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P2X receptors are ATP-activated cation channels involved in a variety of physiological functions. Among the seven subtypes of P2X receptors, the P2X4 subtype plays important roles in both the immune system and the central nervous system, particularly in neuropathic pain. Therefore, P2X4 receptors are of increasing interest as potential drug targets, and several P2X4 subtype-specific inhibitors have been developed. However, the mechanism of allosteric inhibition of P2X4 receptors remains largely unclear due to the lack of structural information.Here, we report the cryo-EM structures of the zebrafish P2X4 receptor in complex with two P2X4 subtype-specific antagonists, BX430 and BAY-1797. Both antagonists bind to the same allosteric site located at the subunit interface at the top of the extracellular domain. Structure-based mutational analysis by electrophysiology identified the important residues for the allosteric inhibition of both zebrafish and human P2X4 receptors.Interestingly, in the previously reported apo structure, the binding pocket is closed and too narrow to accommodate allosteric modulators. Structural comparison revealed the ligand-dependent structural rearrangement of the binding pocket to stabilize the binding of allosteric modulators, which in turn would prevent the structural changes of the extracellular domain associated with channel activation. Furthermore, comparison with the previously reported P2X structures of other subtypes provided mechanistic insights into subtype-specific allosteric inhibition.Overall, the present work provides structural insights into the allosteric inhibition mechanism of P2X4 receptors, facilitating the design and optimization of specific modulators for P2X4 receptors.

Transient receptor potential vanilloid member 1 (TRPV1) is a heat and capsaicin receptor that allows cations to permeate and cause pain. As the molecular basis for temperature sensing, the heat capacity (Δ C p ) model [D. E. Clapham, C. Miller, Proc. Natl. Acad. Sci. U.S.A. 108 , 19492–19497 (2011).] has been proposed and experimentally supported. Theoretically, heat capacity is proportional to a variance in enthalpy, presumably related to structural fluctuation; however, the fluctuation of TRPV1 has not been directly visualized. In this study, we directly visualized single-molecule structural fluctuations of the TRPV1 channels in a lipid bilayer with the ligands resiniferatoxin (agonist, 1,000 times hotter than capsaicin) and capsazepine (antagonist) by high-speed atomic force microscopy. We observed the structural fluctuations of TRPV1 in an apo state and found that RTX binding enhances structural fluctuations, while CPZ binding suppresses fluctuations. These ligand-dependent differences in structural fluctuation would play a key role in the gating of TRPV1.

MgtE is a Mg

P2X receptors are ATP-gated trimeric nonselective cation channels that are important for various physiological and pathological processes, including synaptic transmission, pain perception, immune regulation, and apoptosis. Accordingly, they attract a wide range of interest as drug targets, such as those for chronic cough, neuropathic pain, and depression. After the zebrafish P2X4 receptor structure was reported in 2009, various other P2X receptor structures have been reported, extending our understanding of the molecular mechanisms of P2X receptors. This review article describes the recent progress on understanding the structures and mechanisms of P2X receptors, especially of the mechanisms underlying ATP binding and conformational changes during the gating cycle. In addition, since several antagonists for different P2X subtypes have entered into clinical trials, this review also summarizes the binding sites and regulatory mechanisms of these antagonists, which may contribute to new strategies of targeting P2X receptors for drug discovery.

MgtE is a Mg2+-selective ion channel whose orthologs are widely distributed from prokaryotes to eukaryotes, including humans, and play an important role in the maintenance of cellular Mg2+ homeostasis. Previous functional analyses showed that MgtE transports divalent cations with high selectivity for Mg2+ over Ca2+. Whereas the high-resolution structure determination of the MgtE transmembrane (TM) domain in complex with Mg2+ ions revealed a Mg2+ recognition mechanism of MgtE, the previous Ca2+-bound structure of the MgtE TM domain was determined only at moderate resolution (3.2 Å resolution), which was insufficient to visualize the water molecules coordinated to Ca2+ ions. Thus, the structural basis of the ion selectivity of MgtE for Mg2+ over Ca2+ has remained unclear. Here, we showed that the metal-binding site of the MgtE TM domain binds to Mg2+ ∼500-fold more strongly than Ca2+. We then determined the crystal structure of the MgtE TM domain in complex with Ca2+ ions at a higher resolution (2.5 Å resolution), allowing us to reveal hexahydrated Ca2+, which is similarly observed in the previously determined Mg2+-bound structure but with extended metal-oxygen bond lengths. Our structural, biochemical, and computational analyses provide mechanistic insights into the ion selectivity of MgtE for Mg2+ over Ca2+.

