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Dynamic binding mode of a Synaptotagmin-1–SNARE complex in solution

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Brewer, Kyle D, Bacaj, Taulant, Cavalli, Andrea, Camilloni, Carlo, Swarbrick, James D, Liu, Jin, Zhou, Amy, Zhou, Peng +12 more·2015·Nature Structural & Molecular Biology
DOI 10.1038/nsmb.3035PMC PMC4496268
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Abstract

Rapid neurotransmitter release depends on the Ca2+ sensor Synaptotagmin-1 (Syt1) and the SNARE complex formed by synaptobrevin, syntaxin-1 and SNAP-25. How Syt1 triggers release has been unclear, partly because elucidating high-resolution structures of Syt1-SNARE complexes has been challenging. An NMR approach based on lanthanide-induced pseudocontact shifts now reveals a dynamic binding mode in which basic residues in the concave side of the Syt1 C2B-domain β-sandwich interact with a polyacidic region of the SNARE complex formed by syntaxin-1 and SNAP-25. The physiological relevance of this dynamic structural model is supported by mutations in basic residues of Syt1 that markedly impair SNARE-complex binding in vitro and Syt1 function in neurons. Mutations with milder effects on binding have correspondingly milder effects on Syt1 function. Our results support a model whereby dynamic interaction facilitates cooperation between Syt1 and the SNAREs in inducing membrane fusion.

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  • A mutant affecting the polybasic region of Syt1 (R322E, K325E) exhibited a milder phenotype.

    “In contrast, a mutant that affects the polybasic region of Syt1 (R322E, K325E) exhibited a milder phenotype as reported previously30.”

    From Architecture of the synaptotagmin–SNARE machinery for neuronal exocytosis (2015) · cites this paper as [30]

    The Results section (‘Correlation of Syt1-SNARE complex binding with Syt1 function’; page unavailable) identifies R322E K325E as a polybasic-region double mutant and describes its impairment of neurotransmitter release as partial. It also says this mutant strongly impaired rescue of evoked release, while other double mutants had much milder effects. The source therefore supports an impairment, but not the unqualified description of this mutant’s phenotype as milder. (LLM, conf=0.82)

    Experimental — accuracy not yet measured

  • Syt1 interacts with the neuronal SNARE complex.

    “Syt1 also interacts with the neuronal SNARE complex based on immunoprecipitation and pull-downs24–27, single molecule fluorescence resonance energy transfer (smFRET)28, and nuclear magnetic resonance29,30 experiments.”

    From Architecture of the synaptotagmin–SNARE machinery for neuronal exocytosis (2015) · cites this paper as [30]

  • Covalent lanthanide labels attached to SNAP-25 residues 41 and 166 would likely disrupt the primary interface, so the crystal structures cannot be compared with that NMR study.

    “In a recent study, lanthanide labels were covalently attached to SNAP-25 residues 41 and 166 for pseudocontact chemical shift NMR measurements in the presence of 125 thiocyanate30; these covalent labels would likely disrupt the primary interface (Fig. 2c), so it is not possible to compare our crystal structures with this NMR study.”

    From Architecture of the synaptotagmin–SNARE machinery for neuronal exocytosis (2015) · cites this paper as [30]

  • The effect of the Syt1 R322E, K325E mutant and reported observations suggest a functional role for the polybasic region.

    “The effect of the Syt1 R322E, K325E mutant (Fig. 4) as well as reported observations29,30,35,44,45 suggests a functional role for the polybasic region.”

    From Architecture of the synaptotagmin–SNARE machinery for neuronal exocytosis (2015) · cites this paper as [30]

  • Other dynamic or approximate models of the C2AB-SNARE complex obtained by NMR cannot be readily compared with the previous smFRET studies and the results presented here because of differences in conditions, covalent attachment of lanthanide labels, and lack of atomic resolution.

    “Other dynamic or approximate models of the C2AB-SNARE complex were obtained by nuclear magnetic resonance (NMR)29,30, but cannot be readily compared with the previous smFRET studies28 and the results presented here because of differences in conditions, particular covalent attachment of lanthanide labels30, and lack of atomic resolution.”

    From Architecture of the synaptotagmin–SNARE machinery for neuronal exocytosis (2015) · cites this paper as [30]

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Authors
Brewer, K. D., Bacaj, T., Cavalli, A., Camilloni, C., Swarbrick, J. D., Liu, J., Zhou, A., Zhou, P., Barlow, N., Xu, J., Seven, A. B., Prinslow, E. A., Voleti, R., Häussinger, D., Bonvin, A. M. J. J., Tomchick, D. R., Vendruscolo, M., Graham, B., Südhof, T. C., & Rizo, J.
Affiliations
  • The University of Texas Southwestern Medical Center (US)
  • Howard Hughes Medical Institute (US)
  • University of Cambridge (GB)
  • Monash University (AU)
  • Southern Methodist University (US)
  • University of Basel (CH)
  • Utrecht University (NL)
Corresponding author
Rizo, Josep
ORCID
Venue
Nature Structural & Molecular Biology
Publisher
Springer Science and Business Media LLC
Published
2015-06-01
Volume
22
Issue
7
Pages
555-564
ISSN
  • 1545-9985 (ISSN-L)
  • 1545-9993
DOI
10.1038/nsmb.3035
PMC
PMC4496268
OpenAlex
W1913308306
Cited by
142
References
2
Citations by year
2026: 2 · 2025: 4 · 2024: 5 · 2023: 9 · 2022: 26 · 2021: 12 · 2020: 10 · 2019: 15 · 2018: 16 · 2017: 19 · 2016: 17 · 2015: 7
FWCI
10.48
Type
journal article
Language
en
Open access
Licence
www.springer.com/tdm (tdm)
Locations
  • Nature Structural & Molecular Biology (publishedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
  • Monash University Research Portal (Monash University) (submittedVersion)
  • Archivio Istituzionale della Ricerca (Universita Degli Studi Di Milano) (submittedVersion, other-oa)
  • Utrecht University Repository (Utrecht University) (submittedVersion, other-oa)
Funders
  • National Science Foundation (S10RR026461)
  • Welch Foundation
  • University of Texas Southwestern Medical Center
  • University of Texas at Austin
  • Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (130263, 200021)
  • National Institutes of Health
  • Australian Research Council (FT130100838, DP150100383)
  • National Institute of General Medical Sciences (T32GM008297)
  • National Institute of Neurological Disorders and Stroke (K99NS087086, NS040944, R01NS040944)
  • National Center for Research Resources (S10RR026461)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Lipid Membrane Structure and Behavior
Keywords
synaptotagmin-1, SNARE complex, neurotransmitter release, C2B domain, syntaxin-1, SNAP-25, synaptobrevin, calcium sensor, membrane fusion, basic residues, pseudocontact shifts
MeSH terms
Animals, Cells, Cultured, Humans, Mice, Inbred C57BL, Models, Molecular, Neurons, Protein Binding, Protein Structure, Tertiary, Nuclear Magnetic Resonance, Biomolecular, SNARE Proteins, Synaptotagmin I, Rats

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