Visualizing structural transitions of ligand-dependent gating of the TRPM2 channel

Abstract

The transient receptor potential melastatin 2 (TRPM2) channel plays a key role in redox sensation in many cell types. Channel activation requires binding of both ADP-ribose (ADPR) and Ca²⁺. The recently published TRPM2 structures from Danio rerio in the ligand-free and the ADPR/Ca²⁺-bound conditions represent the channel in closed and open states, which uncovered substantial tertiary and quaternary conformational rearrangements. However, it is unclear how these rearrangements are achieved within the tetrameric channel during channel gating. Here we report the cryo-electron microscopy structures of Danio rerio TRPM2 in the absence of ligands, in complex with Ca²⁺ alone, and with both ADPR and Ca²⁺, resolved to ~4.3 Å, ~3.8 Å, and ~4.2 Å, respectively. In contrast to the published results, our studies capture ligand-bound TRPM2 structures in two-fold symmetric intermediate states, offering a glimpse of the structural transitions that bridge the closed and open conformations.

Fig. 1

Overall architecture of TRPM2_DR structures. Cryo-EM reconstructions of TRPM2_{DR_Apo-C4}, TRPM2_{DR_Ca2+}, and TRPM2_{DR_ADPR/Ca2+} structures viewed within the membrane bilayer (a) and from the cytosolic side (b). For TRPM2_{DR_ADPR/Ca2+}, the NUDT9H domain is shown at 0.018 thresholding to emphasize conformational distinctions, while the rest of the molecule is shown at 0.033 thresholding. c Cartoon representations of the TRPM2_{DR_Ca2+} (left) and TRPM2_{DR_ADPR/Ca2+} (right) structures with the same orientation as in a. d Detailed view of the green-colored protomer of TRPM2_{DR_Ca2+} structure in c. The NUDT9H domain is highlighted with a red surface representation. Dashed lines indicate loops and helices that were not modeled in the structure

Fig. 2

Comparison of TRPM2_DR structures in the ligand-free condition. a Alignment (cartoon representation) of a protomer from the TRPM2_{DR_Apo-C4} structure (wheat) and from the TRPM2_closed structure (silver, PDB 6DRK). b Alignment of protomers A (light purple) and B (purple) of the TRPM2_{DR_Apo-pseudo C4} structure. c Alignment of a protomer from the TRPM2_{DR_Apo-C4} structure (wheat) with protomers A/C (left, light purple) and B/D (right, purple) from the TRPM2_{DR_Apo-pseudo C4} structure, respectively

Fig. 3

TRPM2_DR structures in the intermediate states. a Extracellular views sliced through the middle layer of the CDs comprised of the MHR1/2 and MHR3 domains in the TRPM2_closed, TRPM2_{DR_Ca2+}, TRPM2_{DR_ADPR/Ca2+}, and TRPM2_open structures. Structural alignment (cartoon representation) shows that protomer A in the TRPM2_{DR_Ca2+} structure (yellow) resembles the closed conformation in the TRPM2_closed structure (silver, PDB 6DRK) (b), while the protomer B (teal) resembles the open conformation in the TRPM2_open structure (violet, PDB 6DRJ) (c)

Fig. 4

Flexible junctions enable conformational heterogeneity within the channel. a Cartoon representation showing alignment between protomer A (yellow) and protomer B (teal) from the TRPM2_{DR_Ca2+} structure. b Schematic diagram showing the three flexible junctions: NUDT9H-MHR1/2, MHR1/2- MHR3, and VSLD-pore (in green), and the static region: MHR3-MHR4-VSLD and the pore domain (in gray). The pre-S1 domain, TRP domain, and C-terminal helices are omitted for simplicity. c Protomers A (left) and B (right) of the TRPM2_{DR_Ca2+} structure aligned at MHR3 and MHR4 (gray). Side-by-side surface representations indicate that the rotations of MHR1/2 and NUDT9H in protomer A around individual axes (left) lead to the orientations observed in protomer B (right). D90 (yellow) and Q137 (green) from MHR1/2 and V1372 (green) and A1467 (yellow) from NUDT9H are denoted as dots in both protomers. d Protomers A (yellow) and B (teal) of the TRPM2_{DR_Ca2+} structure aligned at VSLD. Close-up view shows the structural divergence occurring at the S4b and the S4–S5 linker regions, giving rise to different configurations of the pore domain

