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Prefusion structure of syntaxin-1A suggests pathway for folding into neuronal trans-SNARE complex fusion intermediate.

ABSTRACT: The assembly of the three neuronal soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) proteins synaptobrevin 2, syntaxin-1A, and SNAP-25 is the key step that leads to exocytotic fusion of synaptic vesicles. In the fully assembled SNARE complex, these three proteins form a coiled-coil four-helix bundle structure by interaction of their respective SNARE motifs. Although biochemical and mutational analyses strongly suggest that the heptad-repeat SNARE motifs zipper into the final structure, little is known about the prefusion state of individual membrane-bound SNAREs and how they change conformation from the unzippered prefusion to the zippered postfusion state in a membrane environment. We have solved the solution NMR structure of micelle-bound syntaxin-1A in its prefusion conformation. In addition to the transmembrane helix, the SNARE motif consists of two well-ordered, membrane-bound helices separated by the "0-layer" residue Gln226. This unexpected structural order of the N- and C-terminal halves of the uncomplexed SNARE motif suggests the formation of partially zippered SNARE complex intermediates, with the 0-layer serving as a proofreading site for correct SNARE assembly. Interferometric fluorescence measurements in lipid bilayers confirm that the open SNARE motif helices of syntaxin interact with lipid bilayers and that association with the other target-membrane SNARE SNAP-25 lifts the SNARE motif off the membrane as a critical prerequisite for SNARE complex assembly and membrane fusion.

SUBMITTER: Liang B

PROVIDER: S-EPMC3845119 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

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Prefusion structure of syntaxin-1A suggests pathway for folding into neuronal trans-SNARE complex fusion intermediate.

Liang Binyong B Kiessling Volker V Tamm Lukas K LK

Proceedings of the National Academy of Sciences of the United States of America 20131111 48

The assembly of the three neuronal soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) proteins synaptobrevin 2, syntaxin-1A, and SNAP-25 is the key step that leads to exocytotic fusion of synaptic vesicles. In the fully assembled SNARE complex, these three proteins form a coiled-coil four-helix bundle structure by interaction of their respective SNARE motifs. Although biochemical and mutational analyses strongly suggest that the heptad-repeat SNARE motifs zipper ...[more]

PMID: 24218570

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Project description:Chlamydia trachomatis, an obligate intracellular pathogen, grows inside of a vacuole, termed the inclusion. Within the inclusion, the organisms differentiate from the infectious elementary body (EB) into the reticulate body (RB). The RB communicates with the host cell through the inclusion membrane to obtain the nutrients necessary to divide, thus expanding the chlamydial population. At late time points within the developmental cycle, the RBs respond to unknown molecular signals to redifferentiate into infectious EBs to perpetuate the infection cycle. One strategy for Chlamydia to obtain necessary nutrients and metabolites from the host is to intercept host vesicular trafficking pathways. In this study we demonstrate that a trans-Golgi soluble N-ethylmaleimide-sensitive factor attachment protein (SNARE), syntaxin 10, and/or syntaxin 10-associated Golgi elements colocalize with the chlamydial inclusion. We hypothesized that Chlamydia utilizes the molecular machinery of syntaxin 10 at the inclusion membrane to intercept specific vesicular trafficking pathways in order to create and maintain an optimal intra-inclusion environment. To test this hypothesis, we used siRNA knockdown of syntaxin 10 to examine the impact of the loss of syntaxin 10 on chlamydial growth and development. Our results demonstrate that loss of syntaxin 10 leads to defects in normal chlamydial maturation including: variable inclusion size with fewer chlamydial organisms per inclusion, fewer infectious progeny, and delayed or halted RB-EB differentiation. These defects in chlamydial development correlate with an overabundance of NBD-lipid retained by inclusions cultured in syntaxin 10 knockdown cells. Overall, loss of syntaxin 10 at the inclusion membrane negatively affects Chlamydia. Understanding host machinery involved in maintaining an optimal inclusion environment to support chlamydial growth and development is critical toward understanding the molecular signals involved in successful progression through the chlamydial developmental cycle.

Dataset Information

Prefusion structure of syntaxin-1A suggests pathway for folding into neuronal trans-SNARE complex fusion intermediate.

Publications

Prefusion structure of syntaxin-1A suggests pathway for folding into neuronal trans-SNARE complex fusion intermediate.

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