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Re-visiting the trans insertion model for complexin clamping.

Krishnakumar SS, Li F, Coleman J, Schauder CM, Kümmel D, Pincet F, Rothman JE, Reinisch KM - Elife (2015)

Bottom Line: We have previously proposed that complexin cross-links multiple pre-fusion SNARE complexes via a trans interaction to function as a clamp on SNARE-mediated neurotransmitter release.A recent NMR study was unable to detect the trans clamping interaction of complexin and therefore questioned the previous interpretation of the fluorescence resonance energy transfer and isothermal titration calorimetry data on which the trans clamping model was originally based.Here we present new biochemical data that underscore the validity of our previous interpretation and the continued relevancy of the trans insertion model for complexin clamping.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Yale University School of Medicine, New Haven, United States.

ABSTRACT
We have previously proposed that complexin cross-links multiple pre-fusion SNARE complexes via a trans interaction to function as a clamp on SNARE-mediated neurotransmitter release. A recent NMR study was unable to detect the trans clamping interaction of complexin and therefore questioned the previous interpretation of the fluorescence resonance energy transfer and isothermal titration calorimetry data on which the trans clamping model was originally based. Here we present new biochemical data that underscore the validity of our previous interpretation and the continued relevancy of the trans insertion model for complexin clamping.

No MeSH data available.


Related in: MedlinePlus

Interaction of complexin accessory helix (CPXacc) with the t-SNARE groove for full-length and truncated (residue 26–83) CPX characterized by isothermal titration calorimetry.Full-length (A) or CPX26–83 (B) were titrated into pre-fusion SNAREΔ60 complex with the CPX central helix (CPXcen) binding site blocked with CPX-48 to exclusively measure the CPXacc–t-SNARE clamping interaction. The solid lines represent the best fit to the corresponding data points using non-linear least squares fit with one-set-of-sites-model and results of the fit are shown in Table 1. All experiments were conducted in triplicate and a representative thermogram is shown.DOI:http://dx.doi.org/10.7554/eLife.04463.005
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fig2: Interaction of complexin accessory helix (CPXacc) with the t-SNARE groove for full-length and truncated (residue 26–83) CPX characterized by isothermal titration calorimetry.Full-length (A) or CPX26–83 (B) were titrated into pre-fusion SNAREΔ60 complex with the CPX central helix (CPXcen) binding site blocked with CPX-48 to exclusively measure the CPXacc–t-SNARE clamping interaction. The solid lines represent the best fit to the corresponding data points using non-linear least squares fit with one-set-of-sites-model and results of the fit are shown in Table 1. All experiments were conducted in triplicate and a representative thermogram is shown.DOI:http://dx.doi.org/10.7554/eLife.04463.005

Mentions: To restore confidence in our ITC data reported in Kümmel et al. (2011), we also repeated the ITC binding experiments using 2.5–3-fold molar excess of CPX-48 to completely block the CPXcen binding (≥99%). We found that CPX binds to the blocked SNAREΔ60 with a binding affinity of 15.2 ± 1.4 µM (Figure 2A, Table 1), matching well the Kd ∼16 µM reported in Kümmel et al. (2011). We note that in these experiments as well as those reported in Kümmel et al. we titrated full-length CPX (residues 1–134) into the blocked SNAREΔ60, and not the minimal functional domain (residues 26–83) as we had implied (“we used a complexin construct comprising both the central and accessory helices [residues 26–83]” [Kümmel et al., 2011]), and we apologize for this reporting error. We have now additionally carried out the ITC experiments with the minimal functional domain (CPX26–83) and find that this truncated version also binds to the blocked SNAREΔ60, albeit with slightly weaker affinity (Kd = 23.9 ± 0.1 µM) compared with full-length CPX (Figure 2B, Table 1). Taken together, the data strongly support our earlier conclusion that the ITC binding studies carried out with CPX titrated into blocked SNAREΔ60 correctly reflect the binding of CPXacc to t-SNARE. Consistent with this, we have recently also been able to characterize the binding of mammalian CPXacc to Drosophila t-SNAREs using blocked Drosophila pre-fusion SNARE complex (Cho et al., 2014). Mutations in the CPXacc predicted to enhance or decrease the binding of CPXacc to t-SNARE exhibit corresponding binding profiles in ITC experiments (Cho et al., 2014) in support of the trans insertion model (Kümmel et al., 2011).10.7554/eLife.04463.005Figure 2.Interaction of complexin accessory helix (CPXacc) with the t-SNARE groove for full-length and truncated (residue 26–83) CPX characterized by isothermal titration calorimetry.


