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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

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 helix (position #1). (B). Average domain helicity from (A) using AH (orange, residues −19 to 0) and CH (blue, residues 1 to 25) domains for the four species shown in A. (C) Percent sequence identity relative to human mCpx1 for the AH (orange) and CH (blue) domains for the three invertebrate complexins. Note that the placazoan Trichoplax does not possess a nervous system.DOI:http://dx.doi.org/10.7554/eLife.04553.005
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fig1s2: 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 helix (position #1). (B). Average domain helicity from (A) using AH (orange, residues −19 to 0) and CH (blue, residues 1 to 25) domains for the four species shown in A. (C) Percent sequence identity relative to human mCpx1 for the AH (orange) and CH (blue) domains for the three invertebrate complexins. Note that the placazoan Trichoplax does not possess a nervous system.DOI:http://dx.doi.org/10.7554/eLife.04553.005

Mentions: Why compare complexins from multiple divergent species? Several features of the AH domain appear to be conserved across more than half a billion years of evolution based on the species examined here, so an evolutionary comparison provides insight into critical aspects of the underlying mechanisms. Beyond their primary sequence, the two most striking conserved features of the AH domain are its high negative charge density and the distribution and orientation of its hydrophobic residues (Figure 3—figure supplements 1–2). Although the deep conservation of charge density and hydrophobic moment suggests that they play a role in complexin function, the inhibition of spontaneous fusion does not require either feature in worm (this study) and hydrophobicity is superfluous in fly as well (Cho et al., 2014). Perhaps other roles of complexin utilize these properties. In contrast to charge and hydrophobicity, a stable helix is both deeply conserved and required for complexin inhibitory function. How conserved is helicity vs primary protein sequence? The SNARE-binding central helix is the defining motif of complexin homologs based on primary protein sequences (Pabst et al., 2000; Brose, 2008). Two of the most distantly related complexin genes reported belong to the placozoan Trichoplax adherens (Ta) and the ctenophore Mnemiopsis leidyi (Ml) (Flicek et al., 2014). Interestingly, the CH domains of these representatives of basal animal phyla, share 44% identity with the human Cpx1 CH. However, whereas the AH domain of Ta shares 40% identity with the human AH domain, there is only 10% sequence conservation between Ml and human AH domains. The predicted AH and CH structures for human and Ml both contain extended regions of stable helix propagation similar to worm, whereas Ta is predicted to have only a modest degree of stable helical structure on its own (Figure 1—figure supplement 2). This provides an evolutionary example of how primary sequence and helicity do not necessarily change in parallel in the AH domain. A speculative explanation for the noticeable increase in AH domain helical stability progressing from ctenophore (36%) to worm (54%) to mammal (>80% in human) is the higher body temperatures of warm-blooded animals compared to soil- and marine invertebrates. Helical stability is highly dependent on ambient temperature, so the higher body temperatures of mammals and many other vertebrates may necessitate more stable helical sequences (Privalov, 1982; Scholtz and Baldwin, 1992).


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)

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 helix (position #1). (B). Average domain helicity from (A) using AH (orange, residues −19 to 0) and CH (blue, residues 1 to 25) domains for the four species shown in A. (C) Percent sequence identity relative to human mCpx1 for the AH (orange) and CH (blue) domains for the three invertebrate complexins. Note that the placazoan Trichoplax does not possess a nervous system.DOI:http://dx.doi.org/10.7554/eLife.04553.005
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Related In: Results  -  Collection

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fig1s2: 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 helix (position #1). (B). Average domain helicity from (A) using AH (orange, residues −19 to 0) and CH (blue, residues 1 to 25) domains for the four species shown in A. (C) Percent sequence identity relative to human mCpx1 for the AH (orange) and CH (blue) domains for the three invertebrate complexins. Note that the placazoan Trichoplax does not possess a nervous system.DOI:http://dx.doi.org/10.7554/eLife.04553.005
Mentions: Why compare complexins from multiple divergent species? Several features of the AH domain appear to be conserved across more than half a billion years of evolution based on the species examined here, so an evolutionary comparison provides insight into critical aspects of the underlying mechanisms. Beyond their primary sequence, the two most striking conserved features of the AH domain are its high negative charge density and the distribution and orientation of its hydrophobic residues (Figure 3—figure supplements 1–2). Although the deep conservation of charge density and hydrophobic moment suggests that they play a role in complexin function, the inhibition of spontaneous fusion does not require either feature in worm (this study) and hydrophobicity is superfluous in fly as well (Cho et al., 2014). Perhaps other roles of complexin utilize these properties. In contrast to charge and hydrophobicity, a stable helix is both deeply conserved and required for complexin inhibitory function. How conserved is helicity vs primary protein sequence? The SNARE-binding central helix is the defining motif of complexin homologs based on primary protein sequences (Pabst et al., 2000; Brose, 2008). Two of the most distantly related complexin genes reported belong to the placozoan Trichoplax adherens (Ta) and the ctenophore Mnemiopsis leidyi (Ml) (Flicek et al., 2014). Interestingly, the CH domains of these representatives of basal animal phyla, share 44% identity with the human Cpx1 CH. However, whereas the AH domain of Ta shares 40% identity with the human AH domain, there is only 10% sequence conservation between Ml and human AH domains. The predicted AH and CH structures for human and Ml both contain extended regions of stable helix propagation similar to worm, whereas Ta is predicted to have only a modest degree of stable helical structure on its own (Figure 1—figure supplement 2). This provides an evolutionary example of how primary sequence and helicity do not necessarily change in parallel in the AH domain. A speculative explanation for the noticeable increase in AH domain helical stability progressing from ctenophore (36%) to worm (54%) to mammal (>80% in human) is the higher body temperatures of warm-blooded animals compared to soil- and marine invertebrates. Helical stability is highly dependent on ambient temperature, so the higher body temperatures of mammals and many other vertebrates may necessitate more stable helical sequences (Privalov, 1982; Scholtz and Baldwin, 1992).

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