Magnesium ions (Mg

Gefapixant/AF-219, a selective inhibitor of the P2X3 receptor, is the first new drug other than dextromethorphan to be approved for the treatment of refractory chronic cough (RCC) in nearly 60 years. To date, seven P2X subtypes (P2X1-7) activated by extracellular ATP have been cloned, and subtype selectivity of P2X inhibitors is a prerequisite for reducing side effects. We previously identified the site and mechanism of action of Gefapixant/AF-219 on the P2X3 receptor, which occupies a pocket consisting of the left flipper (LF) and lower body (LB) domains. However, the mechanism by which AF-219 selectively acts on the P2X3 receptor is unknown. Here, we combined mutagenesis, chimera construction, molecular simulations, covalent occupation and chemical synthesis, and find that the negative allosteric site of AF-219 at P2X3 is also present in other P2X subtypes, at least for P2X1, P2X2 and P2X4. By constructing each chimera of AF-219 sensitive P2X3 and insensitive P2X2 subtypes, the insensitive P2X2 subtype was made to acquire the inhibitory properties of AF-219 and AF-353, an analog of AF-219 with higher affinity. Our results suggest that the selectivity of AF-219/AF-353 for P2X3 over the other P2X subtypes is determined by a combination of the accessibility of P2X3 binding site and the internal shape of this pocket, a finding that could provide new perspectives for drug design against P2X3-mediated diseases such as RCC, idiopathic pulmonary fibrosis, hypertension and overactive bladder disorder.

P2X receptors are a class of nonselective cation channels widely distributed in the immune and nervous systems, and their dysfunction is a significant cause of tumors, inflammation, leukemia, and immune diseases. P2X7 is a unique member of the P2X receptor family with many properties that differ from other subtypes in terms of primary sequence, the architecture of N- and C-terminals, and channel function. Here, we suggest that the observed lengthened β2- and β3-sheets and their linker (loop β2,3), encoded by redundant sequences, play an indispensable role in the activation of the P2X7 receptor. We show that deletion of this longer structural element leads to the loss of P2X7 function. Furthermore, by combining mutagenesis, chimera construction, surface expression, and protein stability analysis, we found that the deletion of the longer β2,3-loop affects P2X7 surface expression but, more importantly, that this loop affects channel gating of P2X7. We propose that the longer β2,3-sheets may have a negative regulatory effect on a loop on the head domain and on the structural element formed by E171 and its surrounding regions. Understanding the role of the unique structure of the P2X7 receptor in the gating process will aid in the development of selective drugs targeting this subtype.

Glycosylphosphatidylinositol (GPI) anchor modification is a posttranslational modification of proteins that has been conserved in eukaryotes. The biosynthesis and transfer of GPI to proteins are carried out in the endoplasmic reticulum. Attachment of GPI to proteins is mediated by the GPI-transamidase (GPI-TA) complex, which recognizes and cleaves the C-terminal GPI attachment signal of precursor proteins. Then, GPI is transferred to the newly exposed C-terminus of the proteins. GPI-TA consists of five subunits: PIGK, GPAA1, PIGT, PIGS, and PIGU, and the absence of any subunit leads to the loss of activity. Here, we analyzed functionally important residues of the five subunits of GPI-TA by comparing conserved sequences among homologous proteins. In addition, we optimized the purification method for analyzing the structure of GPI-TA. Using purified GPI-TA, preliminary single particle images were obtained. Our results provide guidance for the structural and functional analysis of GPI-TA.

Summary The CorC/CNNM family of Na+-dependent Mg2+ transporters is ubiquitously conserved from bacteria to humans. CorC, the bacterial CorC/CNNM family of proteins, is involved in resistance to antibiotic exposure and in the survival of pathogenic microorganisms in their host environment. The CorC/CNNM family proteins possess a cytoplasmic region containing the regulatory ATP-binding site. CorC and CNNM have attracted interest as therapeutic targets, whereas inhibitors targeting the ATP-binding site have not been identified. Here, we performed a virtual screening of CorC by targeting its ATP-binding site, identified a compound named IGN95a with inhibitory effects on ATP binding and Mg2+ export, and determined the cytoplasmic domain structure in complex with IGN95a. Furthermore, a chemical cross-linking experiment indicated that with ATP bound to the cytoplasmic domain, the conformational equilibrium of CorC was shifted more toward the inward-facing state of the transmembrane domain. In contrast, IGN95a did not induce such a shift., Graphical abstract, Highlights • A compound, IGN95a, inhibited ATP binding and Mg2+ export of the CorC Mg2+ transporter • The CorC cytoplasmic domain structure in complex with IGN95a was determined • ATP shifted the CorC conformational equilibrium toward the inward-facing state • In contrast, IGN95a binding did not induce such a shift, Chemical Compound ; Membranes ; Structural Biology

The CorC/CNNM family of Na+-dependent Mg2+ transporters is ubiquitously conserved from bacteria to humans. CorC, the bacterial member of the CorC/CNNM family of proteins, is involved in resistance to antibiotic exposure and in the survival of pathogenic microorganisms in their host environment. The CorC/CNNM family proteins possess a cytoplasmic region containing the regulatory ATP-binding site. While CorC and CNNM have attracted interest as therapeutic targets, inhibitors targeting the ir regulatory ATP-binding site have not yet been identified.Here, we performed a virtual screening of CorC by targeting its regulatory ATP-binding site, identified a chemical compound named IGN95a with inhibitory effects on both ATP binding and Mg2+ export, and determined the cytoplasmic domain structure in complex with IGN95a. Furthermore, a chemical cross-linking experiment indicated that with ATP bound to the cytoplasmic domain, the conformational equilibrium of CorC was shifted more towards the inward-facing state of the transmembrane domain. In contrast, IGN95a did not induce such a shift. Our results provide a structural basis for the further design and optimization of chemical compounds targeting the regulatory ATP-binding site of CorC as well as mechanistic insights into how ATP and chemical compounds modulate the transport activity of CorC.

