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Structure, dynamics and implied gating mechanism of a human cyclic nucleotide-gated channel.

Gofman Y, Schärfe C, Marks DS, Haliloglu T, Ben-Tal N - PLoS Comput. Biol. (2014)

Bottom Line: Next, we used elastic network analysis of the model structure to investigate global motions of the channel and to elucidate its gating mechanism.We found the following: (i) In the main mode of motion, the TM and cytosolic domains counter-rotated around the membrane normal.We related this motion to gating, a proposition that is supported by previous experimental data, and by comparison to the known gating mechanism of the bacterial KirBac channel. (ii) The VSDs could facilitate gating (supplementing the pore gate), explaining their presence in such 'voltage-insensitive' channels. (iii) Our elastic network model analysis of the CNGA3 channel supports a modular model of allosteric gating, according to which protein domains are quasi-independent: they can move independently, but are coupled to each other allosterically.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Tel Aviv, Israel.

ABSTRACT
Cyclic nucleotide-gated (CNG) ion channels are nonselective cation channels, essential for visual and olfactory sensory transduction. Although the channels include voltage-sensor domains (VSDs), their conductance is thought to be independent of the membrane potential, and their gating regulated by cytosolic cyclic nucleotide-binding domains. Mutations in these channels result in severe, degenerative retinal diseases, which remain untreatable. The lack of structural information on CNG channels has prevented mechanistic understanding of disease-causing mutations, precluded structure-based drug design, and hampered in silico investigation of the gating mechanism. To address this, we built a 3D model of the cone tetrameric CNG channel, based on homology to two distinct templates with known structures: the transmembrane (TM) domain of a bacterial channel, and the cyclic nucleotide-binding domain of the mouse HCN2 channel. Since the TM-domain template had low sequence-similarity to the TM domains of the CNG channels, and to reconcile conflicts between the two templates, we developed a novel, hybrid approach, combining homology modeling with evolutionary coupling constraints. Next, we used elastic network analysis of the model structure to investigate global motions of the channel and to elucidate its gating mechanism. We found the following: (i) In the main mode of motion, the TM and cytosolic domains counter-rotated around the membrane normal. We related this motion to gating, a proposition that is supported by previous experimental data, and by comparison to the known gating mechanism of the bacterial KirBac channel. (ii) The VSDs could facilitate gating (supplementing the pore gate), explaining their presence in such 'voltage-insensitive' channels. (iii) Our elastic network model analysis of the CNGA3 channel supports a modular model of allosteric gating, according to which protein domains are quasi-independent: they can move independently, but are coupled to each other allosterically.

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Evolutionary coupling (EC) calculations support the model.Contact maps of top-ranked predicted ECs (red) overlaid on monomer (light grey) and intermonomer (dark grey) contacts from (A) the model structure of the TM domain of CNGA3; (B) the model structure of the cytosolic domain of CNGA3. The insets show the contact maps of alternative CNGA3 model structures; the orange arrows point to evolutionary couplings between amino acid pairs that are not in contact in the alternative models, but are in contact in our final model. Clearly, the overlay of the ECs with the contact map is far better for the chosen model structure than for the alternatives.
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pcbi-1003976-g003: Evolutionary coupling (EC) calculations support the model.Contact maps of top-ranked predicted ECs (red) overlaid on monomer (light grey) and intermonomer (dark grey) contacts from (A) the model structure of the TM domain of CNGA3; (B) the model structure of the cytosolic domain of CNGA3. The insets show the contact maps of alternative CNGA3 model structures; the orange arrows point to evolutionary couplings between amino acid pairs that are not in contact in the alternative models, but are in contact in our final model. Clearly, the overlay of the ECs with the contact map is far better for the chosen model structure than for the alternatives.

Mentions: We further evaluated the predicted model structure of the CNGA3 monomer and tetramer using evolutionary couplings calculated separately for the TM domain and for the cytosolic domain. To this end, we used the EVcouplings algorithm [30], [31]. For each domain we carried out the comparison using the 2L/3 residues with the greatest coupling strength, where L is the sequence length; L = 240 in the TM region and L = 197 in the cytoplasmic region; see Methods. In both domains the overlay of the calculated evolutionary couplings and the contacts derived from the model structure was remarkable (Fig. 3): among the residue pairs used in the evaluations of the TM and cytosolic domains, 75% and 92%, respectively, were in contact. A control calculation using the templates revealed similar ratios of contacting evolutionary couplings in the two domains (Figure S5). A more detailed analysis of the couplings detected between residues that were not in contact according to the model structure (or the templates) suggested that most of these are related to flexibility in the loop regions (Text S1).