Re-visiting the trans insertion model for complexin clamping.

Krishnakumar SS, Li F, Coleman J, Schauder CM, Kümmel D, Pincet F, Rothman JE, Reinisch KM - Elife (2015)

Interaction of complexin accessory helix (CPXacc) with the t-SNARE groove for full-length and truncated (residue 26–83) CPX characterized by isothermal titration calorimetry.Full-length (A) or CPX26–83 (B) were titrated into pre-fusion SNAREΔ60 complex with the CPX central helix (CPXcen) binding site blocked with CPX-48 to exclusively measure the CPXacc–t-SNARE clamping interaction. The solid lines represent the best fit to the corresponding data points using non-linear least squares fit with one-set-of-sites-model and results of the fit are shown in Table 1. All experiments were conducted in triplicate and a representative thermogram is shown.DOI:http://dx.doi.org/10.7554/eLife.04463.005
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Related In: Results  -  Collection

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fig2: Interaction of complexin accessory helix (CPXacc) with the t-SNARE groove for full-length and truncated (residue 26–83) CPX characterized by isothermal titration calorimetry.Full-length (A) or CPX26–83 (B) were titrated into pre-fusion SNAREΔ60 complex with the CPX central helix (CPXcen) binding site blocked with CPX-48 to exclusively measure the CPXacc–t-SNARE clamping interaction. The solid lines represent the best fit to the corresponding data points using non-linear least squares fit with one-set-of-sites-model and results of the fit are shown in Table 1. All experiments were conducted in triplicate and a representative thermogram is shown.DOI:http://dx.doi.org/10.7554/eLife.04463.005
Mentions: To restore confidence in our ITC data reported in Kümmel et al. (2011), we also repeated the ITC binding experiments using 2.5–3-fold molar excess of CPX-48 to completely block the CPXcen binding (≥99%). We found that CPX binds to the blocked SNAREΔ60 with a binding affinity of 15.2 ± 1.4 µM (Figure 2A, Table 1), matching well the Kd ∼16 µM reported in Kümmel et al. (2011). We note that in these experiments as well as those reported in Kümmel et al. we titrated full-length CPX (residues 1–134) into the blocked SNAREΔ60, and not the minimal functional domain (residues 26–83) as we had implied (“we used a complexin construct comprising both the central and accessory helices [residues 26–83]” [Kümmel et al., 2011]), and we apologize for this reporting error. We have now additionally carried out the ITC experiments with the minimal functional domain (CPX26–83) and find that this truncated version also binds to the blocked SNAREΔ60, albeit with slightly weaker affinity (Kd = 23.9 ± 0.1 µM) compared with full-length CPX (Figure 2B, Table 1). Taken together, the data strongly support our earlier conclusion that the ITC binding studies carried out with CPX titrated into blocked SNAREΔ60 correctly reflect the binding of CPXacc to t-SNARE. Consistent with this, we have recently also been able to characterize the binding of mammalian CPXacc to Drosophila t-SNAREs using blocked Drosophila pre-fusion SNARE complex (Cho et al., 2014). Mutations in the CPXacc predicted to enhance or decrease the binding of CPXacc to t-SNARE exhibit corresponding binding profiles in ITC experiments (Cho et al., 2014) in support of the trans insertion model (Kümmel et al., 2011).10.7554/eLife.04463.005Figure 2.Interaction of complexin accessory helix (CPXacc) with the t-SNARE groove for full-length and truncated (residue 26–83) CPX characterized by isothermal titration calorimetry.

Bottom Line: We have previously proposed that complexin cross-links multiple pre-fusion SNARE complexes via a trans interaction to function as a clamp on SNARE-mediated neurotransmitter release.A recent NMR study was unable to detect the trans clamping interaction of complexin and therefore questioned the previous interpretation of the fluorescence resonance energy transfer and isothermal titration calorimetry data on which the trans clamping model was originally based.Here we present new biochemical data that underscore the validity of our previous interpretation and the continued relevancy of the trans insertion model for complexin clamping.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Yale University School of Medicine, New Haven, United States.

ABSTRACT
We have previously proposed that complexin cross-links multiple pre-fusion SNARE complexes via a trans interaction to function as a clamp on SNARE-mediated neurotransmitter release. A recent NMR study was unable to detect the trans clamping interaction of complexin and therefore questioned the previous interpretation of the fluorescence resonance energy transfer and isothermal titration calorimetry data on which the trans clamping model was originally based. Here we present new biochemical data that underscore the validity of our previous interpretation and the continued relevancy of the trans insertion model for complexin clamping.

No MeSH data available.


Related in: MedlinePlus