Crystal structures of the CorC Mg2+ transporter revealed the unique Mg2+ binding site associated with genetic diseases., The CNNM/CorC family proteins are Mg2+ transporters that are widely distributed in all domains of life. In bacteria, CorC has been implicated in the survival of pathogenic microorganisms. In humans, CNNM proteins are involved in various biological events, such as body absorption/reabsorption of Mg2+ and genetic disorders. Here, we determined the crystal structure of the Mg2+-bound CorC TM domain dimer. Each protomer has a single Mg2+ binding site with a fully dehydrated Mg2+ ion. The residues at the Mg2+ binding site are strictly conserved in both human CNNM2 and CNNM4, and many of these residues are associated with genetic diseases. Furthermore, we determined the structures of the CorC cytoplasmic region containing its regulatory ATP-binding domain. A combination of structural and functional analyses not only revealed the potential interface between the TM and cytoplasmic domains but also showed that ATP binding is important for the Mg2+ export activity of CorC.

Membrane proteins play numerous physiological roles and are thus of tremendous interest in pharmacology. Nevertheless, stable and homogeneous sample preparation is one of the bottlenecks in biophysical and pharmacological studies of membrane proteins because membrane proteins are typically unstable and poorly expressed. To overcome such obstacles, GFP fusion-based Fluorescence-detection Size-Exclusion Chromatography (FSEC) has been widely employed for membrane protein expression screening for over a decade. However, fused GFP itself may occasionally affect the expression and/or stability of the targeted membrane protein, leading to both false-positive and false-negative results in expression screening. Furthermore, GFP fusion technology is not well suited for some membrane proteins depending on their membrane topology. Here, we developed an FSEC assay utilizing nanobody (Nb) technology, named FSEC-Nb, in which targeted membrane proteins are fused to a small peptide tag and recombinantly expressed. The whole-cell extracts are solubilized, mixed with anti-peptide Nb fused to GFP and applied to a size-exclusion chromatography column attached to a fluorescence detector for FSEC analysis. FSEC-Nb enables one to evaluate the expression, monodispersity and thermostability of membrane proteins without the need of purification by utilizing the benefits of the GFP fusion-based FSEC method, but does not require direct GFP fusion to targeted proteins. We applied FSEC-Nb to screen zinc-activated ion channel (ZAC) family proteins in the Cys-loop superfamily and membrane proteins from SARS-CoV-2 as examples of the practical application of FSEC-Nb. We successfully identified a ZAC ortholog with high monodispersity but moderate expression levels that could not be identified with the previously developed GFP fusion-free FSEC method. Consistent with the results of FSEC-Nb screening, the purified ZAC ortholog showed monodispersed particles by both negative staining EM and cryo-EM. Furthermore, we identified two membrane proteins from SARS-CoV-2 with high monodispersity and expression level by FSEC-Nb, which may facilitate structural and functional studies of SARS-CoV-2. Overall, our results show FSEC-Nb as a powerful tool for membrane protein expression screening that can provide further opportunity to prepare well-behaved membrane proteins for structural and functional studies.

MgtE is a Mg2+ channel conserved in organisms ranging from prokaryotes to eukaryotes, including humans, and plays an important role in Mg2+ homeostasis. The previously determined MgtE structures in the Mg2+-bound, closed state and structure-based functional analyses of MgtE revealed that the binding of Mg2+ ions to the MgtE cytoplasmic domain induces channel inactivation to maintain Mg2+ homeostasis. However, due to the lack of a structure of the MgtE channel, including its transmembrane domain in Mg2+-free conditions, the pore-opening mechanism of MgtE has remained unclear.Here, we determined the cryoelectron microscopy (cryo-EM) structure of the MgtE-Fab complex in the absence of Mg2+ ions. The Mg2+-free MgtE transmembrane domain structure and its comparison with the Mg2+-bound, closed-state structure, together with functional analyses, showed the Mg2+-dependent pore opening of MgtE on the cytoplasmic side and revealed the kink motions of the TM2 and TM5 helices at the glycine residues, which are important for channel activity. Overall, our work provides structure-based mechanistic insights into the channel gating of MgtE.

