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

Brewer KD, Bacaj T, Cavalli A, Camilloni C, Swarbrick JD, Liu J, Zhou A, Zhou P, Barlow N, Xu J, Seven AB, Prinslow EA, Voleti R, Häussinger D, Bonvin AM, Tomchick DR, Vendruscolo M, Graham B, Südhof TC, Rizo J - Nat. Struct. Mol. Biol. (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, Texas, USA. [2] Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas, USA. [3] Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

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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Disruption of Syt1 function correlates with impairment of Syt1-SNARE complex binding. (a,d) Sample traces of evoked inhibitory postsynaptic currents (eIPSCs) observed in cultured Syt1 KO neurons without or with lentiviral expression of wild type (WT) or mutant Syt1 as indicated. Stimulus onset is indicated by the tick mark. (b,c,e,f) Summary graphs of the eIPSC amplitudes and charge transfers observed in the rescue experiments with WT and mutant Syt1. (g,j) Sample traces of spontaneous release in excitatory (g) or inhibitory (j) neurons from Syt1 KO mice without or with lentiviral expression of WT Syt1 or selected Syt1 double mutants as indicated. (h,i,k,l) Summary graphs of spontaneous miniature EPSC (mEPSC) (h,i) and mIPSC (k,l) frequencies and amplitudes. All data are means ± SEM; numbers in bars indicate number of neurons/independent cultures analyzed. Statistical significance was assessed by one-way ANOVA (***, p<0.001; n.s., not significant).
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Figure 7: Disruption of Syt1 function correlates with impairment of Syt1-SNARE complex binding. (a,d) Sample traces of evoked inhibitory postsynaptic currents (eIPSCs) observed in cultured Syt1 KO neurons without or with lentiviral expression of wild type (WT) or mutant Syt1 as indicated. Stimulus onset is indicated by the tick mark. (b,c,e,f) Summary graphs of the eIPSC amplitudes and charge transfers observed in the rescue experiments with WT and mutant Syt1. (g,j) Sample traces of spontaneous release in excitatory (g) or inhibitory (j) neurons from Syt1 KO mice without or with lentiviral expression of WT Syt1 or selected Syt1 double mutants as indicated. (h,i,k,l) Summary graphs of spontaneous miniature EPSC (mEPSC) (h,i) and mIPSC (k,l) frequencies and amplitudes. All data are means ± SEM; numbers in bars indicate number of neurons/independent cultures analyzed. Statistical significance was assessed by one-way ANOVA (***, p<0.001; n.s., not significant).

Mentions: To investigate the functional relevance of the Syt1-SNARE complex binding mode described above, we performed electrophysiological rescue experiments. As previously described49, lentiviral expression of WT Syt1 rescued evoked release in Syt1 KO neurons (Figs. 7a–c). Three single Syt1 mutants with substitutions in residues from the polybasic region (K313E, R322E and K326E) rescued evoked release almost as efficiently as WT Syt1 (Figs. 7a–c), in correlation with the finding that such mutations do not markedly impair C2AB-SNARE complex binding (Supplementary Figs. 6a,b). Importantly however, two double mutations in the concave side of C2B (K313E K325E and R322E K325E) strongly impaired rescue of evoked release in Syt1 KO neurons, whereas much milder effects were observed for the control double mutant (K354E R388E) and the mutant with substitutions in residues of the polybasic region that are not in the concave side (K324E K326E) (Figs. 7d–f). These differences in rescue activities do not arise from inefficient protein overexpression, as all double mutants were overexpressed at similar levels (Supplementary Fig. 7). These results establish a striking correlation between the disruption of Syt1 function in neurons and the impairment of C2AB-SNARE complex binding (Fig. 6b) caused by the double mutations.


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

Brewer KD, Bacaj T, Cavalli A, Camilloni C, Swarbrick JD, Liu J, Zhou A, Zhou P, Barlow N, Xu J, Seven AB, Prinslow EA, Voleti R, Häussinger D, Bonvin AM, Tomchick DR, Vendruscolo M, Graham B, Südhof TC, Rizo J - Nat. Struct. Mol. Biol. (2015)

Disruption of Syt1 function correlates with impairment of Syt1-SNARE complex binding. (a,d) Sample traces of evoked inhibitory postsynaptic currents (eIPSCs) observed in cultured Syt1 KO neurons without or with lentiviral expression of wild type (WT) or mutant Syt1 as indicated. Stimulus onset is indicated by the tick mark. (b,c,e,f) Summary graphs of the eIPSC amplitudes and charge transfers observed in the rescue experiments with WT and mutant Syt1. (g,j) Sample traces of spontaneous release in excitatory (g) or inhibitory (j) neurons from Syt1 KO mice without or with lentiviral expression of WT Syt1 or selected Syt1 double mutants as indicated. (h,i,k,l) Summary graphs of spontaneous miniature EPSC (mEPSC) (h,i) and mIPSC (k,l) frequencies and amplitudes. All data are means ± SEM; numbers in bars indicate number of neurons/independent cultures analyzed. Statistical significance was assessed by one-way ANOVA (***, p<0.001; n.s., not significant).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4496268&req=5

Figure 7: Disruption of Syt1 function correlates with impairment of Syt1-SNARE complex binding. (a,d) Sample traces of evoked inhibitory postsynaptic currents (eIPSCs) observed in cultured Syt1 KO neurons without or with lentiviral expression of wild type (WT) or mutant Syt1 as indicated. Stimulus onset is indicated by the tick mark. (b,c,e,f) Summary graphs of the eIPSC amplitudes and charge transfers observed in the rescue experiments with WT and mutant Syt1. (g,j) Sample traces of spontaneous release in excitatory (g) or inhibitory (j) neurons from Syt1 KO mice without or with lentiviral expression of WT Syt1 or selected Syt1 double mutants as indicated. (h,i,k,l) Summary graphs of spontaneous miniature EPSC (mEPSC) (h,i) and mIPSC (k,l) frequencies and amplitudes. All data are means ± SEM; numbers in bars indicate number of neurons/independent cultures analyzed. Statistical significance was assessed by one-way ANOVA (***, p<0.001; n.s., not significant).
Mentions: To investigate the functional relevance of the Syt1-SNARE complex binding mode described above, we performed electrophysiological rescue experiments. As previously described49, lentiviral expression of WT Syt1 rescued evoked release in Syt1 KO neurons (Figs. 7a–c). Three single Syt1 mutants with substitutions in residues from the polybasic region (K313E, R322E and K326E) rescued evoked release almost as efficiently as WT Syt1 (Figs. 7a–c), in correlation with the finding that such mutations do not markedly impair C2AB-SNARE complex binding (Supplementary Figs. 6a,b). Importantly however, two double mutations in the concave side of C2B (K313E K325E and R322E K325E) strongly impaired rescue of evoked release in Syt1 KO neurons, whereas much milder effects were observed for the control double mutant (K354E R388E) and the mutant with substitutions in residues of the polybasic region that are not in the concave side (K324E K326E) (Figs. 7d–f). These differences in rescue activities do not arise from inefficient protein overexpression, as all double mutants were overexpressed at similar levels (Supplementary Fig. 7). These results establish a striking correlation between the disruption of Syt1 function in neurons and the impairment of C2AB-SNARE complex binding (Fig. 6b) caused by the double mutations.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, Texas, USA. [2] Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas, USA. [3] Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

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.

Show MeSH
Related in: MedlinePlus