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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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Analysis of the C2B-SNARE complex by MD simulations. (a) Ribbon diagrams of the SNARE complex and C2B in the positions corresponding to the 166-manual model (gray), the 166-HADDOCK model (purple) and the 166-MD model (orange). (b) Ribbon diagram of the SNARE complex and stick models showing Cα traces of C2B in a range of orientations visited during the MD simulation started from the 166-HADDOCK model (purple). One of the structures from the end of the simulation (in orange) is represented in panel (a) and is referred to as 166-MD model. (c) Ribbon diagrams of the SNARE complex and C2B in the positions corresponding to the 166-MD model (orange) and the 41-manual model (cyan). (d) Ribbon diagram of the SNARE complex and stick models showing the Cα traces of C2B in a range of representative orientations visited during MD simulations incorporating chemical shift restraints. The structure of the CpxI(26–83)-SNARE complex (PDB code 1KIL) has been superimposed to show that CpxI would bump with C2B in some of the positions in the MD simulations. N represents the N-terminus of the SNARE complex in (a–d). N and C represent the N- and C-termini of CpxI(26–83) in (d). (e,f) Correlations between experimental C2B PCSs induced by SC166Dy (e) or SC41Dy (f) and PCSs calculated as ensemble averages using different populations of structures from the 73 clusters visited during the chemical-shift restrained MD simulations. Correlation coefficients (r) and slopes (m) are indicated.
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Figure 4: Analysis of the C2B-SNARE complex by MD simulations. (a) Ribbon diagrams of the SNARE complex and C2B in the positions corresponding to the 166-manual model (gray), the 166-HADDOCK model (purple) and the 166-MD model (orange). (b) Ribbon diagram of the SNARE complex and stick models showing Cα traces of C2B in a range of orientations visited during the MD simulation started from the 166-HADDOCK model (purple). One of the structures from the end of the simulation (in orange) is represented in panel (a) and is referred to as 166-MD model. (c) Ribbon diagrams of the SNARE complex and C2B in the positions corresponding to the 166-MD model (orange) and the 41-manual model (cyan). (d) Ribbon diagram of the SNARE complex and stick models showing the Cα traces of C2B in a range of representative orientations visited during MD simulations incorporating chemical shift restraints. The structure of the CpxI(26–83)-SNARE complex (PDB code 1KIL) has been superimposed to show that CpxI would bump with C2B in some of the positions in the MD simulations. N represents the N-terminus of the SNARE complex in (a–d). N and C represent the N- and C-termini of CpxI(26–83) in (d). (e,f) Correlations between experimental C2B PCSs induced by SC166Dy (e) or SC41Dy (f) and PCSs calculated as ensemble averages using different populations of structures from the 73 clusters visited during the chemical-shift restrained MD simulations. Correlation coefficients (r) and slopes (m) are indicated.

Mentions: The large slopes in Figs. 3g–j can be attributed to a highly dynamic structure where the C2B domain binds to the SNARE complex in multiple orientations at the same or nearby sites. This dynamic nature leads to averaging of the PCSs to smaller values than those expected for a static structure, and is also manifested in the different shapes of the tensors derived from PCSs measured on C2B and the SNARE complex (Supplementary Figs. 3a,b). Indeed, attempts to derive single C2B-SNARE complex structures consistent with the SC166Dy-induced PCSs using HADDOCK-PCS42 yielded structures where C2B was ‘pushed away’ from the center of the SC166 tensor, an expected effect of dynamic averaging of PCSs43 (illustrated in Fig. 4a for a representative structure, referred to as 166-HADDOCK model). Moreover, the HADDOCK-PCS structures exhibited few salt bridges between C2B and the SNAREs, and the pattern of positive-negative PCSs did not match the SC166 tensor lobes well (e.g. Supplementary Fig. 4b). Interestingly, in unrestrained molecular dynamics (MD) simulations started with the 166-HADDOCK model, C2B moved naturally toward the position of the 166-manual model (Figs. 4a,b). A representative structure from the end of the simulation (referred to as 166-MD model) exhibits abundant C2B-SNARE salt bridges (see below), a relatively good correlation between calculated and measured PCSs (Supplementary Fig. 4c), and a good match of positive-negative PCS patterns with the SC166 tensor lobes (Supplementary Fig. 4d). Note that the position of C2B domain in the 166-MD model is also close to that observed in the 41-manual model (Fig. 4c).


