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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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Modeling of the full-length cone channel in its resting state, on the basis of two separate templates.(A) Side view of the TM region of the bacterial channel MlotiK1 in a closed state, PDB entry 3BEH [10]. This structure was the template for modeling the TM region of the cone channel. (B) Side view of the mouse HCN2 CNBDs in a resting state, PDB entry 1Q3E [15]. This structure served as a template for the cytosolic domain of the cone channel. (A, B) The regions of conflict between the templates are in red. (C) Side view of the resultant model structure of the human cone channel, shown in cartoon representation. CNGA3 subunits are colored cyan (light and dark); CNGB3 subunits are colored orange (light and dark).
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pcbi-1003976-g001: Modeling of the full-length cone channel in its resting state, on the basis of two separate templates.(A) Side view of the TM region of the bacterial channel MlotiK1 in a closed state, PDB entry 3BEH [10]. This structure was the template for modeling the TM region of the cone channel. (B) Side view of the mouse HCN2 CNBDs in a resting state, PDB entry 1Q3E [15]. This structure served as a template for the cytosolic domain of the cone channel. (A, B) The regions of conflict between the templates are in red. (C) Side view of the resultant model structure of the human cone channel, shown in cartoon representation. CNGA3 subunits are colored cyan (light and dark); CNGB3 subunits are colored orange (light and dark).

Mentions: We modeled by homology the 3D structure of the cone CNG channel using two distinct templates: the TM region structure of the bacterial channel MlotiK1, and the cytosolic domain structure of the mouse HCN2 channel (Fig. 1). We projected ConSurf [40] conservation scores of the CNGA3 and CNGB3 subunits, composing the cone channel, onto the resultant model to evaluate it (Fig. 2). The evolutionary conservation profile has previously been demonstrated to be a valuable tool for assessing the quality of model structures; the assessment approach is based on the observation that the protein core is typically conserved, whereas residues that face the surroundings, either lipids or water, are variable [29]. Our model structure was compatible with the anticipated evolutionary profile (Fig. 2). The model structure of the TM domain was also compatible with the expected hydrophobicity profile: hydrophobic residues were exposed to the lipid, and charged and polar amino acids were buried in the protein core or located in the loop regions (Figure S1). The model correlates well with the experimental data available for the closely homologous bovine channel CNGA1 (Text S1; Figures S2 and S3). Furthermore, it suggests molecular interpretations for the effects of known clinical mutations (Text S1; Tables S1 and S2; Figure S4).


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)

Modeling of the full-length cone channel in its resting state, on the basis of two separate templates.(A) Side view of the TM region of the bacterial channel MlotiK1 in a closed state, PDB entry 3BEH [10]. This structure was the template for modeling the TM region of the cone channel. (B) Side view of the mouse HCN2 CNBDs in a resting state, PDB entry 1Q3E [15]. This structure served as a template for the cytosolic domain of the cone channel. (A, B) The regions of conflict between the templates are in red. (C) Side view of the resultant model structure of the human cone channel, shown in cartoon representation. CNGA3 subunits are colored cyan (light and dark); CNGB3 subunits are colored orange (light and dark).
© Copyright Policy
Related In: Results  -  Collection

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

pcbi-1003976-g001: Modeling of the full-length cone channel in its resting state, on the basis of two separate templates.(A) Side view of the TM region of the bacterial channel MlotiK1 in a closed state, PDB entry 3BEH [10]. This structure was the template for modeling the TM region of the cone channel. (B) Side view of the mouse HCN2 CNBDs in a resting state, PDB entry 1Q3E [15]. This structure served as a template for the cytosolic domain of the cone channel. (A, B) The regions of conflict between the templates are in red. (C) Side view of the resultant model structure of the human cone channel, shown in cartoon representation. CNGA3 subunits are colored cyan (light and dark); CNGB3 subunits are colored orange (light and dark).
Mentions: We modeled by homology the 3D structure of the cone CNG channel using two distinct templates: the TM region structure of the bacterial channel MlotiK1, and the cytosolic domain structure of the mouse HCN2 channel (Fig. 1). We projected ConSurf [40] conservation scores of the CNGA3 and CNGB3 subunits, composing the cone channel, onto the resultant model to evaluate it (Fig. 2). The evolutionary conservation profile has previously been demonstrated to be a valuable tool for assessing the quality of model structures; the assessment approach is based on the observation that the protein core is typically conserved, whereas residues that face the surroundings, either lipids or water, are variable [29]. Our model structure was compatible with the anticipated evolutionary profile (Fig. 2). The model structure of the TM domain was also compatible with the expected hydrophobicity profile: hydrophobic residues were exposed to the lipid, and charged and polar amino acids were buried in the protein core or located in the loop regions (Figure S1). The model correlates well with the experimental data available for the closely homologous bovine channel CNGA1 (Text S1; Figures S2 and S3). Furthermore, it suggests molecular interpretations for the effects of known clinical mutations (Text S1; Tables S1 and S2; Figure S4).

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