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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.


Isothermal titration calorimetry indicates multivalent interactions between SNAREΔ60 and CPX.(A) Calorimetric titration of super-clamp complexin (scCPX; residues 1–134, with D27L, E34F, R37A mutations) into pre-fusion SNAREΔ60 complex describes a multi-site interaction of CPX. The solid lines represent the predicted binding thermogram assuming that both scCPX and truncated SNAREΔ60 are bivalent with well-defined independent thermodynamic parameters describing CPX central helix and CPX accessory helix binding (B). (C) Representative thermogram of full-length wild-type CPX (residues 1–134) titrated into unblocked SNAREΔ60.DOI:http://dx.doi.org/10.7554/eLife.04463.006
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fig3: Isothermal titration calorimetry indicates multivalent interactions between SNAREΔ60 and CPX.(A) Calorimetric titration of super-clamp complexin (scCPX; residues 1–134, with D27L, E34F, R37A mutations) into pre-fusion SNAREΔ60 complex describes a multi-site interaction of CPX. The solid lines represent the predicted binding thermogram assuming that both scCPX and truncated SNAREΔ60 are bivalent with well-defined independent thermodynamic parameters describing CPX central helix and CPX accessory helix binding (B). (C) Representative thermogram of full-length wild-type CPX (residues 1–134) titrated into unblocked SNAREΔ60.DOI:http://dx.doi.org/10.7554/eLife.04463.006

Mentions: Further, to directly monitor the multiple binding modes of CPX to the pre-fusion SNAREpin, we carried out new ITC experiments where we titrated full-length CPX into unblocked SNAREΔ60. As our initial ITC data had suggested that the accessory helix of super clamp CPX (residues 1–134 with D27L, E34F, R37A; scCPX) binds to SNAREΔ60 with ∼10× higher affinity (Table 1) than wild-type (Kümmel et al., 2011), we used scCPX for this analysis. As shown in Figure 3A, titration of scCPX into the unblocked SNAREΔ60 results in a thermal graph characteristic of a reaction involving multiple binding sites, demonstrating that CPX has more than one binding site per SNAREΔ60 complex. The data can be best approximated using the independent thermodynamic parameters for the CPXcen and CPXacc interaction, with the assumption that both the truncated SNAREΔ60 and scCPX are bivalent (Figure 3B). We observed a qualitatively similar titration curve for wild-type CPX (Figure 3C) but, since the CPXacc interaction with SNAREΔ60 is much weaker, the fitting with multiple binding sites was not resolved in detail.10.7554/eLife.04463.006Figure 3.Isothermal titration calorimetry indicates multivalent interactions between SNAREΔ60 and CPX.


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)

Isothermal titration calorimetry indicates multivalent interactions between SNAREΔ60 and CPX.(A) Calorimetric titration of super-clamp complexin (scCPX; residues 1–134, with D27L, E34F, R37A mutations) into pre-fusion SNAREΔ60 complex describes a multi-site interaction of CPX. The solid lines represent the predicted binding thermogram assuming that both scCPX and truncated SNAREΔ60 are bivalent with well-defined independent thermodynamic parameters describing CPX central helix and CPX accessory helix binding (B). (C) Representative thermogram of full-length wild-type CPX (residues 1–134) titrated into unblocked SNAREΔ60.DOI:http://dx.doi.org/10.7554/eLife.04463.006
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4384536&req=5

fig3: Isothermal titration calorimetry indicates multivalent interactions between SNAREΔ60 and CPX.(A) Calorimetric titration of super-clamp complexin (scCPX; residues 1–134, with D27L, E34F, R37A mutations) into pre-fusion SNAREΔ60 complex describes a multi-site interaction of CPX. The solid lines represent the predicted binding thermogram assuming that both scCPX and truncated SNAREΔ60 are bivalent with well-defined independent thermodynamic parameters describing CPX central helix and CPX accessory helix binding (B). (C) Representative thermogram of full-length wild-type CPX (residues 1–134) titrated into unblocked SNAREΔ60.DOI:http://dx.doi.org/10.7554/eLife.04463.006
Mentions: Further, to directly monitor the multiple binding modes of CPX to the pre-fusion SNAREpin, we carried out new ITC experiments where we titrated full-length CPX into unblocked SNAREΔ60. As our initial ITC data had suggested that the accessory helix of super clamp CPX (residues 1–134 with D27L, E34F, R37A; scCPX) binds to SNAREΔ60 with ∼10× higher affinity (Table 1) than wild-type (Kümmel et al., 2011), we used scCPX for this analysis. As shown in Figure 3A, titration of scCPX into the unblocked SNAREΔ60 results in a thermal graph characteristic of a reaction involving multiple binding sites, demonstrating that CPX has more than one binding site per SNAREΔ60 complex. The data can be best approximated using the independent thermodynamic parameters for the CPXcen and CPXacc interaction, with the assumption that both the truncated SNAREΔ60 and scCPX are bivalent (Figure 3B). We observed a qualitatively similar titration curve for wild-type CPX (Figure 3C) but, since the CPXacc interaction with SNAREΔ60 is much weaker, the fitting with multiple binding sites was not resolved in detail.10.7554/eLife.04463.006Figure 3.Isothermal titration calorimetry indicates multivalent interactions between SNAREΔ60 and CPX.

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.