Cryo-EM reveals ligand induced allostery underlying InsP3R channel gating

Abstract

Inositol-1,4,5-trisphosphate receptors (InsP₃Rs) are cation channels that mobilize Ca²⁺ from intracellular stores in response to a wide range of cellular stimuli. The paradigm of InsP₃R activation is the coupled interplay between binding of InsP₃ and Ca²⁺ that switches the ion conduction pathway between closed and open states to enable the passage of Ca²⁺ through the channel. However, the molecular mechanism of how the receptor senses and decodes ligand-binding signals into gating motion remains unknown. Here, we present the electron cryo-microscopy structure of InsP₃R1 from rat cerebellum determined to 4.1 Å resolution in the presence of activating concentrations of Ca²⁺ and adenophostin A (AdA), a structural mimetic of InsP₃ and the most potent known agonist of the channel. Comparison with the 3.9 Å-resolution structure of InsP₃R1 in the Apo-state, also reported herein, reveals the binding arrangement of AdA in the tetrameric channel assembly and striking ligand-induced conformational rearrangements within cytoplasmic domains coupled to the dilation of a hydrophobic constriction at the gate. Together, our results provide critical insights into the mechanistic principles by which ligand-binding allosterically gates InsP₃R channel.

Fig. 1

The cryo-EM structure of InsP₃R1 channel bound with AdA. a The cryo-EM density map of the InsP₃R1-AdA complex is viewed from cytosol along its four-fold axis (left) and along the membrane plane (right). The map is filtered to 4.1 Å and corrected with a temperature factor of −100 Ų. The domains within each protomer are delineated by different colours. Densities corresponding to AdA are coloured magenta. b Zoomed-in view of the AdA density present in the ligand-binding pocket. AdA-InsP₃R1 structure is overlaid on the corresponding density map and coloured by domain; the AdA molecule is fit in the density adjoining the LBDs. c AdA molecule is shown in the InsP₃R1 binding pocket and is overlaid with densities (mesh) from the difference map (4σ). Residues within 5 Å distance from the docked AdA molecule are labeled and coloured magenta

Fig. 2

Structural rearrangements in the ligand-binding pocket of InsP₃R1. a Shown is an overlay of ribbon diagrams of the ARM1 and β-TF2 domains from Apo-InsP₃R1 (light purple) and AdA-InsP₃R1 (green). The structure of AdA molecule is colour-coded by element (phosphorous - orange; oxygen - red; nitrogen - blue; carbon - grey) and shown within the binding pocket; 2′-, 3′′- and 4′′- phosphates are labeled. Distances between Cα atoms at the narrowest point within the ligand binding pocket for AdA-bound (between T266 and K569) and Apo-InsP₃R1 (between G268 and K569) are indicated. b Overlay of the AdA- (green) and Apo- (light purple) InsP₃R1 structures zoomed in at the β-TF1/β-TF2′ interface. Y167 is depicted as a sphere. c The Cα RMS deviations calculated between Apo- and AdA-bound structures are shown in the AdA-bound LBD structure colour-coded based on RMS deviation - ribbon color/thickness denotes lowest RMSD (blue/thinnest) to highest RMSD (orange/thickest). The most variable residues contributing to intra- and inter-domain contacts are labeled. Wide arrows point to the interacting domains of neighboring subunits. The prime symbol (′) is used to differentiate subunits

Fig. 3

Conformational changes in the ion conduction pathway upon ligand binding. a Solvent-accessible pathway along the pore mapped using the program HOLE for Apo- (light purple) and AdA-bound (green) InsP₃R1. A series of residues within the ion conduction pathway of the channel pore are labeled. b Comparison of pore diameter for Apo- (light purple) and AdA-bound (green) InsP₃R1. c–d Sections of density maps perpendicular to the four-fold axis at the position of F2586 (c) and I2590 (d) are shown overlaid with their corresponding models and viewed from the cytosol; Apo-InsP₃R1 model is shown in light purple, AdA-InsP₃R1 is shown in green; distances between the sidechains from two opposing TM6 helices are indicated; corresponding side-chain rotations are indicated in the right panels. e Zoomed-in views of the bulge in TM6 seen in AdA-InsP₃R1 (green) overlaid on TM6 from Apo-InsP₃R1 (light purple). The lower panel represents a ~ 40° rotation in view from the upper panel

