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The accessory helix of complexin functions by stabilizing central helix secondary structure.

Radoff DT, Dong Y, Snead D, Bai J, Eliezer D, Dittman JS - Elife (2014)

Bottom Line: The mouse AH fully restored function when substituted into worm CPX suggesting its mechanism is evolutionarily conserved.CPX inhibitory function was impaired when helix propagation into the CH was disrupted whereas replacing the AH with a non-native helical sequence restored CPX function.We propose that the AH operates by stabilizing CH secondary structure rather than through protein or lipid interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Weill Cornell Medical College, New York, United States.

ABSTRACT
The presynaptic protein complexin (CPX) is a critical regulator of synaptic vesicle fusion, but the mechanisms underlying its regulatory effects are not well understood. Its highly conserved central helix (CH) directly binds the ternary SNARE complex and is required for all known CPX functions. The adjacent accessory helix (AH) is not conserved despite also playing an important role in CPX function, and numerous models for its mechanism have been proposed. We examined the impact of AH mutations and chimeras on CPX function in vivo and in vitro using C. elegans. The mouse AH fully restored function when substituted into worm CPX suggesting its mechanism is evolutionarily conserved. CPX inhibitory function was impaired when helix propagation into the CH was disrupted whereas replacing the AH with a non-native helical sequence restored CPX function. We propose that the AH operates by stabilizing CH secondary structure rather than through protein or lipid interactions.

No MeSH data available.


Related in: MedlinePlus

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 Agadir for wild-type (black), and R43P complexin (blue). (B) Average secondary chemical shift for wild-type (black) and R43P (blue) complexin either over the entire peptide (residues 1–77, left), AH domain (37–49, middle), or CH domain (50–74, right). The average helical content was estimated by dividing the chemical shift by 3.4 (average shift of 100% helical peptide). The helical content was also measured by CD spectroscopy (red arrowheads). (C) Helical content for wild type (black) and R43P (blue) complexin was measured by CD spectroscopy for increasing concentrations of 2,2,2-trifluoroethanol (TFE). The resulting dose–response data was fit to a simple equilibrium binding curve with equilibrium constants and Hill coefficients indicated on the graph. Average spontaneous EPSC Rate (D) and EPSC amplitude (E) for wild-type, cpx-1, and either the ΔAHshort or R43P rescuing transgene expressed in cpx-1 as indicated. (F) Sensitivity to aldicarb was quantified by the average time to 50% paralysis and then normalized to wild-type and cpx-1 mutant animals. On this scale, rescue with ΔAHshort or R43P variants of CPX-1 partially restored wild-type aldicarb sensitivity. Data are mean ± SEM and the number of independent assays is indicated for each genotype. Using Tukey–Kramer statistics for multiple comparisons, ** denotes significantly different from wild type, * significantly different from both wild type and cpx-1 (p < 0.01), n.s. is not significant.DOI:http://dx.doi.org/10.7554/eLife.04553.011
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fig4: 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 Agadir for wild-type (black), and R43P complexin (blue). (B) Average secondary chemical shift for wild-type (black) and R43P (blue) complexin either over the entire peptide (residues 1–77, left), AH domain (37–49, middle), or CH domain (50–74, right). The average helical content was estimated by dividing the chemical shift by 3.4 (average shift of 100% helical peptide). The helical content was also measured by CD spectroscopy (red arrowheads). (C) Helical content for wild type (black) and R43P (blue) complexin was measured by CD spectroscopy for increasing concentrations of 2,2,2-trifluoroethanol (TFE). The resulting dose–response data was fit to a simple equilibrium binding curve with equilibrium constants and Hill coefficients indicated on the graph. Average spontaneous EPSC Rate (D) and EPSC amplitude (E) for wild-type, cpx-1, and either the ΔAHshort or R43P rescuing transgene expressed in cpx-1 as indicated. (F) Sensitivity to aldicarb was quantified by the average time to 50% paralysis and then normalized to wild-type and cpx-1 mutant animals. On this scale, rescue with ΔAHshort or R43P variants of CPX-1 partially restored wild-type aldicarb sensitivity. Data are mean ± SEM and the number of independent assays is indicated for each genotype. Using Tukey–Kramer statistics for multiple comparisons, ** denotes significantly different from wild type, * significantly different from both wild type and cpx-1 (p < 0.01), n.s. is not significant.DOI:http://dx.doi.org/10.7554/eLife.04553.011