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)

Analysis of the C2B-SNARE complex by MD simulations. (a) Ribbon diagrams of the SNARE complex and C2B in the positions corresponding to the 166-manual model (gray), the 166-HADDOCK model (purple) and the 166-MD model (orange). (b) Ribbon diagram of the SNARE complex and stick models showing Cα traces of C2B in a range of orientations visited during the MD simulation started from the 166-HADDOCK model (purple). One of the structures from the end of the simulation (in orange) is represented in panel (a) and is referred to as 166-MD model. (c) Ribbon diagrams of the SNARE complex and C2B in the positions corresponding to the 166-MD model (orange) and the 41-manual model (cyan). (d) Ribbon diagram of the SNARE complex and stick models showing the Cα traces of C2B in a range of representative orientations visited during MD simulations incorporating chemical shift restraints. The structure of the CpxI(26–83)-SNARE complex (PDB code 1KIL) has been superimposed to show that CpxI would bump with C2B in some of the positions in the MD simulations. N represents the N-terminus of the SNARE complex in (a–d). N and C represent the N- and C-termini of CpxI(26–83) in (d). (e,f) Correlations between experimental C2B PCSs induced by SC166Dy (e) or SC41Dy (f) and PCSs calculated as ensemble averages using different populations of structures from the 73 clusters visited during the chemical-shift restrained MD simulations. Correlation coefficients (r) and slopes (m) are indicated.
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Figure 4: Analysis of the C2B-SNARE complex by MD simulations. (a) Ribbon diagrams of the SNARE complex and C2B in the positions corresponding to the 166-manual model (gray), the 166-HADDOCK model (purple) and the 166-MD model (orange). (b) Ribbon diagram of the SNARE complex and stick models showing Cα traces of C2B in a range of orientations visited during the MD simulation started from the 166-HADDOCK model (purple). One of the structures from the end of the simulation (in orange) is represented in panel (a) and is referred to as 166-MD model. (c) Ribbon diagrams of the SNARE complex and C2B in the positions corresponding to the 166-MD model (orange) and the 41-manual model (cyan). (d) Ribbon diagram of the SNARE complex and stick models showing the Cα traces of C2B in a range of representative orientations visited during MD simulations incorporating chemical shift restraints. The structure of the CpxI(26–83)-SNARE complex (PDB code 1KIL) has been superimposed to show that CpxI would bump with C2B in some of the positions in the MD simulations. N represents the N-terminus of the SNARE complex in (a–d). N and C represent the N- and C-termini of CpxI(26–83) in (d). (e,f) Correlations between experimental C2B PCSs induced by SC166Dy (e) or SC41Dy (f) and PCSs calculated as ensemble averages using different populations of structures from the 73 clusters visited during the chemical-shift restrained MD simulations. Correlation coefficients (r) and slopes (m) are indicated.
Mentions: The large slopes in Figs. 3g–j can be attributed to a highly dynamic structure where the C2B domain binds to the SNARE complex in multiple orientations at the same or nearby sites. This dynamic nature leads to averaging of the PCSs to smaller values than those expected for a static structure, and is also manifested in the different shapes of the tensors derived from PCSs measured on C2B and the SNARE complex (Supplementary Figs. 3a,b). Indeed, attempts to derive single C2B-SNARE complex structures consistent with the SC166Dy-induced PCSs using HADDOCK-PCS42 yielded structures where C2B was ‘pushed away’ from the center of the SC166 tensor, an expected effect of dynamic averaging of PCSs43 (illustrated in Fig. 4a for a representative structure, referred to as 166-HADDOCK model). Moreover, the HADDOCK-PCS structures exhibited few salt bridges between C2B and the SNAREs, and the pattern of positive-negative PCSs did not match the SC166 tensor lobes well (e.g. Supplementary Fig. 4b). Interestingly, in unrestrained molecular dynamics (MD) simulations started with the 166-HADDOCK model, C2B moved naturally toward the position of the 166-manual model (Figs. 4a,b). A representative structure from the end of the simulation (referred to as 166-MD model) exhibits abundant C2B-SNARE salt bridges (see below), a relatively good correlation between calculated and measured PCSs (Supplementary Fig. 4c), and a good match of positive-negative PCS patterns with the SC166 tensor lobes (Supplementary Fig. 4d). Note that the position of C2B domain in the 166-MD model is also close to that observed in the 41-manual model (Fig. 4c).

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