Green fluorescent proteins (GFPs) are widely used in biological research. Although GFP can be visualized easily, its precise manipulation through binding partners is still burdensome because of the limited availability of high-affinity binding partners and related structural information. Here, we report the crystal structure of GFPuv in complex with the anti-GFP nanobody LaG16 at 1.67 Å resolution, revealing the details of the binding between GFPuv and LaG16. The LaG16 binding site was on the opposite side of the GFP β-barrel from the binding site of the GFP-enhancer, another anti-GFP nanobody, indicating that the GFP-enhancer and LaG16 can bind to GFP together. Thus, we further designed 3 linkers of different lengths to fuse LaG16 and GFP-enhancer together, and the GFP binding of the three constructs was further tested by ITC. The construct with the (GGGGS)4 linker had the highest affinity with a KD of 0.5 nM. The GFP-enhancer-(GGGGS)4-LaG16 chimeric nanobody was further covalently linked to NHS-activated agarose and then used in the purification of a GFP-tagged membrane protein, GFP-tagged zebrafish P2X4, resulting in higher yield than purification with the GFP-enhancer nanobody alone. This work provides a proof of concept for the design of ultra-high-affinity binders of target proteins through dimerized nanobody chimaeras, and this strategy may also be applied to link interesting target protein nanobodies without overlapping binding surfaces.

The cyclin M (CNNM) family of Mg2+ transporters is reported to promote tumour progression by binding to phosphatase of regenerating liver (PRL) proteins. Here, we established an assay for detection of the binding between the cystathionine-beta-synthase (CBS) domain of human CNNM3 (a region responsible for PRL binding) and human PRL2 using fluorescence resonance energy transfer (FRET) techniques. By fusing YPet to the C-terminus of the CNNM3 CBS domain and CyPet to the N-terminus of PRL2, we performed a FRET-based binding assay with purified proteins in multiwell plates and successfully detected the changes in fluorescence intensity derived from FRET with a reasonable Kd. We then confirmed that the addition of non-YPet-tagged CNNM3 and non-CyPet-tagged PRL proteins inhibited the changes in FRET intensity, whereas non-YPet-tagged CNNM3 with a mutation at the PRL2-binding site did not exhibit such inhibition. Furthermore, newly synthesized peptides derived from the CNNM loop region, with the PRL-binding sequences of the CNNM3 CBS domain, inhibited the interactions between CNNM3 and PRL2. Overall, these results showed that this method can be used for screening to identify inhibitors of CNNM-PRL interactions, potentially for novel anticancer therapy.

Maintenance of cell volume against osmotic change is crucial for proper cell functions. Leucine-rich repeat-containing 8 proteins are anion-selective channels that extrude anions to decrease the cell volume on cellular swelling. Here, we present the structure of human leucine-rich repeat-containing 8A, determined by single-particle cryo-electron microscopy. The structure shows a hexameric assembly, and the transmembrane region features a topology similar to gap junction channels. The LRR region, with 15 leucine-rich repeats, forms a long, twisted arc. The channel pore is located along the central axis and constricted on the extracellular side, where highly conserved polar and charged residues at the tip of the extracellular helix contribute to permeability to anions and other osmolytes. Two structural populations were identified, corresponding to compact and relaxed conformations. Comparing the two conformations suggests that the LRR region is flexible and mobile, with rigid-body motions, which might be implicated in structural transitions on pore opening.

Magnesium is an essential ion for numerous physiological processes. MgtE is a Mg2+ selective channel involved in the maintenance of intracellular Mg2+ homeostasis, whose gating is regulated by intracellular Mg2+ levels. Here, we report that ATP binds to MgtE, regulating its Mg2+-dependent gating. Crystal structures of MgtE–ATP complex show that ATP binds to the intracellular CBS domain of MgtE. Functional studies support that ATP binding to MgtE enhances the intracellular domain affinity for Mg2+ within physiological concentrations of this divalent cation, enabling MgtE to function as an in vivo Mg2+ sensor. ATP dissociation from MgtE upregulates Mg2+ influx at both high and low intracellular Mg2+ concentrations. Using site-directed mutagenesis and structure based-electrophysiological and biochemical analyses, we identify key residues and main structural changes involved in the process. This work provides the molecular basis of ATP-dependent modulation of MgtE in Mg2+ homeostasis. MgtE is an Mg2+ transporter involved in Mg2+ homeostasis. Here, the authors report that ATP regulates the Mg+2-dependent gating of MgtE and use X-ray crystallography combined with functional studies to propose the molecular mechanisms involved in this process.

Ca2+ release from the sarcoplasmic reticulum (SR) and endoplasmic reticulum (ER) is crucial for muscle contraction, cell growth, apoptosis, learning and memory. The trimeric intracellular cation (TRIC) channels were recently identified as cation channels balancing the SR and ER membrane potentials, and are implicated in Ca2+ signaling and homeostasis. Here we present the crystal structures of prokaryotic TRIC channels in the closed state and structure-based functional analyses of prokaryotic and eukaryotic TRIC channels. Each trimer subunit consists of seven transmembrane (TM) helices with two inverted repeated regions. The electrophysiological, biochemical and biophysical analyses revealed that TRIC channels possess an ion-conducting pore within each subunit, and that the trimer formation contributes to the stability of the protein. The symmetrically related TM2 and TM5 helices are kinked at the conserved glycine clusters, and these kinks are important for the channel activity. Furthermore, the kinks of the TM2 and TM5 helices generate lateral fenestrations at each subunit interface. Unexpectedly, these lateral fenestrations are occupied with lipid molecules. This study provides the structural and functional framework for the molecular mechanism of this ion channel superfamily.

