Structures and gating mechanism of human TRPM2.

INTRODUCTION Transient receptor potential (TRP) melastatin 2 (TRPM2) is a Ca 2+ -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 Ca 2+ . 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 Ca 2+ were determined to 3.6-, 6.1-, and 6.4-A 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 Ca 2+ –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 Ca 2+ , 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 Ca 2+ 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 Ca 2+ , 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.