Mentions: Previous studies on mouse complexin suggested that the helical structure of the AH domain is important for stabilizing the CH domain (Pabst et al., 2000; Chen et al., 2002) and for the inhibitory function of mCpx1 (Xue et al., 2007), but the reason for this requirement remains unclear. While several potential roles for the AH in mediating protein–protein interactions have been proposed, the AH may simply serve to nucleate and propagate helical structure into the CH region (Chen et al., 2002), but this idea has never been tested. To explore this possibility, a helix-breaking proline was inserted into the AH domain (R43P), and the effects on AH domain secondary structure of a recombinant truncated form of the mutant protein missing its C-terminal domain (ΔCT) were examined by NMR spectroscopy (Figure 4A). Furthermore, because conversion from random coil to alpha helix is a highly cooperative process, helicity in the CH domain is also predicted to decrease for the R43P mutant (Munoz and Serrano, 1997). Indeed, decreased NMR carbon secondary shifts were observed throughout the AH and extending well into the CH domain, confirming decreases in both nucleation and propagation of the helical conformation (Figure 4B). Circular dichroism (CD) spectroscopy provides another measure of overall alpha helical structure (Greenfield and Fasman, 1969; Saxena and Wetlaufer, 1971; Rohl and Baldwin, 1997). Absorption at 222 nm was monitored in recombinant ΔCT protein while titrating in 2,2,2-trifluoroethanol (TFE), a co-solvent known to stabilize alpha helices in solution (Nelson and Kallenbach, 1986; Segawa et al., 1991; Shiraki et al., 1995). The increase in alpha helical structure with increasing concentration of TFE can be used to measure the stability and cooperativity of alpha helix formation. While some of the cooperativity arises from coordination of multiple TFE molecules (Berkessel et al., 2006), intramolecular propagation of helical structure will also contribute. As shown in Figure 4C, the R43P variant displayed a lower propensity for helix formation with a lower cooperativity, consistent with both decreased helix nucleation and propagation, as also evident from both the computational predictions and the NMR data. In living worms, inserting a proline into the AH domain completely eliminated AH function since the CPX(R43P) rescue was indistinguishable from the ΔAHshort rescue in both electrophysiological (Figure 4D,E) and behavioral (Figure 4F) assays of synaptic function. Thus, inhibition of spontaneous fusion requires a helical AH domain in vivo.10.7554/eLife.04553.011Figure 4.Disrupting AH helix stability impairs CPX-1 inhibitory function.


The accessory helix of complexin functions by stabilizing central helix secondary structure.

Radoff DT, Dong Y, Snead D, Bai J, Eliezer D, Dittman JS - Elife (2014)

