Limits...
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.

Show MeSH

Related in: MedlinePlus

PCSs induced by the SC166Dy and SC41Dy on the Syt1 C2B domain. (a,b) 1H-13C HMQC spectra of 30 μM 15N,2H-ILV-13CH3-C2B R398Q R399Q mutant in the presence of 30 μM SC166Dy (a) or SC41Dy (b) before (red contours) or after (black contours) tag removal. (c,d) Ribbon diagrams of Syt1 C2B showing PCSs induced by SC166Dy (c) or SC41Dy (d). Amide hydrogens and methyl carbons are shown as spheres color-coded according to the measured PCSs (dark blue, > 0.06 ppm; blue, 0.04 to 0.06 ppm; cyan, 0.02 to 0.04 ppm; pale cyan, 0.008 to 0.02 ppm; red, −0.04 to −0.06 ppm; salmon, −0.02 to −0.04 ppm; light pink, −0.008 to −0.02 ppm). Yellow spheres represent Ca2+ ions. (e,f) Models of C2B bound to the SNARE complex built manually to match the C2B PCSs with the SC166 (e) and SC41 (f) tensors represented by isosurfaces as in Figs. 2c,f. (g–j) Correlations between experimental PCSs induced on C2B by SC166Dy (g,h) or SC41Dy (i,j) and PCSs calculated with the 166- and 41-manual models using the optimized SC166 (g) and SC41 (i) tensors (illustrated in Figs. 2c,f, respectively) or slightly modified tensors (h,j) (see Supplementary note 7). Correlation coefficients (r) and slopes (m) are indicated.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4496268&req=5

Figure 3: PCSs induced by the SC166Dy and SC41Dy on the Syt1 C2B domain. (a,b) 1H-13C HMQC spectra of 30 μM 15N,2H-ILV-13CH3-C2B R398Q R399Q mutant in the presence of 30 μM SC166Dy (a) or SC41Dy (b) before (red contours) or after (black contours) tag removal. (c,d) Ribbon diagrams of Syt1 C2B showing PCSs induced by SC166Dy (c) or SC41Dy (d). Amide hydrogens and methyl carbons are shown as spheres color-coded according to the measured PCSs (dark blue, > 0.06 ppm; blue, 0.04 to 0.06 ppm; cyan, 0.02 to 0.04 ppm; pale cyan, 0.008 to 0.02 ppm; red, −0.04 to −0.06 ppm; salmon, −0.02 to −0.04 ppm; light pink, −0.008 to −0.02 ppm). Yellow spheres represent Ca2+ ions. (e,f) Models of C2B bound to the SNARE complex built manually to match the C2B PCSs with the SC166 (e) and SC41 (f) tensors represented by isosurfaces as in Figs. 2c,f. (g–j) Correlations between experimental PCSs induced on C2B by SC166Dy (g,h) or SC41Dy (i,j) and PCSs calculated with the 166- and 41-manual models using the optimized SC166 (g) and SC41 (i) tensors (illustrated in Figs. 2c,f, respectively) or slightly modified tensors (h,j) (see Supplementary note 7). Correlation coefficients (r) and slopes (m) are indicated.

Mentions: To analyze the Syt1-SNARE complex binding mode, we used 15N,2H-labeled Syt1 fragments specifically 1H,13C-labeled at Ile, Leu and Val methyl groups (15N,2H-ILV-13CH3-labeling) for optimal relaxation properties36. Most of the PCSs induced by SC41Dy or SC166Dy on Syt1 C2AB were observed for cross-peaks from C2B, and very similar PCSs were observed using the isolated C2B domain, or the C2B domain with an R398Q R399Q mutation that hinders aggregation with the SNARE complex34 (Figs. 3a,b and Supplementary Figs. 2a,b,d,e; see section on Measurement of PCSs in Online Methods). Since the latter yielded the best-quality data and Arg398-Arg399 might mediate binding modes that promote aggregation, structural analyses were performed with PCS data obtained from 1H-15N TROSY-TROSY HSQC and 1H-13C heteronuclear multiple quantum coherence (HMQC) spectra of 15N,2H-ILV-13CH3-C2B domain bearing the R398Q R399Q mutation (below referred to as C2B for simplicity). These included 149 and 151 PCSs induced by SC41Dy or SC166Dy, respectively, on the C2B domain. C2B binding did not affect the PCSs within SC166Dy but caused slight alterations of PCSs within SC41Dy (5–20%; Supplementary Figs. 2f,g), showing that C2B does not contact residue 166 but is close to residue 41. Nevertheless, we still analyzed the SC41Dy data to examine the consistency with the SC166Dy data. We also note that addition of CpxI caused no or very small changes in the PCSs induced by SC166Dy (Supplementary Fig. 2c), indicating that the binding sites for Syt1 and CpxI on the SNARE complex in solution are distinct but proximal.