MgtE is a Mg2+ channel conserved in organisms ranging from prokaryotes to eukaryotes, including humans, and plays an important role in Mg2+ homeostasis. The previously determined MgtE structures in the Mg2+-bound, closed-state, and structure-based functional analyses of MgtE revealed that the binding of Mg2+ ions to the MgtE cytoplasmic domain induces channel inactivation to maintain Mg2+ homeostasis. There are no structures of the transmembrane (TM) domain for MgtE in Mg2+-free conditions, and the pore-opening mechanism has thus remained unclear. Here, we determined the cryo-electron microscopy (cryo-EM) structure of the MgtE-Fab complex in the absence of Mg2+ ions. The Mg2+-free MgtE TM domain structure and its comparison with the Mg2+-bound, closed-state structure, together with functional analyses, showed the Mg2+-dependent pore opening of MgtE on the cytoplasmic side and revealed the kink motions of the TM2 and TM5 helices at the glycine residues, which are important for channel activity. Overall, our work provides structure-based mechanistic insights into the channel gating of MgtE., MgtE is a magnesium-selective ion channel whose gating is regulated by cytoplasmic magnesium concentration; this cryo-EM study reveals how MgtE undergoes magnesium-dependent structural changes to open the pore on the cytoplasmic side.

Ligand-gated ion channels are partially activated by their ligands, resulting in currents lower than the currents evoked by the physiological full agonists. In the case of P2X purinergic receptors, a cation-selective pore in the transmembrane region expands upon ATP binding to the extracellular ATP-binding site, and the currents evoked by α,β-methylene ATP are lower than the currents evoked by ATP. However, the mechanism underlying the partial activation of the P2X receptors is unknown although the crystal structures of zebrafish P2X4 receptor in the apo and ATP-bound states are available. Here, we observed the NMR signals from M339 and M351, which were introduced in the transmembrane region, and the endogenous alanine and methionine residues of the zebrafish P2X4 purinergic receptor in the apo, ATP-bound, and α,β-methylene ATP-bound states. Our NMR analyses revealed that, in the α,β-methylene ATP-bound state, M339, M351, and the residues that connect the ATP-binding site and the transmembrane region, M325 and A330, exist in conformational equilibrium between closed and open conformations, with slower exchange rates than the chemical shift difference (

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Transmembrane channel-like (TMC) 1 and 2 are required for the mechanotransduction of mouse inner ear hair cells and localize to the site of mechanotransduction in mouse hair cell stereocilia. However, it remains unclear whether TMC1 and TMC2 are indeed ion channels and whether they can sense mechanical force directly. Here we express TMC1 from the green sea turtle (CmTMC1) and TMC2 from the budgerigar (MuTMC2) in insect cells, purify and reconstitute the proteins, and show that liposome-reconstituted CmTMC1 and MuTMC2 proteins possess ion channel activity. Furthermore, by applying pressure to proteoliposomes, we demonstrate that both CmTMC1 and MuTMC2 proteins can indeed respond to mechanical stimuli. In addition, CmTMC1 mutants corresponding to human hearing loss mutants exhibit reduced or no ion channel activity. Taken together, our results show that the CmTMC1 and MuTMC2 proteins are pore-forming subunits of mechanosensitive ion channels, supporting TMC1 and TMC2 as hair cell transduction channels.

Maintenance of cell volume against osmotic change is crucial for proper cell functions, such as cell proliferation and migration. The leucine-rich repeat-containing 8 (LRRC8) proteins are anion selective channels, and were recently identified as pore components of the volume-regulated anion channels (VRACs), which extrude anions to decrease the cell volume upon cell-swelling. Here, we present the human LRRC8A structure, determined by a single-particle cryo-electron microscopy analysis. The sea anemone-like structure represents a trimer of dimers assembly, rather than a symmetrical hexameric assembly. The four-spanning transmembrane region has a gap junction channel-like membrane topology, while the LRR region containing 15 leucine-rich repeats forms a long twisted arc. The channel pore is along the central axis and constricted on the extracellular side, where the highly conserved polar and charged residues at the tip of the extracellular helix contribute to the anion and other osmolyte permeability. Comparing the two structural populations facilitated the identification of both compact and relaxed conformations, suggesting that the LRR region is flexible and mobile with rigid-body motions, which might be implicated in structural transitions upon pore opening. Overall, our structure provides a framework for understanding the molecular mechanisms of this unique class of ion channels.

