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NMR analysis of the closed conformation of syntaxin-1

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Abstract

The Sec1/Munc18 (SM) protein Munc18-1 and the SNAREs syntaxin-1, SNAP-25 and synaptobrevin form the core of the membrane fusion machinery that triggers neurotransmitter release. Munc18-1 binds to syntaxin-1 folded into a closed conformation and to the SNARE complex formed by the three SNAREs, which involves an open syntaxin-1 conformation. The former interaction is likely specialized for neurotransmitter release, whereas SM protein/SNARE complex interactions are likely key for all types of intracellular membrane fusion. It is currently unclear whether the closed conformation is highly or only marginally populated in isolated syntaxin-1, and whether Munc18-1 stabilizes the close conformation or helps to open it to facilitate SNARE complex formation. A detailed NMR analysis now suggests that the closed conformation is almost quantitatively populated in isolated syntaxin-1 in the absence of oligomerization, and indicates that its structure is very similar to that observed previously in the crystal structure of the Munc18-1/syntaxin-1 complex. Moreover, we demonstrate that Munc18-1 binding prevents opening of the syntaxin-1 closed conformation. These results support a model whereby the closed conformation constitutes a key intrinsic property of isolated syntaxin-1 and Munc18-1 binding stabilizes this conformation; in this model, Munc18-1 plays in addition an active role in downstream events after another factor(s) helps to open the syntaxin-1 conformation.

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Abbreviations

COSY:

Correlation spectroscopy

FRET:

Fluorescence resonance energy transfer

HSQC:

Heteronuclear single quantum coherence

NOESY:

Nuclear Overhauser effect spectroscopy

NSF:

N-ethylmaleimide sensitive factor

SM protein:

Sec1/Munc18 protein

SNAP:

Soluble NSF attachment protein

SNAP-25:

Synaptosomal associated protein of 25 kDa

SNARE:

SNAP receptor

TMAO:

Trimethylamine N-oxide

TOCSY:

Total correlation spectroscopy

TROSY:

Transverse relaxation optimized spectroscopy

VAMP:

Vesicle associated membrane protein

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Acknowledgements

We thank Josep Ubach, Imma Fernandez and Iryna Huryeva for preparation of recombinant proteins, Lewis Kay for providing all pulse sequences used in this study, and Thomas Südhof for fruitful discussions. This work was supported by grant I-1304 from the Welch Foundation and NIH grant NS37200 (to J.R.).

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Author notes

  1. Jun Lu

    Present address: Merck & C. Inc., 126 E. Lincoln Ave, P.O. Box 2000, Rahway, NJ, 07065, USA

  2. Irina Dulubova

    Present address: Reata Pharmaceuticals, Inc., 2801 Gateway Drive # 150, Irving, TX, 75063, USA

Authors and Affiliations

  1. Departments of Biochemistry and Pharmacology, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, TX, 75390, USA

    Xiaocheng Chen, Jun Lu, Irina Dulubova & Josep Rizo

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  1. Xiaocheng Chen
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  2. Jun Lu
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Correspondence to Josep Rizo.

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Chen, X., Lu, J., Dulubova, I. et al. NMR analysis of the closed conformation of syntaxin-1. J Biomol NMR 41, 43–54 (2008). https://doi.org/10.1007/s10858-008-9239-1

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