Figure 6.
LRT analysis to examine mechanical coupling between C-linker, HCND, and S4 helix in HCN1. (A) Three positions in a HCN1 subunit in which forces in directions of arrows were applied to A425 in C-Linker (arrow a), R112 in b-helix of HCND (arrow b), and M287 on S4 (arrow c). (B) Three-dimensional organization and concerted movement of C-linker (purple), HCND (orange), and S4 (blue). The counterclockwise rotational movement of the C-linker (purple arrows) upon cAMP binding (arrow a in A) causes horizontal translocation of the HCND along tangent of C-linker displacement. Direct and indirect coupling of the HCND to TMDs evokes tilting of S4. The selected arrows show the direction of movements of all three domains after perturbing the elbow (arrow a in A). (C) Kink formation in S4 domain of HCN1 in response to negative voltage and ligand binding. (C i–iv) S4 domain (in blue) from cryo-EM HCN1 structures in depolarized (PDB 5U6O) (Ci) and hyperpolarized (PDB 6UQF (C ii) condition with kink formation around S272 (orange) (Lee and MacKinnon, 2017; Lee and MacKinnon, 2019). (C iii) S4 domain from same perspective as in C i/C ii with overlay of S4 conformation in apo-like (blue) and holo-like state (cyan). Structures predicted from LRT analysis of HCN1 following application of force to A425 in the C-linker (arrow a in A), which mimics cAMP binding (PDB 5U6O) (Groß et al., 2018; Porro et al., 2019). For comparison of LRT data in C iii with structures in C i/C ii, the S4 structures were normalized to the amino acids 270–272 in the hinge (in orange). (C iv) Apo-like structure from C iii with displacement vectors evoked by force application to A425 (arrow a in A). (D) LRT analysis to examine reciprocity of mechanical coupling between C-linker, HCND, and S4 helix in HCN1. HCN1 was perturbed in three different domains (arrows a–c in A) to elicit movement of C-linker and HCND. Images D i–D iii show C-linkers (in orange) and HCNDs (in red) as tetramers from extracellular perspective in response different perturbations. Irrespectively on whether the force was applied at the elbow by arrow a (D i), at the HCND by arrow b (D ii), or on the S4 domain by arrow c (D iii), the C-linker and the HCND exhibit the same rotational movement as apparent from the direction of the displacement vectors. See caption Fig. 5 for the magnitude/lengths of the arrows. Refer to the image caption for details. Panel A shows three selected sites within an H C N 1 subunit where directional perturbations are applied, including the C-linker, H C N D region, and S 4 helix, to probe their structural responses. Panel B shows the spatial arrangement of the C-linker, H C N D, and S 4 helix, illustrating coordinated movements between these domains, with arrows indicating relative directions of displacement following perturbation. Panel C shows conformational variations of the S 4 helix across different states and conditions, including comparisons of structural bending and alignment, along with predicted conformations derived from analysis. Panel D shows the collective displacement patterns of the C-linker and H C N D across multiple perturbation conditions, demonstrating consistent rotational-like movements irrespective of the initial site of force application.

LRT analysis to examine mechanical coupling between C-linker, HCND, and S4 helix in HCN1. (A) Three positions in a HCN1 subunit in which forces in directions of arrows were applied to A425 in C-Linker (arrow a), R112 in b-helix of HCND (arrow b), and M287 on S4 (arrow c). (B) Three-dimensional organization and concerted movement of C-linker (purple), HCND (orange), and S4 (blue). The counterclockwise rotational movement of the C-linker (purple arrows) upon cAMP binding (arrow a in A) causes horizontal translocation of the HCND along tangent of C-linker displacement. Direct and indirect coupling of the HCND to TMDs evokes tilting of S4. The selected arrows show the direction of movements of all three domains after perturbing the elbow (arrow a in A). (C) Kink formation in S4 domain of HCN1 in response to negative voltage and ligand binding. (C i–iv) S4 domain (in blue) from cryo-EM HCN1 structures in depolarized (PDB 5U6O) (Ci) and hyperpolarized (PDB 6UQF (C ii) condition with kink formation around S272 (orange) (Lee and MacKinnon, 2017; Lee and MacKinnon, 2019). (C iii) S4 domain from same perspective as in C i/C ii with overlay of S4 conformation in apo-like (blue) and holo-like state (cyan). Structures predicted from LRT analysis of HCN1 following application of force to A425 in the C-linker (arrow a in A), which mimics cAMP binding (PDB 5U6O) (Groß et al., 2018; Porro et al., 2019). For comparison of LRT data in C iii with structures in C i/C ii, the S4 structures were normalized to the amino acids 270–272 in the hinge (in orange). (C iv) Apo-like structure from C iii with displacement vectors evoked by force application to A425 (arrow a in A). (D) LRT analysis to examine reciprocity of mechanical coupling between C-linker, HCND, and S4 helix in HCN1. HCN1 was perturbed in three different domains (arrows a–c in A) to elicit movement of C-linker and HCND. Images D i–D iii show C-linkers (in orange) and HCNDs (in red) as tetramers from extracellular perspective in response different perturbations. Irrespectively on whether the force was applied at the elbow by arrow a (D i), at the HCND by arrow b (D ii), or on the S4 domain by arrow c (D iii), the C-linker and the HCND exhibit the same rotational movement as apparent from the direction of the displacement vectors. See caption Fig. 5 for the magnitude/lengths of the arrows.

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