Allosteric modulation provides exciting opportunities for drug discovery of enzymes, ion channels, and G protein-coupled receptors. As cation channels gated by extracellular ATP, P2X receptors have attracted wide attention as new drug targets. Although small molecules targeting P2X receptors have entered into clinical trials for rheumatoid arthritis, cough, and pain, negative allosteric modulation of these receptors remains largely unexplored. Here, combining X-ray crystallography, computational modeling, and functional studies of channel mutants, we identified a negative allosteric site on P2X3 receptors, fostered by the left flipper (LF), lower body (LB), and dorsal fin (DF) domains. Using two structurally analogous subtype-specific allosteric inhibitors of P2X3, AF-353 and AF-219, the latter being a drug candidate under phase II clinical trials for refractory chronic cough and idiopathic pulmonary fibrosis, we defined the molecular interactions between the drugs and receptors and the mechanism by which allosteric changes in the LF, DF, and LB domains modulate ATP activation of P2X3. Our detailed characterization of this druggable allosteric site should inspire new strategies to develop P2X3-specific allosteric modulators for clinical use.

Magnesium ions (Mg2+) are divalent cations essential for various cellular functions. Mg2+ homeostasis is maintained through Mg2+ channels such as MgtE, a prokaryotic Mg2+ channel whose gating is regulated by intracellular Mg2+ levels. Our previous crystal structure of MgtE in the Mg2+-bound, closed state revealed the existence of seven crystallographically-independent Mg2+-binding sites, Mg1–Mg7. The role of Mg2+-binding to each site in channel closure remains unknown. Here, we investigated Mg2+-dependent changes in the structure and dynamics of MgtE using nuclear magnetic resonance spectroscopy. Mg2+-titration experiments, using wild-type and mutant forms of MgtE, revealed that the Mg2+ binding sites Mg1, Mg2, Mg3, and Mg6, exhibited cooperativity and a higher affinity for Mg2+, enabling the remaining Mg2+ binding sites, Mg4, Mg5, and Mg7, to play important roles in channel closure. This study revealed the role of each Mg2+-binding site in MgtE gating, underlying the mechanism of cellular Mg2+ homeostasis.

P2X receptors are trimeric ATP-gated cation channels involved in diverse physiological processes, ranging from muscle contraction to nociception. Despite the recent structure determination of the ATP-bound P2X receptors, the molecular mechanism of the nucleotide base specificity has remained elusive. Here, we present the crystal structure of zebrafish P2X4 in complex with a weak affinity agonist, CTP, together with structure-based electrophysiological and spectroscopic analyses. The CTP-bound structure revealed a hydrogen bond, between the cytosine base and the side chain of the basic residue in the agonist binding site, which mediates the weak but significant affinity for CTP. The cytosine base is further recognized by two main chain atoms, as in the ATP-bound structure, but their bond lengths seem to be extended in the CTP-bound structure, also possibly contributing to the weaker affinity for CTP over ATP. This work provides the structural insights for the nucleotide base specificity of P2X receptors.

P2X receptors are non-selective cation channels gated by extracellular ATP, and the P2X7 receptor subtype plays a crucial role in the immune and nervous systems. Altered expression and dysfunctions of P2X7 receptors caused by genetic deletions, mutations, and polymorphic variations have been linked to various diseases, such as rheumatoid arthritis and hypertension. Despite the availability of crystal structures of P2X receptors, the mechanism of competitive antagonist action for P2X receptors remains controversial. Here, we determine the crystal structure of the chicken P2X7 receptor in complex with the competitive P2X antagonist, TNP-ATP. The structure reveals an expanded, incompletely activated conformation of the channel, and identified the unique recognition manner of TNP-ATP, which is distinct from that observed in the previously determined human P2X3 receptor structure. A structure-based computational analysis furnishes mechanistic insights into the TNP-ATP-dependent inhibition. Our work provides structural insights into the functional mechanism of the P2X competitive antagonist., P2X receptors are nonselective cation channels that are gated by extracellular ATP. Here the authors present the crystal structure of chicken P2X7 with its bound competitive antagonist TNP-ATP and give mechanistic insights into TNP-ATP dependent inhibition through further computational analysis and electrophysiology measurements.

Multidrug and toxic compound extrusion (MATE) family transporters are conserved in the three primary domains of life (Archaea, Bacteria and Eukarya), and export xenobiotics using an electrochemical gradient of H+ or Na+ across the membrane1, 2. MATE transporters confer multidrug resistance to bacterial pathogens3, 4, 5, 6 and cancer cells7, thus causing critical reductions in the therapeutic efficacies of antibiotics and anti-cancer drugs, respectively. Therefore, the development of MATE inhibitors has long been awaited in the field of clinical medicine8, 9. Here we present the crystal structures of the H+-driven MATE transporter from Pyrococcus furiosus in two distinct apo-form conformations, and in complexes with a derivative of the antibacterial drug norfloxacin and three in vitro selected thioether-macrocyclic peptides, at 2.1?3.0?A resolutions. The structures, combined with functional analyses, show that the protonation of Asp?41 on the amino (N)-terminal lobe induces the bending of TM1, which in turn collapses the N-lobe cavity, thereby extruding the substrate drug to the extracellular space. Moreover, the macrocyclic peptides bind the central cleft in distinct manners, which correlate with their inhibitory activities. The strongest inhibitory peptide that occupies the N-lobe cavity may pave the way towards the development of efficient inhibitors against MATE transporters.