Fig. 4

Detailed views of the selectivity filter in InsP₃R1. a Sequence alignment for the P-loop region of selected InsP₃R channels; the secondary structure elements are given above sequences; highly conserved residues within the signature sequence of the SF are coloured cyan; residues undergoing significant conformational changes are coloured yellow; D2551 within the SF, for which mutations can abolish Ca²⁺ conductance, is colored red; b EM densities with the corresponding models for the SF of Apo- (light purple) and AdA-InsP₃R1 (green) are viewed from the cytosolic side. Cα atoms within the SF are depicted as spheres with the same colour code as in (a). c The SF (wire representation) from two opposing subunits is viewed from the side in Apo- (light purple) and AdA-InsP₃R1 (green); distances between Cα atoms (spheres, colour-coded as in (a)) along the SF are indicated; d The surface electrostatic potential in the SF. Top panels show cross-sections along the 4-fold axis through the ion conduction pathway in Apo- (left) and AdA-InsP₃R1 (right); lumenal entrance at the bottom. Bottom panels slices through the channel pore perpendicular to the 4-fold axis at the positions indicated with dashed lines in upper panels (viewed from lumen)

Fig. 5

Inter-subunit contacts within cytoplasmic region. a Superimposition of interfaces between β-TF1 and ARM3′ domains in AdA-bound (green) and Apo- (light purple) InsP₃R1 structures; helices are rendered as cylinders. The lower panel shows the ATP-binding consensus (GXGXXG, indicated in blue) within the ARM3 domain. b Interface between β-TF2 and CTD′′′ domains in AdA-bound (green) and Apo- (light purple) InsP₃R1 structures is shown in view parallel to the membrane plane. β-TF2 residues within 5 Å of the CTD are coloured blue and labeled. Right panel shows the overlay of AdA- (green) and Apo- (grey) InsP₃R1 models. Notable structural changes are indicated. In the AdA-InsP₃R1, the CTD helix exhibits pronounced rotation compared to its position in the Apo-structure

Fig. 6

Domain rearrangements at the cytosolic-membrane interface. Two orthogonal views of domains at the cytosolic-membrane interface in the Apo- (left) and AdA-bound (right) InsP₃R1 structures (colour-coded by domain). Structural differences are notable in the CTD helices (red), which are connected to the LNK domain (orange). Ligand-triggered conformational changes are ultimately funneled to the TMDs (purple) through the interface comprised of the ILD/LNK sandwich. The lower panels show two opposing subunits for clarity. Inserts show zoomed-in views (indicated with dashed-lines) of domains from one subunit of AdA-InsP₃R1 that are colour-coded based on the Cα RMS deviations calculated between Apo- and AdA-structures: ribbon color/thickness denotes lowest RMS (blue/thinnest) to highest RMS (orange/thickest) deviations

Fig. 7

Schematic of ligand-induced conformational changes underlying activation of InsP₃R1. Top view of the channel along the 4-fold axis from the cytosolic side (left panel). Depicted are the LBDs and CTDs for each InsP₃R1 protomer coloured by subunit. Section of tetrameric InsP₃R1 through its 4-fold axis is viewed parallel to the membrane plane (right panel); regions of constriction and SF are indicted with red circles, Ca²⁺ ion - yellow sphere. Ligand-evoked domain motions are indicated with arrows. Presumably, conformational changes evoked by binding of AdA between the β-TF2 and ARM1 domains are propagated via several inter-subunit interfaces (β-TF1/β-TF2, CTD/β-TF2 and β-TF1/ARM3) in the cytoplasmic solenoid structure to the ILD/LNK assembly that can exert force directly on the TMDs to open the channel gate