeruginosa. As a member of the LuxR family of proteins, VqsR shares the common motif of a helix–turn–helix (HTH)-type DNA-binding domain at the C-terminus, while the function of its N-terminal domain remains obscure. Here, the crystal structure of the N-terminal domain of VqsR (VqsR-N; residues 1–193) was determined at a resolution of 2.1 Å. The structure is folded into a regular α–β–α sandwich topology, which is similar to the ligand-binding domain (LBD) of the LuxR-type QS receptors. Although their sequence similarity is very low, structural comparison reveals that VqsR-N has a conserved enclosed cavity which could recognize acyl-homoserine lactones (AHLs) as in other LuxR-type AHL receptors. The structure suggests that VqsR could be a potential AHL receptor.

scholarly journals Cell surface-mediated activation of progelatinase A: demonstration of the involvement of the C-terminal domain of progelatinase A in cell surface binding and activation of progelatinase A by primary fibroblasts

1994 ◽  
Vol 304 (1) ◽  
pp. 263-269 ◽  
Author(s):  
R V Ward ◽  
S J Atkinson ◽  
J J Reynolds ◽  
G Murphy
Keyword(s):  
Cell Surface ◽  
Concanavalin A ◽  
Plasma Membranes ◽  
Scatchard Analysis ◽  
Gelatinase A ◽  
Surface Binding ◽  
Binding Studies ◽  
Cell Surface Binding ◽  
Terminal Domain ◽  
Primary Fibroblasts

We report that the isolated C-terminal domain of progelatinase A is inhibitory to the activation of this proenzyme by primary skin fibroblast plasma membranes but is unable to inhibit organomercurial-induced self-cleavage and activation. Ligand binding studies demonstrate that fibroblasts stimulated with concanavalin A to activate progelatinase A have a significantly enhanced level of cell surface-associated progelatinase A. Tissue inhibitor of metalloproteinases-2 (TIMP-2), an effective inhibitor of membrane-mediated progelatinase A activation, is able to abolish the enhanced level of cell surface-associated progelatinase A that occurs following stimulation. TIMP-1, a poor inhibitor of membrane activation, is unable to inhibit the cell surface binding of progelatinase A. The enhancement in the binding of 125I-progelatinase A to fibroblasts following concanavalin A stimulation can be blocked by the inclusion of excess C-terminal gelatinase A but not by a truncated form of gelatinase A lacking the C-terminal domain. Scatchard analysis of the binding of 125I-progelatinase A to concanavalin A-stimulated fibroblasts has identified 950,000 gelatinase binding sites per cell with a Kd of 1.3 x 10(-8) M. Analysis of non-stimulated fibroblasts has identified 500,000 sites per cell with a Kd of 2.6 x 10(-8) M. We propose that membrane-mediated activation of progelatinase A involves binding of the proenzyme through its C-terminal domain to the cell surface and that TIMP-2 can inhibit activation by interaction with progelatinase A through the C-terminal domain, thus preventing binding of the proenzyme.

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