Magnesium is an essential ion for numerous physiological processes. MgtE is a Mg2+ selective channel involved in the maintenance of intracellular Mg2+ homeostasis, whose gating is regulated by intracellular Mg2+ levels. Here, we report that ATP binds to MgtE, regulating its Mg2+-dependent gating. Crystal structures of MgtE–ATP complex show that ATP binds to the intracellular CBS domain of MgtE. Functional studies support that ATP binding to MgtE enhances the intracellular domain affinity for Mg2+ within physiological concentrations of this divalent cation, enabling MgtE to function as an in vivo Mg2+ sensor. ATP dissociation from MgtE upregulates Mg2+ influx at both high and low intracellular Mg2+ concentrations. Using site-directed mutagenesis and structure based-electrophysiological and biochemical analyses, we identify key residues and main structural changes involved in the process. This work provides the molecular basis of ATP-dependent modulation of MgtE in Mg2+ homeostasis., Author summary MgtE is an Mg2+ transporter involved in Mg2+ homeostasis. Here, the authors report that ATP regulates the Mg+2-dependent gating of MgtE and use X-ray crystallography combined with functional studies to propose the molecular mechanisms involved in this process.

SummaryOptimization of membrane protein stability under different solution conditions is essential for obtaining crystals that diffract to high resolution. Traditional methods that evaluate protein stability require large amounts of material and are, therefore, ill suited for medium- to high-throughput screening of membrane proteins. Here we present a rapid and efficient fluorescence-detection size-exclusion chromatography-based thermostability assay (FSEC-TS). In this method, the target protein is fused to GFP. Heated protein samples, treated with a panel of additives, are then analyzed by FSEC. FSEC-TS allows one to evaluate the thermostability of nanogram-to-microgram amounts of the target protein under a variety of conditions without purification. We applied this method to the Danio rerio P2X4 receptor and Caenorhabditis elegans GluCl to screen ligands, ions, and lipids, including newly designed cholesterol derivatives. In the case of GluCl, the screening results were used to obtain crystals of the receptor in the presence of lipids.

P2X receptors are trimeric ATP-activated ion channels permeable to Na+, K+ and Ca2+. The seven P2X receptor subtypes are implicated in physiological processes that include modulation of synaptic transmission, contraction of smooth muscle, secretion of chemical transmitters and regulation of immune responses. Despite the importance of P2X receptors in cellular physiology, the three-dimensional composition of the ATP-binding site, the structural mechanism of ATP-dependent ion channel gating and the architecture of the open ion channel pore are unknown. Here we report the crystal structure of the zebrafish P2X4 receptor in complex with ATP and a new structure of the apo receptor. The agonist-bound structure reveals a previously unseen ATP-binding motif and an open ion channel pore. ATP binding induces cleft closure of the nucleotide-binding pocket, flexing of the lower body β-sheet and a radial expansion of the extracellular vestibule. The structural widening of the extracellular vestibule is directly coupled to the opening of the ion channel pore by way of an iris-like expansion of the transmembrane helices. The structural delineation of the ATP-binding site and the ion channel pore, together with the conformational changes associated with ion channel gating, will stimulate development of new pharmacological agents.

Summary The FeoB family proteins are widely distributed prokaryotic membrane proteins involved in Fe 2+ uptake. FeoB consists of N-terminal cytosolic and C-terminal transmembrane domains. The N-terminal region of the cytosolic domain is homologous to small GTPase (G) proteins and is considered to regulate Fe 2+ uptake. The spacer region connecting the G and TM domains reportedly functions as a GDP dissociation inhibitor (GDI)–like domain that stabilizes the GDP-binding state. However, the function of the G and GDI-like domains in iron uptake remains unclear. Here, we report the structural and functional analyses of the FeoB cytosolic domain from Thermotoga maritima. The structure-based mutational analysis indicated that the interaction between the G and GDI-like domains is important for both the GDI and Fe 2+ uptake activities. On the basis of these results, we propose a regulatory mechanism of Fe 2+ uptake.

