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Modification of a hydrophobic layer by a point mutation in syntaxin 1A regulates the rate of synaptic vesicle fusion.

Lagow RD, Bao H, Cohen EN, Daniels RW, Zuzek A, Williams WH, Macleod GT, Sutton RB, Zhang B - PLoS Biol. (2007)

Bottom Line: At present, little is known about how the SNARE complexes mediating these two distinct pathways differ in structure.Syntaxin 1A molecules share a highly conserved threonine in the C-terminal +7 layer near the transmembrane domain.Mutation of this threonine to isoleucine results in a structural change that more closely resembles those found in syntaxins ascribed to the constitutive secretory pathway.

View Article: PubMed Central - PubMed

Affiliation: Section of Neurobiology and Institute for Neuroscience, University of Texas at Austin, Austin, Texas, United States of America.

ABSTRACT
Both constitutive secretion and Ca(2+)-regulated exocytosis require the assembly of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes. At present, little is known about how the SNARE complexes mediating these two distinct pathways differ in structure. Using the Drosophila neuromuscular synapse as a model, we show that a mutation modifying a hydrophobic layer in syntaxin 1A regulates the rate of vesicle fusion. Syntaxin 1A molecules share a highly conserved threonine in the C-terminal +7 layer near the transmembrane domain. Mutation of this threonine to isoleucine results in a structural change that more closely resembles those found in syntaxins ascribed to the constitutive secretory pathway. Flies carrying the I254 mutant protein have increased levels of SNARE complexes and dramatically enhanced rate of both constitutive and evoked vesicle fusion. In contrast, overexpression of the T254 wild-type protein in neurons reduces vesicle fusion only in the I254 mutant background. These results are consistent with molecular dynamics simulations of the SNARE core complex, suggesting that T254 serves as an internal brake to dampen SNARE zippering and impede vesicle fusion, whereas I254 favors fusion by enhancing intermolecular interaction within the SNARE core complex.

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Structural Modeling Suggests That the T254I Mutation in Syntaxin 1A Increases Direct Molecular Interactions within the +7 Layer(A) The core complex layers of the synaptic SNARE complex (1SFFC), consisting of two α-helical bundles from SNAP-25 (SN1 and SN2) and one bundle each from syntaxin 1A (Syx) and synaptobrevin (Syb), are shown (adapted from [6]). Although initially obtained as cis complexes with truncated SNAREs [6], these layers of the core complex are most likely found in pre-fusion trans SNARE complexes.(B) Crystal structures of +1 and +7 layers of the synaptic core complex (1SFC [6]) show tightly and loosely packed bundles, respectively. Note the void space within the +7 layer. Our structural modeling shows that the mutation of the hydrophilic threonine at position 251 (which is equivalent to position 254 in Drosophila syntaxin 1A) to a hydrophobic isoleucine results in a relatively tightly packed +7 layer. This may allow direct molecular interactions between syntaxin 1A with its neighboring bundles from SNAP-25 and synaptobrevin. It is hypothesized that the T254I mutation in syx3–69 stimulates vesicle fusion by lowering the energy barrier for zippering of the SNARE complex.(C) Representative +7 layer abstracted from the crystal structure of the endosomal SNARE (1GL2 [20]). Note that this layer is tightly packed and similar to the T251I mutant layer. Given the evolutionary conservation of hydrophobic residues at the +7 layer among “constitutive” syntaxin orthologs (Figure 1B), this structural resemblance suggests that the T254I mutant syntaxin 1A may function as a constitutive syntaxin to promote vesicle fusion.
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pbio-0050072-g005: Structural Modeling Suggests That the T254I Mutation in Syntaxin 1A Increases Direct Molecular Interactions within the +7 Layer(A) The core complex layers of the synaptic SNARE complex (1SFFC), consisting of two α-helical bundles from SNAP-25 (SN1 and SN2) and one bundle each from syntaxin 1A (Syx) and synaptobrevin (Syb), are shown (adapted from [6]). Although initially obtained as cis complexes with truncated SNAREs [6], these layers of the core complex are most likely found in pre-fusion trans SNARE complexes.(B) Crystal structures of +1 and +7 layers of the synaptic core complex (1SFC [6]) show tightly and loosely packed bundles, respectively. Note the void space within the +7 layer. Our structural modeling shows that the mutation of the hydrophilic threonine at position 251 (which is equivalent to position 254 in Drosophila syntaxin 1A) to a hydrophobic isoleucine results in a relatively tightly packed +7 layer. This may allow direct molecular interactions between syntaxin 1A with its neighboring bundles from SNAP-25 and synaptobrevin. It is hypothesized that the T254I mutation in syx3–69 stimulates vesicle fusion by lowering the energy barrier for zippering of the SNARE complex.(C) Representative +7 layer abstracted from the crystal structure of the endosomal SNARE (1GL2 [20]). Note that this layer is tightly packed and similar to the T251I mutant layer. Given the evolutionary conservation of hydrophobic residues at the +7 layer among “constitutive” syntaxin orthologs (Figure 1B), this structural resemblance suggests that the T254I mutant syntaxin 1A may function as a constitutive syntaxin to promote vesicle fusion.

