Re-examining how complexin inhibits neurotransmitter release
- Charité-Universitätsmedizin Berlin, Germany
- University of Texas Southwestern Medical Center, United States
Figures
Figure 1 with 2 supplements
Models of complexin function.
(A) Domain diagram of CpxI and ribbon diagram of the crystal structure of the CpxI(26-83)/SNARE complex (PDB code 1KIL) (Chen et al., 2002). Selected residue numbers are indicated above the ribbon …
Figure 1—figure supplement 1
The interface between CpxI and the SNARE complex.
A ribbon diagram of the crystal structure of the CpxI(26-83)/SNARE complex(PDB code 1KIL) (Chen et al., 2002) is shown, with synaptobrevin colored in red, syntaxin-1 in yellow, SNAP-25 in blue and …
Figure 1—figure supplement 2
High B-factors in the accessory helix in the crystal structure of the CpxI(26-83) superclamp mutant bound to SCΔ60.
(A) Electron density images of the crystal structure of CpxI(26-83) superclamp mutant bound to SCΔ60 (PDB accession code 3RK3; Kummel et al., 2011). Superimposed on the coordinates from 3RK3 is the …
Figure 2 with 2 supplements
NMR analysis of interactions between 2H,15N-labeled CpxI fragments and synaptobrevin-truncated SNARE complexes.
(A–C) Expansions of 1H-15N TROSY-HSQC spectra of 2H,15N-CpxI(26-83) free (black contours), or bound to non-truncated SNARE complex (red contours), to SCΔ68 (blue contours) or SCΔ62 (green contours). …
Figure 2—figure supplement 1
Additional analysis of the interaction between 2H,15N-labeled CpxI fragments and synaptobrevin-truncated SNARE complexes.
(A) Chemical shift changes in the CpxI central helix of SNARE complex-bound 2H,15N-CpxI(26- 83) caused by truncation of synaptobrevin to residue 68, normalized by the changes caused by binding of …
Figure 2—figure supplement 2
NMR analysis of interactions between 2H,15N-labeled CpxI superclamp mutant fragments and synaptobrevin-truncated SNARE complexes.
(A) Expansions of 1H-15N TROSY-HSQC spectra of D27L, E34F, R37A superclamp mutant (supcl) 2H,15N-CpxI(26-48) in the absence (black contours) or presence (red contours) of SCΔ60. Because the red and …
Figure 3 with 1 supplement
NMR analysis of interactions between 2H,15N-labeled synaptobrevin-truncated SNARE complexes and CpxI fragments.
(A and B) Expansions of 1H-15N TROSY-HSQC spectra of the non-truncated 2H,15N-SNC-SC (black contours), 2H,15N-SNC-SCΔ76 (green contours) and 2H,15N-SNC-SCΔ62 (red contours). (C and D) Expansions of 1…
Figure 3—figure supplement 1
Additional NMR analyses of interactions between 2H,15N-labeled synaptobrevin-truncated SNARE complexes and CpxI fragments.
Expansions of 1H-15N TROSY-HSQC spectra of 2H,15N-SNCΔ62 (A and B) or 2H,15N-SNCΔ60 (C and D) in the absence (red contours) or presence (blue contours) of CpxI(26-83) D27L, E34F, R37A superclamp …
Figure 4 with 1 supplement
ITC analysis of binding of CpxI fragments to SNARE complexes.
(A and B) Direct titrations of non-truncated SNARE complex (SC; A) or SCΔ60 (B) with CpxI(47-134). (C–E) Competition assays where samples containing SCΔ60 and 1.5 equivalents of CpxI(47-134) were …
Figure 4—figure supplement 1
Additional ITC analysis of binding of CpxI fragments to SNARE complexes.
(A) Competition assay where a sample containing SCΔ60 and 3.0 equivalents of CpxI(47-134) was titrated with WT CpxI(26-83). (B) Composite diagram where the ITC data shown in panel (A) (shown here as …
Figure 5 with 1 supplement
NMR analysis of interactions between 2H,15N-labeled syntaxin-1 truncated SNARE complexes and CpxI fragments.
(A and B) Expansions of 1H-15N TROSY-HSQC spectra of free 2H,15N-labeled synaptobrevin SNARE motif (2H,15N-Syb, green contours), the non-truncated 2H,15N-Syb-SC (black contours) and 2H,15N-Syb-SCΔ236…
Figure 5—figure supplement 1
Additional NMR analyses of interactions between 2H,15N-labeled syntaxin-1 truncated SNARE complexes and CpxI fragments.
(A) Expansion of a 1H-15N TROSY-HSQC spectrum of free 2H,15N-labeled synaptobrevin SNARE motif (2H,15N-Syb). Cross-peak assignments are based on those obtained for synaptobrevin(1-96) (Hazzard et …
Figure 6
NMR analysis of interactions between 2H,15N-labeled CpxI fragments and syntaxin-1-truncated SNARE complexes.
(A and B) Expansions of 1H-15N TROSY-HSQC spectra of 2H,15N-CpxI(26-83) bound to nontruncated SNARE complex (SC; red contours), to SCΔ236 (blue contours), SCΔ232 (green contours) or SCΔ228 (purple …
Figure 7 with 1 supplement
Rescue of the complexin KO phenotype with CpxI-superclamp, but not with a clamping deficient CpxI.
(A) Overview of the introduced mutations in CpxI. (B–C) Representative traces and summary data of evoked EPSC (B) and synaptic responses to hypertonic sucrose solution (RRP) (C) of T-KO, K26A, D27L …
Figure 7—figure supplement 1
Expression of Cpx variants in hippocampal CPXI-III triple KO neurons by lentiviral transduction.
(A) Immunocytochemical detection of cytosolic complexin-FLAG tagged protein variants within presynaptic compartments illustrated by VGlut1 colocalisation. Arrowheads point towards examples of …
Figure 8
Inhibition of spontaneous release by the accessory alpha helix of dmCpx.
(A) Overview of the replacement of the accessory helix of CpxI with the accessory helix sequence of drosophila Cpx. (B) Representative traces and summary data of evoked EPSC (B) and synaptic …
Figure 9
Inhibitory effect of the accessory alpha helix is charge dependent.
(A) Overview of the CpxI accessory alpha helix sequence and the introduced mutations resulting in more positively charged (CpxI-3R) or more negatively charged (CpxI-5E) accessory alpha helix. (B–C) …
Figure 10
Fusogenicity of synaptic vesicles is influenced by the charge of the accessory alpha helix of CpxI.
(A) Average traces of synaptic responses induced by 250 mM sucrose solution (T-KO n = 33, CpxI-3R n = 49, CpxI-5E n = 39, WT CpxI n = 50). (B) Summary data of 250 mM sucrose solution-induced …
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