Fig. 5

Addition of ADPR converts protomer configurations at the CD. Cartoon representations showing structural alignment of protomer B in the TRPM2_{DR_Ca2+} structure (teal) and protomer B in the TRPM2_{DR_ADPR/Ca2+} structure (blue) (a), protomer A in the TRPM2_{DR_Ca2+} structure (yellow) and protomer A in the TRPM2_{DR_ADPR/Ca2+} structure (red) (b), and protomer A (red) and protomer B (blue) in the TRPM2_{DR_ADPR/Ca2+} structure (c). Protomers are aligned at MHR4 domains. TMDs and C-terminal domains are colored in gray

Fig. 6

Alternating quaternary structure rearrangements in the cytoplasmic domain (CD). Comparison of the interaction networks between the NUDT9H domain and the neighboring MHR1/2 domain in the TRPM2_closed (a, PDB 6DRK), TRPM2_{DR_Ca2+} (b), and TRPM2_open (c, PDB 6DRJ) structures, respectively. Close-up views show that the NUDT9H domain in the TRPM2_closed structure makes contact solely with MHR1/2 from the same subunit (a), while the core subdomain of the NUDT9H in the TRPM2_open structure (c) makes additional interactions with the MHR1/2 from the adjacent protomer. More importantly, the TRPM2_{DR_Ca2+} structure adopts both interactions mediated by the NUDT9H and MHR1/2 domains (b). Viewed from the intracellular side, cartoon and transparent surface representations compare the conformational changes at the bottom layer of the CD in the TRPM2_closed (d), TRPM2_{DR_Ca2+} (e) and TRPM2_{DR_ADPR/Ca2+} (f) from the current study, and TRPM2_open (g) structures, respectively. Arrows indicate the domain movements observed in the TRPM2_{DR_Ca2+} (e) and TRPM2_{DR_ADPR/Ca2+} (f) structures relative to the channel in the closed conformation (d) and en route to the open conformation (g). Dashed circles highlight the detachment (green) and association (red) between the different domains as a result of structural rearrangements

Fig. 7

Subunit–subunit interfaces in the middle layer of CD. Comparison of the interaction networks between the neighboring subunits mediated by MHR1/2 and MHR3 domains in the TRPM2_closed (a, PDB 6DRK), TRPM2_{DR_Ca2+} (b), and TRPM2_open (c, PDB 6DRJ) structures. Close-up views of regions highlighted by dashed squares in a–c correspondingly. In the TRPM2_{DR_Ca2+} structure, the interface between MHR1/2 in protomer A and MHR3 in protomer D (equivalent to B) (e, left panel) resembles the interfacial network in the TRPM2_closed structure (d); the interface between MHR1/2 in protomer B and MHR3 in protomer A (e, right panel) is similar to that in the TRPM2_open structure (f)

Fig. 8

A trajectory of conformational changes during in the TRPM2_DR channel gating. Viewed from the extracellular side, surface and cartoon representations of the middle layer of CD sliced through TRPM2_closed (a), TRPM2_{DR_Ca2+} (b), TRPM2_{DR_ADPR/Ca2+} (c), and TRPM2_open (d) structures. Individual protomers are highlighted in black frames. The protomer configurations are indicated by type “A” or “B” and also depicted as cartoon diagrams in insets. Cα atoms of the E233 residues are shown as black dots, and distances between Cα atoms are indicated (Å). Viewed from the extracellular side, surface and cartoon representations of the TMD of TRPM2_closed (e), TRPM2_{DR_Ca2+} (f), TRPM2_{DR_ADPR/Ca2+} (g), and TRPM2_open (h) structures. Individual pore domains are highlighted in black frames. The protomer configurations are indicated by type “A” or “B” and also depicted as cartoon diagrams in insets. Cα atoms of the P839 residues are shown as black dots, and distances between Cα atoms are indicated (Å). i Cartoon diagram of alternating quaternary structural rearrangements in TRPM2 channel gating