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 Agadir for wild-type (black), and R43P complexin (blue). (B) Average secondary chemical shift for wild-type (black) and R43P (blue) complexin either over the entire peptide (residues 1–77, left), AH domain (37–49, middle), or CH domain (50–74, right). The average helical content was estimated by dividing the chemical shift by 3.4 (average shift of 100% helical peptide). The helical content was also measured by CD spectroscopy (red arrowheads). (C) Helical content for wild type (black) and R43P (blue) complexin was measured by CD spectroscopy for increasing concentrations of 2,2,2-trifluoroethanol (TFE). The resulting dose–response data was fit to a simple equilibrium binding curve with equilibrium constants and Hill coefficients indicated on the graph. Average spontaneous EPSC Rate (D) and EPSC amplitude (E) for wild-type, cpx-1, and either the ΔAHshort or R43P rescuing transgene expressed in cpx-1 as indicated. (F) Sensitivity to aldicarb was quantified by the average time to 50% paralysis and then normalized to wild-type and cpx-1 mutant animals. On this scale, rescue with ΔAHshort or R43P variants of CPX-1 partially restored wild-type aldicarb sensitivity. Data are mean ± SEM and the number of independent assays is indicated for each genotype. Using Tukey–Kramer statistics for multiple comparisons, ** denotes significantly different from wild type, * significantly different from both wild type and cpx-1 (p < 0.01), n.s. is not significant.DOI:http://dx.doi.org/10.7554/eLife.04553.011
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fig4: 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 Agadir for wild-type (black), and R43P complexin (blue). (B) Average secondary chemical shift for wild-type (black) and R43P (blue) complexin either over the entire peptide (residues 1–77, left), AH domain (37–49, middle), or CH domain (50–74, right). The average helical content was estimated by dividing the chemical shift by 3.4 (average shift of 100% helical peptide). The helical content was also measured by CD spectroscopy (red arrowheads). (C) Helical content for wild type (black) and R43P (blue) complexin was measured by CD spectroscopy for increasing concentrations of 2,2,2-trifluoroethanol (TFE). The resulting dose–response data was fit to a simple equilibrium binding curve with equilibrium constants and Hill coefficients indicated on the graph. Average spontaneous EPSC Rate (D) and EPSC amplitude (E) for wild-type, cpx-1, and either the ΔAHshort or R43P rescuing transgene expressed in cpx-1 as indicated. (F) Sensitivity to aldicarb was quantified by the average time to 50% paralysis and then normalized to wild-type and cpx-1 mutant animals. On this scale, rescue with ΔAHshort or R43P variants of CPX-1 partially restored wild-type aldicarb sensitivity. Data are mean ± SEM and the number of independent assays is indicated for each genotype. Using Tukey–Kramer statistics for multiple comparisons, ** denotes significantly different from wild type, * significantly different from both wild type and cpx-1 (p < 0.01), n.s. is not significant.DOI:http://dx.doi.org/10.7554/eLife.04553.011
Mentions: Previous studies on mouse complexin suggested that the helical structure of the AH domain is important for stabilizing the CH domain (Pabst et al., 2000; Chen et al., 2002) and for the inhibitory function of mCpx1 (Xue et al., 2007), but the reason for this requirement remains unclear. While several potential roles for the AH in mediating protein–protein interactions have been proposed, the AH may simply serve to nucleate and propagate helical structure into the CH region (Chen et al., 2002), but this idea has never been tested. To explore this possibility, a helix-breaking proline was inserted into the AH domain (R43P), and the effects on AH domain secondary structure of a recombinant truncated form of the mutant protein missing its C-terminal domain (ΔCT) were examined by NMR spectroscopy (Figure 4A). Furthermore, because conversion from random coil to alpha helix is a highly cooperative process, helicity in the CH domain is also predicted to decrease for the R43P mutant (Munoz and Serrano, 1997). Indeed, decreased NMR carbon secondary shifts were observed throughout the AH and extending well into the CH domain, confirming decreases in both nucleation and propagation of the helical conformation (Figure 4B). Circular dichroism (CD) spectroscopy provides another measure of overall alpha helical structure (Greenfield and Fasman, 1969; Saxena and Wetlaufer, 1971; Rohl and Baldwin, 1997). Absorption at 222 nm was monitored in recombinant ΔCT protein while titrating in 2,2,2-trifluoroethanol (TFE), a co-solvent known to stabilize alpha helices in solution (Nelson and Kallenbach, 1986; Segawa et al., 1991; Shiraki et al., 1995). The increase in alpha helical structure with increasing concentration of TFE can be used to measure the stability and cooperativity of alpha helix formation. While some of the cooperativity arises from coordination of multiple TFE molecules (Berkessel et al., 2006), intramolecular propagation of helical structure will also contribute. As shown in Figure 4C, the R43P variant displayed a lower propensity for helix formation with a lower cooperativity, consistent with both decreased helix nucleation and propagation, as also evident from both the computational predictions and the NMR data. In living worms, inserting a proline into the AH domain completely eliminated AH function since the CPX(R43P) rescue was indistinguishable from the ΔAHshort rescue in both electrophysiological (Figure 4D,E) and behavioral (Figure 4F) assays of synaptic function. Thus, inhibition of spontaneous fusion requires a helical AH domain in vivo.10.7554/eLife.04553.011Figure 4.Disrupting AH helix stability impairs CPX-1 inhibitory function.

Bottom Line: The mouse AH fully restored function when substituted into worm CPX suggesting its mechanism is evolutionarily conserved.CPX inhibitory function was impaired when helix propagation into the CH was disrupted whereas replacing the AH with a non-native helical sequence restored CPX function.We propose that the AH operates by stabilizing CH secondary structure rather than through protein or lipid interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Weill Cornell Medical College, New York, United States.

ABSTRACT
The presynaptic protein complexin (CPX) is a critical regulator of synaptic vesicle fusion, but the mechanisms underlying its regulatory effects are not well understood. Its highly conserved central helix (CH) directly binds the ternary SNARE complex and is required for all known CPX functions. The adjacent accessory helix (AH) is not conserved despite also playing an important role in CPX function, and numerous models for its mechanism have been proposed. We examined the impact of AH mutations and chimeras on CPX function in vivo and in vitro using C. elegans. The mouse AH fully restored function when substituted into worm CPX suggesting its mechanism is evolutionarily conserved. CPX inhibitory function was impaired when helix propagation into the CH was disrupted whereas replacing the AH with a non-native helical sequence restored CPX function. We propose that the AH operates by stabilizing CH secondary structure rather than through protein or lipid interactions.

No MeSH data available.


Related in: MedlinePlus