Mentions: To account for the hyperactivity observed in syx3–69 flies, we next examined whether the T254I mutation in syntaxin 1A has any effect on SNARE assembly and synaptic function at permissive temperatures. Upon examination of the available crystal structures of SNARE core complexes [6], we found that many of the central layers are tightly packed with hydrophobic residues contained within the four helical bundles. An example of this tight packing in the +1 layer of the synaptic SNARE core complex is illustrated in Figure 5A and 5B. The interactions of Leu57 and Ile178 from SNAP-25, Ile230 from syntaxin 1A, and Leu60 from synaptobrevin form square-planar geometry typical of the leucine zipper motif. In contrast, the +7 layer containing the wild-type syntaxin 1A is relatively loosely packed due to the presence of a conserved polar threonine residue at position 251 (equivalent to position 254 in Drosophila syntaxin 1A) [6,7,21], which packs against more hydrophobic partners. Results of examination of homologous neuronal SNARE syntaxin proteins implied a similar loosely packed configuration in this layer [7]. Interestingly, the homologous layer of the endosomal SNARE X-ray structure (1GL2) [20] shows more reliance on hydrophobic, branched-chain amino acids, than the synaptic SNARE (Figure 5C). The resulting interaction may contribute more hydrophobic stability of the zippered endosomal complex relative to the wild-type synaptic SNARE complex. We therefore propose that the tightened +7 layer in the SNARE complex containing the T254I mutant syntaxin 1A may mimic the function of the endosomal complex.


Modification of a hydrophobic layer by a point mutation in syntaxin 1A regulates the rate of synaptic vesicle fusion.

Lagow RD, Bao H, Cohen EN, Daniels RW, Zuzek A, Williams WH, Macleod GT, Sutton RB, Zhang B - PLoS Biol. (2007)

Structural Modeling Suggests That the T254I Mutation in Syntaxin 1A Increases Direct Molecular Interactions within the +7 Layer(A) The core complex layers of the synaptic SNARE complex (1SFFC), consisting of two α-helical bundles from SNAP-25 (SN1 and SN2) and one bundle each from syntaxin 1A (Syx) and synaptobrevin (Syb), are shown (adapted from [6]). Although initially obtained as cis complexes with truncated SNAREs [6], these layers of the core complex are most likely found in pre-fusion trans SNARE complexes.(B) Crystal structures of +1 and +7 layers of the synaptic core complex (1SFC [6]) show tightly and loosely packed bundles, respectively. Note the void space within the +7 layer. Our structural modeling shows that the mutation of the hydrophilic threonine at position 251 (which is equivalent to position 254 in Drosophila syntaxin 1A) to a hydrophobic isoleucine results in a relatively tightly packed +7 layer. This may allow direct molecular interactions between syntaxin 1A with its neighboring bundles from SNAP-25 and synaptobrevin. It is hypothesized that the T254I mutation in syx3–69 stimulates vesicle fusion by lowering the energy barrier for zippering of the SNARE complex.(C) Representative +7 layer abstracted from the crystal structure of the endosomal SNARE (1GL2 [20]). Note that this layer is tightly packed and similar to the T251I mutant layer. Given the evolutionary conservation of hydrophobic residues at the +7 layer among “constitutive” syntaxin orthologs (Figure 1B), this structural resemblance suggests that the T254I mutant syntaxin 1A may function as a constitutive syntaxin to promote vesicle fusion.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC1808484&req=5

