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Architecture of the human TRPM2 channel Adenosine diphosphate–ribose (ADPR) mediates calcium (Ca2+) release by activating the transient receptor potential melastatin 2 (TRPM2) channel. Three structures now elucidate the conformational regulation mechanism of TRPM2 gating. Wang et al. describe cryo–electron microscopy structures of human TRPM2 in the apo, ADPR-bound, and ADPR- and Ca2+-bound states. In the apo state, both intra- and intersubunit interactions appeared to lock TRPM2 into a closed and autoinhibited state. ADPR binding disrupted some interactions and dramatically altered the TRPM2 conformation. Binding of Ca2+ further primed the opening of the channel. Science, this issue p. eaav4809 Gating of the TRPM2 (transient receptor potential melastatin 2) cation channel involves transmembrane helix–linked conformational changes. INTRODUCTION Transient receptor potential (TRP) melastatin 2 (TRPM2) is a Ca2+-permeable, nonselective cation channel implicated in the development of many inflammatory and neurodegenerative diseases. Human TRPM2 has a C-terminal NUDT9H domain, which shares similarity to the NUDT9 enzyme that hydrolyzes adenosine diphosphate (ADP)–ribose (ADPR). Previous studies showed that TRPM2 is coactivated by ADPR and Ca2+. However, the molecular mechanism of human TRPM2 activation remains elusive. RATIONALE To address the gating mechanism of human TRPM2, we aimed to resolve the structures of full-length human TRPM2 in different states. By optimizing expression and purification procedures, we obtained homogeneous recombinant TRPM2 samples from human embryonic kidney (HEK) 293F cells. With single-particle cryo–electron microscopy (cryo-EM), the structures of human TRPM2 alone, in complex with ADPR, and in complex with ADPR and Ca2+ were determined to 3.6-, 6.1-, and 6.4-Å resolution, respectively. RESULTS Human TRPM2 assembles into a tetramer with a three-tier architecture, which resembles other structures in the TRPM family (see the figure). The bottom tier is composed of the C-terminal NUDT9H domain, the N-terminal MHR1/2 and MHR3 domains, and the pole helix. The middle tier consists of the MHR4 domain and the rib helix, whereas the top tier comprises the S1 to S6 transmembrane helices and the TRP helices, including TRP H1. One notable feature in the human TRPM2 apo structure is that the NUDT9H domain, which is responsible for sensing ADPR, as shown by binding affinity measurements, folds back to form extensive interactions with the TRPM2 N-terminal domains both in cis and in trans. Upon ADPR binding, the NUDT9H domain and the MHR1/2 domain undergo a 27° rigid-body rotation, which disrupts the trans interaction between NUDT9H and MHR and may prime the channel for opening. Compared with the ADPR-bound structure, the ADPR and Ca2+–doubly bound TRPM2 undergoes a 15° rotation in the cytoplasmic domain, a tilt of the TRP helix, and a twist of the S6 gating helix to open the channel. The structures collectively provide a full depiction for the mechanism of human TRPM2 activation (see the figure). In addition, our structures highlight several differences in the gating mechanism of TRPM2 across species. In contrast with our observation that the NUDT9H domain of human TRPM2 is required for channel coactivation by ADPR and Ca2+, the open-state structure of zebrafish TRPM2 revealed an unexpected ADPR-binding site at the MHR1/2 domains. To resolve this inconsistency, we demonstrated that NUDT9H of human TRPM2 has a substantially higher affinity to ADPR than that of zebrafish TRPM2, and that mutation of MHR1/2 residues in human TRPM2 equivalent to the ADPR-binding residues in zebrafish TRPM2 does not compromise human TRPM2 channel opening. A second major difference is that the P loop of NUDT9H responsible for the trans interaction in human TRPM2 is absent in NUDT9H of zebrafish TRPM2, which does not closely associate with the MHR arm in the apo state. Moreover, in comparison with sea anemone TRPM2, in which the NUDT9H domain hydrolyzes ADPR but does not contribute to channel opening, NUDT9H of human TRPM2 binds ADPR to promote channel opening but does not degrade ADPR. Together, these species-specific features reflect functional and mechanistic complexity in TRPM2 and the TRP superfamily during evolution. CONCLUSION Structures of human TRPM2 alone, in complex with ADPR, and in complex with ADPR and Ca2+ elucidate the mechanism of TRPM2 gating and provide a framework for the understanding of TRPM2-associated diseases. Although it is conserved across species that TRPM2 is coactivated by ADPR and Ca2+, the organization of the NUDT9H domain and how the orthologs respond to ADPR seem to diverge on the basis of our and previously resolved structures of TRPM2. Moreover, our structures reveal an important role of the TRP helix in TRPM2 gating, which may be universal in many other TRP channels. Activation mechanism of the human TRPM2 channel. Cryo-EM structures of full-length human TRPM2 in apo (closed), ADPR-bound (closed), and ADPR- and Ca2+-bound (open) states and corresponding cartoons that illustrate the gating process of the channel. Transient receptor potential (TRP) melastatin 2 (TRPM2) is a cation channel associated with numerous diseases. It has a C-terminal NUDT9 homology (NUDT9H) domain responsible for binding adenosine diphosphate (ADP)–ribose (ADPR), and both ADPR and calcium (Ca2+) are required for TRPM2 activation. Here we report cryo–electron microscopy structures of human TRPM2 alone, with ADPR, and with ADPR and Ca2+. NUDT9H forms both intra- and intersubunit interactions with the N-terminal TRPM homology region (MHR1/2/3) in the apo state but undergoes conformational changes upon ADPR binding, resulting in rotation of MHR1/2 and disruption of the intersubunit interaction. The binding of Ca2+ further engages transmembrane helices and the conserved TRP helix to cause conformational changes at the MHR arm and the lower gating pore to potentiate channel opening. These findings explain the molecular mechanism of concerted TRPM2 gating by ADPR and Ca2+ and provide insights into the gating mechanism of other TRP channels.