Pattern recognition receptors confer plant resistance to pathogen infection by recognizing the conserved pathogen-associated molecular patterns. The cell surface receptor chitin elicitor receptor kinase 1 of Arabidopsis (AtCERK1) directly binds chitin through its lysine motif (LysM)-containing ectodomain (AtCERK1-ECD) to activate immune responses. The crystal structure that we solved of an AtCERK1-ECD complexed with a chitin pentamer reveals that their interaction is primarily mediated by a LysM and three chitin residues. By acting as a bivalent ligand, a chitin octamer induces AtCERK1-ECD dimerization that is inhibited by shorter chitin oligomers. A mutation attenuating chitin-induced AtCERK1-ECD dimerization or formation of nonproductive AtCERK1 dimer by overexpression of AtCERK1-ECD compromises AtCERK1-mediated signaling in plant cells. Together, our data support the notion that chitin-induced AtCERK1 dimerization is critical for its activation.

Brassinosteroids (BRs) are essential phytohormones that play crucial roles in plant growth and development. Perception of BRs requires an active complex of brassinosteroid-insensitive 1 (BRI1) and BRI1-associated kinase 1 (BAK1). Recognized by the extracellular leucine-rich repeat (LRR) domain of BRI1, BRs induce a phosphorylation-mediated cascade to regulate gene expression. Here we present the crystal structures of BRI1-LRR in free and brassinolide (BL)-bound forms. BRI1-LRR exists as a monomer in crystals and solution independent of BL. It comprises a helical solenoid structure that accommodates a separate insertion domain at its concave surface. Sandwiched between them, BL binds to a hydrophobicity-dominating surface groove on BRI1-LRR. BL recognition by BRI1-LRR is through an induced-fit mechanism involving stabilization of two inter-domain loops that creates a pronounced non-polar surface groove for the hormone binding. Together, our results define the molecular mechanisms by which BRI1 recognizes BRs and provide insight into BR-induced BRI1 activation.

Mitochondrial calcium uptake is crucial to the regulation of eukaryotic Ca 2+ homeostasis and is mediated by the mitochondrial calcium uniporter (MCU). While MCU alone can transport Ca 2+ in primitive eukaryotes, metazoans require an essential single membrane-spanning auxiliary component called EMRE to form functional channels; however, the molecular mechanism of EMRE regulation remains elusive. Here, we present the cryo-EM structure of the human MCU-EMRE complex, which defines the interactions between MCU and EMRE as well as pinpoints the juxtamembrane loop of MCU and extended linker of EMRE as the crucial elements in the EMRE-dependent gating mechanism among metazoan MCUs. The structure also features the dimerization of two MCU-EMRE complexes along an interface at the N-terminal domain (NTD) of human MCU that is a hotspot for post-translational modifications. Thus, the human MCU-EMRE complex, which constitutes the minimal channel components among metazoans, provides a framework for future mechanistic studies on MCU.

TRPM4 is a calcium-activated, phosphatidylinositol bisphosphate (PtdInsP2) modulated, non-selective cation channel, and belongs to the family of melastatin-related transient receptor potential (TRPM) channels. Here we present the cryo-EM structures of the mouse TRPM4 channel with and without ATP. TRPM4 consists of multiple transmembrane and cytosolic domains, which assemble into a three-tiered architecture. The N-terminal nucleotide binding domain (NBD) and the C-terminal coiled coil participate in the tetrameric assembly of the channel; ATP binds at NBD and inhibits channel activity. TRPM4 has an exceptionally wide filter but is only permeable to monovalent cations; filter residue Gln973 is essential in defining monovalent selectivity. S1-S4 domain and post-S6 TRP domain form the central gating apparatus that likely house the Ca2+ and PtdInsP2 binding sites. These structures provide an essential starting point for elucidating the complex gating mechanisms of TRPM4 and also reveal the molecular architecture of the TRPM family for the first time.

Organellar two-pore channels (TPCs) function as a homodimer with each subunit containing two homologous Shaker-like 6-TM repeats1. They belong to the voltage-gated ion channel superfamily2 and are ubiquitously expressed in animals and plants3,4. Mammalian TPC1 and TPC2 are localized to the endolysosomal membrane and play critical roles in regulating the physiological functions of these acidic organelles5–7. Here we present the cryo-EM structures of mouse TPC1 (MmTPC1), a voltage-dependent, phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2) activated Na+ selective channel, in both the apo closed and ligand-bound open states which, combined with functional analysis, provide comprehensive structural insights into the selectivity and gating mechanisms of mammalian TPC channels. The channel has a coin slot-shaped ion pathway in the filter that defines the selectivity of mammalian TPCs. Only the voltage sensing domain from the second 6-TM domain confers voltage dependence to MmTPC1. Endolysosome-specific PtdIns(3,5)P2 binds to the first 6-TM domain and activates the channel under depolarizing membrane potential. Structural comparison between the apo and PtdIns(3,5)P2-bound structures elucidates the interplay between voltage and ligand in channel activation. In light of the emerging importance of phosphoinositide regulation of ion channels, the MmTPC1 structures exemplify the lipid binding and regulation in a 6-TM voltage-gated channel.

Transient receptor potential mucolipin 1 (TRPML1) is an endo/lysosomal cation channel ubiquitously expressed in mammalian cells1,2 and its loss-of-function mutations are the direct cause of Type IV mucolipidosis (MLIV), an autosomal recessive lysosomal storage disease3-6. Here we present the single particle cryo-electron microscopy (cryo-EM) structure of the mouse TRPML1 channel embedded in nanodiscs. Combined with mutagenesis, the TRPML1 structure reveals that phosphatidylinositol bisphosphate (PIP2) binds to the N-terminus of the channel – distal from the pore – and the helix-turn-helix extension between S2 and S3 likely couples ligand binding to pore opening. The tightly packed selectivity filter contains multiple ion binding sites and the conserved acidic residues form the luminal Ca2+ blocking site that confers luminal pH and Ca2+ modulation on channel conductance. A luminal linker domain forms a fenestrated canopy atop the channel, providing multiple luminal ion passages to the pore and also creating a negative electrostatic trap – preferably for divalent cations at the luminal entrance. The structure also reveals two equally distributed S4-S5 linker conformations in the closed channel, providing structural implication for the S4-S5 linker-mediated PIP2 gating mechanism among TRPML channels7,8.

Mammalian two-pore channels (TPCs) regulate the physiological functions of the endolysosome. Here we present cryo-EM structures of human TPC2 (HsTPC2), a phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2)-activated, Na+ selective channel, in the ligand-bound and apo states. The apo structure captures the closed conformation, while the ligand-bound form features the channel in both open and closed conformations. Combined with functional analysis, these structures provide insights into the mechanism of PI(3,5)P2-regulated gating of TPC2, which is distinct from that of TPC1. Specifically, the endolysosome-specific PI(3,5)P2 binds at the first 6-TM and activates the channel – independently of the membrane potential – by inducing a structural change at the pore-lining inner helix (IS6), which forms a continuous helix in the open state but breaks into two segments at Gly317 in the closed state. Additionally, structural comparison to the voltage-dependent TPC1 structure allowed us to identify Ile551 as being responsible for the loss of voltage dependence in TPC2.

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