Phosphatidylinositol 4,5-bisphosphate activation mechanism of human KCNQ5

Figures

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  Fig. 1.. Schematic illustration of the activation of KCNQs. The activation process of KCNQ2/3/5 by voltage and PIP2 is shown in the left column, and the corresponding structural models are deciphered in the right column. In the activation process, KCNQ adopts four conformations: (I) closed gate with resting VSD and unbound PIP2, (II) closed gate with activated VSD and unbound PIP2, (III) closed gate with activated VSD and bound PIP2, and (IV) open gate with activated VSD and bound PIP2. While conformations II and IV have been observed in the structures of KCNQ2, conformation III is unknown. In this study, we present structures of KCNQ5 in conformations II, III, and IV. In the right column, VSD with S4 is shown as a gray box with a yellow cylinder; the S4–S5 linker is shown as a dark gray cylinder; PD is in purple; PIP2 is shown as an orange star. The question marks indicate that the PIP2-bound closed KCNQ structure and the PIP2-bound opened KCNQ5 structure are unknown.

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  Fig. 2.. Structures of KCNQ5–CaM in different ligand-bound states. (A) Representative traces of whole-cell currents in CHO cells expressing full-length human KCNQ5 (KCNQ5FL) evoked by membrane depolarization before and after application of 100 nM HN37. (B) The dose–response curve of HN37 on the current amplitudes of KCNQ5FL at −10 mV. (C) Voltage-dependent activation curves of KCNQ5FL before and after application of 100 nM HN37. (D) Representative traces of the truncated KCNQ5 (KCNQ5∆) before and after application of 100 nM HN37. (E) Voltage-dependent activation curves of KCNQ5FL and KCNQ5∆. (F) The activation efficacy (IDrug/IControl) and half-maximal activation voltage shift (∆V1/2) for 100 nM HN37 on KCNQ5FL and KCNQ5∆. (G) Cryo-EM reconstructions of KCNQ5–CaMapo, KCNQ5–CaMPIP2, KCNQ5–CaMPIP2-HN37-C, and KCNQ5–CaMPIP2-HN37-O. In four maps, KCNQ5 subunits are colored pink, blue, slate blue, and yellow; CaM is colored salmon; PIP2 is colored magenta. (H) Pore radii along the ion permeation pathway in different KCNQ5–CaM structures. Key residues forming the two constriction layers of the activation gate in the closed KCNQ5 structures are labeled.

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  Fig. 3.. Structures of KCNQ5–CaMapo. (A) The overall structure of KCNQ5–CaMapo in the side view (Left) and top view (Right). (B) The side view of one subunit of KCNQ5–CaMapo. (C) Topology of KCNQ5 and CaM. (D) The VSD structure of KCNQ5–CaMapo. For clarity, S1 was omitted. Side chains of gating charge residues and residues forming the gating charge transfer center are shown as sticks. (E) The closed activation gate of KCNQ5–CaMapo. The dashed lines show diagonal atom-to-atom distance (in Å) at the constriction-lining residues Ser348 and Leu352.

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  Fig. 4.. Structures of KCNQ5–CaMPIP2. (A) The overall structure of KCNQ5–CaMPIP2 in the side view (Left) and top view (Right). (B) The binding site of PIP2 (P3 site) in KCNQ5–CaMPIP2. Side chains of residues in KCNQ5 involved in the interactions with PIP2 are shown as sticks. (C) Sequence alignments of the S4–S5 linker and the S6 C-terminal segment of KCNQ1-5. The red stars mark residues for mutagenesis studies. (D) Current densities of CHO cells transfected with WT and mutant KCNQ5FL and KCNQ5∆ at −10 mV. (E) Structural comparison of KCNQ5–CaMapo and KCNQ5–CaMPIP2 in the side view. (F) Structural comparison of PDs in KCNQ5–CaMapo and KCNQ5–CaMPIP2 in the bottom view. (G) Structural comparison of the closed gates in KCNQ5–CaMapo and KCNQ5–CaMPIP2 in the side view. The dashed lines show diagonal atom-to-atom distances between constriction-forming residues (in Å). (H) The PIP2-binding site (P1 site) in KCNQ1 in the open conformation in the side view (Left) and top view (Right). (I) The PIP2-binding site (P2 site) in KCNQ2 in the open conformation in the side view (Left) and top view (Right).

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  Fig. 5.. Structures of KCNQ5–CaMPIP2-HN37-C and KCNQ5–CaMPIP2-HN37-O. (A) The overall structure of KCNQ5–CaMPIP2-HN37-C. (B) The HN37-binding site in KCNQ5–CaMPIP2-HN37-C. Side chains of residues in KCNQ5 involved in the interactions with HN37 are shown as sticks. The dashed lines indicate hydrogen bonds. (C) The binding site of PIP2 (P3 site) in KCNQ5–CaMPIP2-HN37-C. Side chains of residues in KCNQ5 involved in the interactions with PIP2 are shown as sticks. (D) The closed activation gate of KCNQ5–CaMPIP2-HN37-C. The dashed lines show diagonal atom-to-atom distance (in Å) at the constriction-lining residues. (E) The overall structure of KCNQ5–CaMPIP2-HN37-O. (F) The HN37-binding site in KCNQ5–CaMPIP2-HN37-O. Side chains of residues in KCNQ5 involved in the interactions with HN37 are shown as sticks. The dashed lines indicate hydrogen bonds. (G) The P2-binding site of PIP2 in KCNQ5–CaMPIP2-HN37-O. Side chains of residues in KCNQ5 involved in the interactions with PIP2 are shown as sticks. (H) The open activation gate of KCNQ5–CaMPIP2-HN37-O. The dashed lines show diagonal atom-to-atom distance (in Å) at the constriction-lining residues. (I) Structural comparison of PDs in…

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  Fig. 6.. The PIP2 activation mechanism of KCNQ5. (A) Structural comparison of KCNQ5–CaMPIP2-HN37-C (slate blue) and KCNQ5–CaMPIP2-HN37-O (yellow) in side and top views. PIP2 bound in the P3 site in KCNQ5–CaMPIP2-HN37-C is shown in magenta; PIP2 in the P3 and P2 sites in KCNQ5–CaMPIP2-HN37-O are shown in cyan and orange, respectively. (B) The PIP2-induced bending of the S6 C-terminal segment results in the folding of the 364EKR366 motif between S6 and HA to an α helix (in red color). PIP2 and Lys361 in S6 are shown as a stick model; Cα atoms of Arg367 in the N terminus of HA and Phe363 in the C terminus of S6 in both closed and open conformations are shown as spheres. (C) The continuous helix of S6/HA in KCNQ5–CaMPIP2-HN37-O would form clashes with the HB-HC linker in KCNQ5–CaMPIP2-HN37-C. (D) Free energy profiles of PIP2 binding at the P2 and P3 sites in the open and closed conformations of KCNQ5–CaM, derived from umbrella sampling simulations. The x-axis represents the reaction coordinate, defined as the progression from the P2 site to the P3 site. The profiles for the open and closed conformations are shown in yellow and blue, respectively, with error bars displayed as transparent…