Structure, dynamics and implied gating mechanism of a human cyclic nucleotide-gated channel.

Gofman Y, Schärfe C, Marks DS, Haliloglu T, Ben-Tal N - PLoS Comput. Biol. (2014)

Evolutionary coupling (EC) calculations support the model.Contact maps of top-ranked predicted ECs (red) overlaid on monomer (light grey) and intermonomer (dark grey) contacts from (A) the model structure of the TM domain of CNGA3; (B) the model structure of the cytosolic domain of CNGA3. The insets show the contact maps of alternative CNGA3 model structures; the orange arrows point to evolutionary couplings between amino acid pairs that are not in contact in the alternative models, but are in contact in our final model. Clearly, the overlay of the ECs with the contact map is far better for the chosen model structure than for the alternatives.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4256070&req=5

pcbi-1003976-g003: Evolutionary coupling (EC) calculations support the model.Contact maps of top-ranked predicted ECs (red) overlaid on monomer (light grey) and intermonomer (dark grey) contacts from (A) the model structure of the TM domain of CNGA3; (B) the model structure of the cytosolic domain of CNGA3. The insets show the contact maps of alternative CNGA3 model structures; the orange arrows point to evolutionary couplings between amino acid pairs that are not in contact in the alternative models, but are in contact in our final model. Clearly, the overlay of the ECs with the contact map is far better for the chosen model structure than for the alternatives.
Mentions: We further evaluated the predicted model structure of the CNGA3 monomer and tetramer using evolutionary couplings calculated separately for the TM domain and for the cytosolic domain. To this end, we used the EVcouplings algorithm [30], [31]. For each domain we carried out the comparison using the 2L/3 residues with the greatest coupling strength, where L is the sequence length; L = 240 in the TM region and L = 197 in the cytoplasmic region; see Methods. In both domains the overlay of the calculated evolutionary couplings and the contacts derived from the model structure was remarkable (Fig. 3): among the residue pairs used in the evaluations of the TM and cytosolic domains, 75% and 92%, respectively, were in contact. A control calculation using the templates revealed similar ratios of contacting evolutionary couplings in the two domains (Figure S5). A more detailed analysis of the couplings detected between residues that were not in contact according to the model structure (or the templates) suggested that most of these are related to flexibility in the loop regions (Text S1).

Bottom Line: Next, we used elastic network analysis of the model structure to investigate global motions of the channel and to elucidate its gating mechanism.We found the following: (i) In the main mode of motion, the TM and cytosolic domains counter-rotated around the membrane normal.We related this motion to gating, a proposition that is supported by previous experimental data, and by comparison to the known gating mechanism of the bacterial KirBac channel. (ii) The VSDs could facilitate gating (supplementing the pore gate), explaining their presence in such 'voltage-insensitive' channels. (iii) Our elastic network model analysis of the CNGA3 channel supports a modular model of allosteric gating, according to which protein domains are quasi-independent: they can move independently, but are coupled to each other allosterically.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Tel Aviv, Israel.

ABSTRACT
Cyclic nucleotide-gated (CNG) ion channels are nonselective cation channels, essential for visual and olfactory sensory transduction. Although the channels include voltage-sensor domains (VSDs), their conductance is thought to be independent of the membrane potential, and their gating regulated by cytosolic cyclic nucleotide-binding domains. Mutations in these channels result in severe, degenerative retinal diseases, which remain untreatable. The lack of structural information on CNG channels has prevented mechanistic understanding of disease-causing mutations, precluded structure-based drug design, and hampered in silico investigation of the gating mechanism. To address this, we built a 3D model of the cone tetrameric CNG channel, based on homology to two distinct templates with known structures: the transmembrane (TM) domain of a bacterial channel, and the cyclic nucleotide-binding domain of the mouse HCN2 channel. Since the TM-domain template had low sequence-similarity to the TM domains of the CNG channels, and to reconcile conflicts between the two templates, we developed a novel, hybrid approach, combining homology modeling with evolutionary coupling constraints. Next, we used elastic network analysis of the model structure to investigate global motions of the channel and to elucidate its gating mechanism. We found the following: (i) In the main mode of motion, the TM and cytosolic domains counter-rotated around the membrane normal. We related this motion to gating, a proposition that is supported by previous experimental data, and by comparison to the known gating mechanism of the bacterial KirBac channel. (ii) The VSDs could facilitate gating (supplementing the pore gate), explaining their presence in such 'voltage-insensitive' channels. (iii) Our elastic network model analysis of the CNGA3 channel supports a modular model of allosteric gating, according to which protein domains are quasi-independent: they can move independently, but are coupled to each other allosterically.

Show MeSH
Related in: MedlinePlus