pbio-0050072-g005: Structural Modeling Suggests That the T254I Mutation in Syntaxin 1A Increases Direct Molecular Interactions within the +7 Layer(A) The core complex layers of the synaptic SNARE complex (1SFFC), consisting of two α-helical bundles from SNAP-25 (SN1 and SN2) and one bundle each from syntaxin 1A (Syx) and synaptobrevin (Syb), are shown (adapted from [6]). Although initially obtained as cis complexes with truncated SNAREs [6], these layers of the core complex are most likely found in pre-fusion trans SNARE complexes.(B) Crystal structures of +1 and +7 layers of the synaptic core complex (1SFC [6]) show tightly and loosely packed bundles, respectively. Note the void space within the +7 layer. Our structural modeling shows that the mutation of the hydrophilic threonine at position 251 (which is equivalent to position 254 in Drosophila syntaxin 1A) to a hydrophobic isoleucine results in a relatively tightly packed +7 layer. This may allow direct molecular interactions between syntaxin 1A with its neighboring bundles from SNAP-25 and synaptobrevin. It is hypothesized that the T254I mutation in syx3–69 stimulates vesicle fusion by lowering the energy barrier for zippering of the SNARE complex.(C) Representative +7 layer abstracted from the crystal structure of the endosomal SNARE (1GL2 [20]). Note that this layer is tightly packed and similar to the T251I mutant layer. Given the evolutionary conservation of hydrophobic residues at the +7 layer among “constitutive” syntaxin orthologs (Figure 1B), this structural resemblance suggests that the T254I mutant syntaxin 1A may function as a constitutive syntaxin to promote vesicle fusion.
Mentions: To account for the hyperactivity observed in syx3–69 flies, we next examined whether the T254I mutation in syntaxin 1A has any effect on SNARE assembly and synaptic function at permissive temperatures. Upon examination of the available crystal structures of SNARE core complexes [6], we found that many of the central layers are tightly packed with hydrophobic residues contained within the four helical bundles. An example of this tight packing in the +1 layer of the synaptic SNARE core complex is illustrated in Figure 5A and 5B. The interactions of Leu57 and Ile178 from SNAP-25, Ile230 from syntaxin 1A, and Leu60 from synaptobrevin form square-planar geometry typical of the leucine zipper motif. In contrast, the +7 layer containing the wild-type syntaxin 1A is relatively loosely packed due to the presence of a conserved polar threonine residue at position 251 (equivalent to position 254 in Drosophila syntaxin 1A) [6,7,21], which packs against more hydrophobic partners. Results of examination of homologous neuronal SNARE syntaxin proteins implied a similar loosely packed configuration in this layer [7]. Interestingly, the homologous layer of the endosomal SNARE X-ray structure (1GL2) [20] shows more reliance on hydrophobic, branched-chain amino acids, than the synaptic SNARE (Figure 5C). The resulting interaction may contribute more hydrophobic stability of the zippered endosomal complex relative to the wild-type synaptic SNARE complex. We therefore propose that the tightened +7 layer in the SNARE complex containing the T254I mutant syntaxin 1A may mimic the function of the endosomal complex.

Bottom Line: At present, little is known about how the SNARE complexes mediating these two distinct pathways differ in structure.Syntaxin 1A molecules share a highly conserved threonine in the C-terminal +7 layer near the transmembrane domain.Mutation of this threonine to isoleucine results in a structural change that more closely resembles those found in syntaxins ascribed to the constitutive secretory pathway.

View Article: PubMed Central - PubMed

Affiliation: Section of Neurobiology and Institute for Neuroscience, University of Texas at Austin, Austin, Texas, United States of America.

ABSTRACT
Both constitutive secretion and Ca(2+)-regulated exocytosis require the assembly of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes. At present, little is known about how the SNARE complexes mediating these two distinct pathways differ in structure. Using the Drosophila neuromuscular synapse as a model, we show that a mutation modifying a hydrophobic layer in syntaxin 1A regulates the rate of vesicle fusion. Syntaxin 1A molecules share a highly conserved threonine in the C-terminal +7 layer near the transmembrane domain. Mutation of this threonine to isoleucine results in a structural change that more closely resembles those found in syntaxins ascribed to the constitutive secretory pathway. Flies carrying the I254 mutant protein have increased levels of SNARE complexes and dramatically enhanced rate of both constitutive and evoked vesicle fusion. In contrast, overexpression of the T254 wild-type protein in neurons reduces vesicle fusion only in the I254 mutant background. These results are consistent with molecular dynamics simulations of the SNARE core complex, suggesting that T254 serves as an internal brake to dampen SNARE zippering and impede vesicle fusion, whereas I254 favors fusion by enhancing intermolecular interaction within the SNARE core complex.

Show MeSH
Related in: MedlinePlus