The accessory helix of complexin functions by stabilizing central helix secondary structure
- Weill Cornell Medical College, United States
- Fred Hutchinson Cancer Research Center, United States
Figures
Figure 1 with 2 supplements
The worm AH domain forms a stable helix and this structure is deeply conserved across phylogeny.
(A) Ribbon diagram of the mammalian complexin-SNARE crystal structure (Chen et al., 2002) using PDB code 1KIL. Cytoplasmic SNARE domains from synaptobrevin (red), Syntaxin (yellow), and SNAP-25 (gree…
Figure 1—figure supplement 1
Alpha helical regions of complexin across phylogeny.
Alignments of the alpha helical regions of complexin across 16 species from seven phyla using Clustal Omega multiple sequence alignment. The accessory helix region (orange) was defined based on …
Figure 1—figure supplement 2
Evolutionary conservation of helicity across >0.5 billion years.
(A) Agadir helicity predictions for human (Hs, blue), ctenophore (Ml, pink), placazoa (Ta, green), and worm (Ce, dashed line) complexin homologs, plotted relative to the first residue of the central …
Figure 2 with 1 supplement
The worm AH contributes to CPX-1 inhibition of spontaneous vesicle fusion.
(A) Two deletions within the worm AH domain were used: ΔAHshort (35–49, red) and ΔAHlong (30–50, aqua). (B) Examples of spontaneous EPSCs in zero external Ca2+ for wild-type, cpx-1, and transgenic …
Figure 2—figure supplement 1
Axonal protein abundance for CPX-1 transgenes.
(A) Schematic of a worm depicting the region imaged for axonal expression (pink box). All rescuing transgenes were quantified by imaging the C-terminal GFP tag in single axons of immobilized intact …
Figure 3 with 2 supplements
Mouse AH functions in worm CPX-1 and neither domain requires hydrophobic residues.
(A) Two chimeric CPX-1 constructs substituting worm AH with the mouse AH. The long form (mouse AH) substitutes worm residues 26–49 with mouse residues 24–47 whereas the short form (mouse AHshort) …
Figure 3—figure supplement 1
Conservation of hydrophobic moments of complexin.
(A) The AH hydrophobic moment was computed with angular orientation relative to the first residue of the CH. (B) Example of a hydrophobic moment for a particular AH (worm). (C) Polar plot of the …
Figure 3—figure supplement 2
Conservation of charge density in the AH domain.
The helical region of the AH domain was defined using Agadir with a criterion of >5% predicted helicity for a continuous region N-terminal to the CH domain. The net formal charge within this stretch …
Figure 4
Disrupting AH helix stability impairs CPX-1 inhibitory function.
(A) NMR derived Cα-Cβ shifts from either wild-type (black) or R43P (blue) worm CPX-1 peptide missing the C-terminal domain. R43P is indicated in red. Below, the predicted helical content using …
Figure 5
Stability of the AH and its propagation into the CH domain are required for CPX-1 inhibitory function.
(A) AH and CH helical content based on Agadir predictions is plotted for wild-type CPX-1 and three variants with a single substitution at residue 43 as indicated. (B) Average helical content of the …
Figure 6 with 2 supplements
The AH domain can be functionally replaced by a non-native helix.
(A) Schematic of the helix substitution strategy. A(EAAK)7A sequences were substituted for residues 26–49 in the worm AH domain. (B) Agadir prediction for helical stability of the 7-turn helix motif …
Figure 6—figure supplement 1
No correlation between charge density and function.
Charge density of the AH domain in seven rescuing CPX-1 variants. Green indicates nearly complete rescue, gray is partial, and red is poor rescue as measured by aldicarb sensitivity. The CPX-1 …
Figure 6—figure supplement 2
Hydrophobic moments of functional and nonfunctional CPX transgenes.
Polar plot of hydrophobic moments for the AH domain of CPX-1 variants. The CPX-1 variants (blue) are mouse AHshort (SM), worm AH with hydrophobic to glutamates (w[h→E]), mouse AH with hydrophobic to …
