Abstract
Transient receptor potential melastatin 2 (TRPM2), a Ca2+-permeable cation channel, is gated by intracellular adenosine diphosphate ribose (ADPR), Ca2+, warm temperature, and oxidative stress. It is critically involved in physiological and pathological processes ranging from inflammation to stroke to neurodegeneration. At present, the channel's gating and ion permeation mechanisms, such as the location and identity of the selectivity filter, remain ambiguous. Here, we report the cryo-electron microscopy (cryo-EM) structure of human TRPM2 in nanodisc in the ligand-free state. Cryo-EM map-guided computational modeling and patch-clamp recording further identify a quadruple-residue motif as the ion selectivity filter, which adopts a restrictive conformation in the closed state and acts as a gate, profoundly contrasting with its widely open conformation in the Nematostella vectensis TRPM2. Our study reveals the gating of human TRPM2 by the filter and demonstrates the feasibility of using cryo-EM in conjunction with computational modeling and functional studies to garner structural information for intrinsically dynamic but functionally important domains.
Highlights
Oxidative stress, arising from excessive generation of reactive oxygen species (ROS) and/or impairment in antioxidant defense and strongly implicated in the pathogenesis of a number of diseases, is linked to ion homeostasis and intracellular signaling pathways by regulating ion channel activity (Coyle and Puttfarcken, 1993; Kietzmann et al, 2000)
Previous functional studies suggest that adenosine diphosphate ribose (ADPR) binds to the Transient receptor potential melastatin 2 (TRPM2)-specific NUDT9-H domain in the distal C terminus (Perraud et al, 2001, 2003), which is supported by subsequent mutagenesis studies that identify several residues crucial for ADPR-induced channel activation (Fliegert et al, 2017; Ku€hn and Lu€ckhoff, 2004; Yu et al, 2017)
The 3.76-Aresolution map of hsTRPM2 in the ligand-free state is of high quality in the bulky cytosolic N-terminal domain (NTD) and C-terminal domain (CTD) and major parts of the transmembrane domain (TMD) (Figures 1B and S1)
Summary
Oxidative stress, arising from excessive generation of reactive oxygen species (ROS) and/or impairment in antioxidant defense and strongly implicated in the pathogenesis of a number of diseases, is linked to ion homeostasis and intracellular signaling pathways by regulating ion channel activity (Coyle and Puttfarcken, 1993; Kietzmann et al, 2000). Recent cryo-electron microscopy (cryo-EM) studies have reported structures of human (hs) TRPM2, as well as non-mammalian Nematostella vectensis (nv) and Danio rerio (dr) TRPM2 channels, in ligand-free state, ADPR-bound state, Ca2+-bound state, and ADPR/Ca2+-bound state (Huang et al, 2018, 2019; Wang et al, 2018; Yin et al, 2019; Zhang et al, 2018) These structures demonstrate two ADPR binding sites in human TRPM2 (hsTRPM2), one in the N-terminal MHR1/2 domain and the other in the C-terminal NUDT9-H domain. One Ca2+ binding site in the intracellular S2-S3 loop is revealed and proposed to mediate Ca2+ binding that induces conformational changes leading the ADPR-bound closed channel to open (Huang et al, 2018, 2019; Wang et al, 2018; Yin et al, 2019; Zhang et al, 2018)
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