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)

PCSs induced by the SC166Dy and SC41Dy on the Syt1 C2B domain. (a,b) 1H-13C HMQC spectra of 30 μM 15N,2H-ILV-13CH3-C2B R398Q R399Q mutant in the presence of 30 μM SC166Dy (a) or SC41Dy (b) before (red contours) or after (black contours) tag removal. (c,d) Ribbon diagrams of Syt1 C2B showing PCSs induced by SC166Dy (c) or SC41Dy (d). Amide hydrogens and methyl carbons are shown as spheres color-coded according to the measured PCSs (dark blue, > 0.06 ppm; blue, 0.04 to 0.06 ppm; cyan, 0.02 to 0.04 ppm; pale cyan, 0.008 to 0.02 ppm; red, −0.04 to −0.06 ppm; salmon, −0.02 to −0.04 ppm; light pink, −0.008 to −0.02 ppm). Yellow spheres represent Ca2+ ions. (e,f) Models of C2B bound to the SNARE complex built manually to match the C2B PCSs with the SC166 (e) and SC41 (f) tensors represented by isosurfaces as in Figs. 2c,f. (g–j) Correlations between experimental PCSs induced on C2B by SC166Dy (g,h) or SC41Dy (i,j) and PCSs calculated with the 166- and 41-manual models using the optimized SC166 (g) and SC41 (i) tensors (illustrated in Figs. 2c,f, respectively) or slightly modified tensors (h,j) (see Supplementary note 7). Correlation coefficients (r) and slopes (m) are indicated.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496268&req=5

Figure 3: PCSs induced by the SC166Dy and SC41Dy on the Syt1 C2B domain. (a,b) 1H-13C HMQC spectra of 30 μM 15N,2H-ILV-13CH3-C2B R398Q R399Q mutant in the presence of 30 μM SC166Dy (a) or SC41Dy (b) before (red contours) or after (black contours) tag removal. (c,d) Ribbon diagrams of Syt1 C2B showing PCSs induced by SC166Dy (c) or SC41Dy (d). Amide hydrogens and methyl carbons are shown as spheres color-coded according to the measured PCSs (dark blue, > 0.06 ppm; blue, 0.04 to 0.06 ppm; cyan, 0.02 to 0.04 ppm; pale cyan, 0.008 to 0.02 ppm; red, −0.04 to −0.06 ppm; salmon, −0.02 to −0.04 ppm; light pink, −0.008 to −0.02 ppm). Yellow spheres represent Ca2+ ions. (e,f) Models of C2B bound to the SNARE complex built manually to match the C2B PCSs with the SC166 (e) and SC41 (f) tensors represented by isosurfaces as in Figs. 2c,f. (g–j) Correlations between experimental PCSs induced on C2B by SC166Dy (g,h) or SC41Dy (i,j) and PCSs calculated with the 166- and 41-manual models using the optimized SC166 (g) and SC41 (i) tensors (illustrated in Figs. 2c,f, respectively) or slightly modified tensors (h,j) (see Supplementary note 7). Correlation coefficients (r) and slopes (m) are indicated.
Mentions: To analyze the Syt1-SNARE complex binding mode, we used 15N,2H-labeled Syt1 fragments specifically 1H,13C-labeled at Ile, Leu and Val methyl groups (15N,2H-ILV-13CH3-labeling) for optimal relaxation properties36. Most of the PCSs induced by SC41Dy or SC166Dy on Syt1 C2AB were observed for cross-peaks from C2B, and very similar PCSs were observed using the isolated C2B domain, or the C2B domain with an R398Q R399Q mutation that hinders aggregation with the SNARE complex34 (Figs. 3a,b and Supplementary Figs. 2a,b,d,e; see section on Measurement of PCSs in Online Methods). Since the latter yielded the best-quality data and Arg398-Arg399 might mediate binding modes that promote aggregation, structural analyses were performed with PCS data obtained from 1H-15N TROSY-TROSY HSQC and 1H-13C heteronuclear multiple quantum coherence (HMQC) spectra of 15N,2H-ILV-13CH3-C2B domain bearing the R398Q R399Q mutation (below referred to as C2B for simplicity). These included 149 and 151 PCSs induced by SC41Dy or SC166Dy, respectively, on the C2B domain. C2B binding did not affect the PCSs within SC166Dy but caused slight alterations of PCSs within SC41Dy (5–20%; Supplementary Figs. 2f,g), showing that C2B does not contact residue 166 but is close to residue 41. Nevertheless, we still analyzed the SC41Dy data to examine the consistency with the SC166Dy data. We also note that addition of CpxI caused no or very small changes in the PCSs induced by SC166Dy (Supplementary Fig. 2c), indicating that the binding sites for Syt1 and CpxI on the SNARE complex in solution are distinct but proximal.

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