Flexible architecture of IP3R1 by Cryo-EM

Abstract

Inositol 1,4,5-trisphosphate receptors (IP3Rs) play a fundamental role in generating Ca2+ signals that trigger many cellular processes in virtually all eukaryotic cells. Thus far, the three-dimensional (3D) structure of these channels has remained extremely controversial. Here, we report a subnanometer resolution electron cryomicroscopy (cryo-EM) structure of a fully functional type 1 IP3R from cerebellum in the closed state. The transmembrane region reveals a twisted bundle of four α helices, one from each subunit, that form a funnel shaped structure around the 4-fold symmetry axis, strikingly similar to the ion-conduction pore of K+ channels. The lumenal face of IP3R1 has prominent densities that surround the pore entrance and similar to the highly structured turrets of Kir channels. 3D statistical analysis of the cryo-EM density map identifies high variance in the cytoplasmic region. This structural variation could be attributed to genuine structural flexibility of IP3R1.

Figure 1. Functional characterization of the purified IP₃R1

(A) IP₃-induced activation of the purified IP₃R1 reconstituted into lipid vesicles. Time-based Ca²⁺ efflux from IP₃R1- liposomes was monitored by measuring the change in Fura-2 fluorescence. Stepwise addition of D-IP₃ induced Ca²⁺ release from the IP₃R1- liposomes up to ~80% from maximum Ca²⁺ available for efflux in the presence of 3 μM ionomycin. (B) Stereo-specific response of the purified IP₃R1 to activation with the D-IP₃ but not to the L-IP₃ confirms pharmacological identity of the purified channel. The slight increase in Ca²⁺ efflux observed with addition of L-IP₃ is due to the presence of contaminating D-IP₃ in the commercially obtained L-IP₃ (per communication with Sigma-Aldrich) and contaminating Ca²⁺ in the solution (see also Figure S1).

Figure 2. Cryo-EM reconstruction of IP₃R1

(A) 200 kV image of IP₃R1 embedded in a vitreous ice. The scale bar is 500 Å. (B) Projections (a) from the 3D reconstruction, corresponding class-averages generated in the final refinement loop (b), selected reference-free class-averages generated by 2D classification without a 3D reference (c), and a selection of corresponding unaligned particles (d). In our preparation, particle orientation was relatively uniform, meaning we expect resolution to be fairly isotropic. Scale bar is 400 Å (see also Figure S2).

Figure 3. Surface representation of a cryo-EM density map of IP₃R1 channel in the closed state

The 3D structure of RyR1 is shown in three orthogonal views: a top view as seen from the cytoplasmic side, a side view and a bottom view as seen from the lumenal side. Subunits identified in the 3D structure of the channel tetramer are individually colored. The contour level corresponds to a protein mass of ~330 kDa per subunit at protein density of 0.81 Da/ų. The grey bars suggest the positions of the cytoplasmic (Cy) and endoplasmic (ER) leaflets of the membrane bilayer. Scale bar is 100 Š(see also Figure S3 and Movie S1).

Figure 4. The internal architecture of IP₃R1

(A) Two opposing subunits are shown in a side view. (B) The 3D statistical variance map of the IP₃R1 structure. The surfaces of individual IP₃R1 subunits are colored according to the variance map. The threshold for color-coding is set to 2.5σ over the mean density in the 3D variance map. Regions of the highest variance are shown in red, the grey regions show no significant variations. Higher variances are observed in the core of the CY region, with the solvent exposed ‘scaffolding’ of the CY region and the TM region being more rigid. The grey bars indicate the putative position of the membrane bilayer (see also Figure S4 and Movie S2).

Figure 5. Close-up view of the TM region

(A) Putative secondary structure elements identified in the TM structure of IP₃R1. TM regions of two opposing IP₃R1 subunits are viewed from the side parallel to the membrane. The densities attributed to α-helices identified are annotated with cylinders and arbitrarily labeled h1–6 for reference. (B) X-ray structure of the eukaryotic Kir2.2 channel (PDB ID: 3JYC) is shown as a ribbon fitted into the cryo-EM density map of IP₃R1; structural elements of the Kir channel pore are indicated. (C) Secondary structure elements identified in the pore region of IP₃R1 are superimposed with the X-ray structure of the Kir2.2 channel: left panel - two subunits are viewed parallel to the membrane plane; right panel - four subunits are viewed along the channel 4-fold axis from the lumenal side, the pore helices of Kir2.2 channel are removed for clarity. The grey bars mark the putative positions of the Cy/In and ER/Out leaflets of the membrane bilayer (see also Figure S5 and Movie S1).