The magnesium ion Mg2+ is a vital element involved in numerous physiological processes. Mg2+ has the largest hydrated radius among all cations, whereas its ionic radius is the smallest. It remains obscure how Mg2+ transporters selectively recognize and dehydrate the large, fully hydrated Mg2+ cation for transport. Recently the crystal structures of the CorA Mg2+ transporter were reported. The MgtE family of Mg2+ transporters is ubiquitously distributed in all phylogenetic domains, and human homologues have been functionally characterized and suggested to be involved in magnesium homeostasis. However, the MgtE transporters have not been thoroughly characterized. Here we determine the crystal structures of the full-length Thermus thermophilus MgtE at 3.5 A resolution, and of the cytosolic domain in the presence and absence of Mg2+ at 2.3 A and 3.9 A resolutions, respectively. The transporter adopts a homodimeric architecture, consisting of the carboxy-terminal five transmembrane domains and the amino-terminal cytosolic domains, which are composed of the superhelical N domain and tandemly repeated cystathionine-beta-synthase domains. A solvent-accessible pore nearly traverses the transmembrane domains, with one potential Mg2+ bound to the conserved Asp 432 within the pore. The transmembrane (TM)5 helices from both subunits close the pore through interactions with the 'connecting helices', which connect the cystathionine-beta-synthase and transmembrane domains. Four putative Mg2+ ions are bound at the interface between the connecting helices and the other domains, and this may lock the closed conformation of the pore. A structural comparison of the two states of the cytosolic domains showed the Mg2+-dependent movement of the connecting helices, which might reorganize the transmembrane helices to open the pore. These findings suggest a homeostasis mechanism, in which Mg2+ bound between cytosolic domains regulates Mg2+ flux by sensing the intracellular Mg2+ concentration. Whether this presumed regulation controls gating of an ion channel or opening of a secondary active transporter remains to be determined.

SummaryP2X receptors are trimeric ATP-gated cation channels involved in physiological processes ranging widely from neurotransmission to pain and taste signal transduction. The modulation of the channel gating, including that by divalent cations, contributes to these diverse physiological functions of P2X receptors. Here, we report the crystal structure of an invertebrate P2X receptor from the Gulf Coast tick Amblyomma maculatum in the presence of ATP and Zn2+ ion, together with electrophysiological and computational analyses. The structure revealed two distinct metal binding sites, M1 and M2, in the extracellular region. The M1 site, located at the trimer interface, is responsible for Zn2+ potentiation by facilitating the structural change of the extracellular domain for pore opening. In contrast, the M2 site, coupled with the ATP binding site, might contribute to regulation by Mg2+. Overall, our work provides structural insights into the divalent cation modulations of P2X receptors.

To achieve accurate aminoacylation of tRNAs with their cognate amino acids, errors in aminoacylation are corrected by the “editing” mechanism in several aminoacyl-tRNA synthetases. Phenylalanyl-tRNA synthetase (PheRS) hydrolyzes, or edits, misformed tyrosyl-tRNA with its editing domain in the β subunit. We report the crystal structure of an N-terminal fragment of the PheRS β subunit (PheRS-β N ) from the archaeon, Pyrococcus horikoshii , at 1.94-Å resolution. PheRS-β N includes the editing domain B3/4, which has archaea/eukarya-specific insertions/deletions and adopts a different orientation relative to other domains, as compared with that of bacterial PheRS. Surprisingly, most residues constituting the editing active-site pocket were substituted between the archaeal/eukaryal and bacterial PheRSs. We prepared Ala-substituted mutants of P. horikoshii PheRS for 16 editing-pocket residues, of which 12 are archaea/eukarya-specific and four are more widely conserved. On the basis of their activities, Tyr-adenosine was modeled on the B3/4-domain structure. First, the mutations of Leu-202, Ser-211, Asp-234, and Thr-236 made the PheRS incorrectly hydrolyze the cognate Phe-tRNA Phe , indicating that these residues participate in the Tyr hydroxy group recognition and are responsible for discrimination against Phe. Second, the mutations of Leu-168 and Arg-223, which could interact with the tRNA 3′-terminal adenosine, reduced Tyr-tRNA Phe deacylation activity. Third, the mutations of archaea/eukarya-specific Gln-126, Glu-127, Arg-137, and Asn-217, which are proximal to the ester bond to be cleaved, also reduced Tyr-tRNA Phe deacylation activity. In particular, the replacement of Asn-217 abolished the activity, revealing its absolute requirement for the catalysis.

Magnesium is the most abundant divalent cation in living cells and is crucial to several biological processes. MgtE is a Mg2+ channel distributed in all domains of life that contributes to the maintenance of cellular Mg2+ homeostasis. Here we report the high-resolution crystal structures of the transmembrane domain of MgtE, bound to Mg2+, Mn2+ and Ca2+. The high-resolution Mg2+-bound crystal structure clearly visualized the hydrated Mg2+ ion within its selectivity filter. Based on those structures and biochemical analyses, we propose a cation selectivity mechanism for MgtE in which the geometry of the hydration shell of the fully hydrated Mg2+ ion is recognized by the side-chain carboxylate groups in the selectivity filter. This is in contrast to the K+-selective filter of KcsA, which recognizes a dehydrated K+ ion. Our results further revealed a cation-binding site on the periplasmic side, which regulate channel opening and prevents conduction of near-cognate cations., MgtE is a magnesium ion-selective channel conserved in all domains of life that contributes to the maintenance of cellular Mg2+ homeostasis. Here, the authors provide high-resolution crystal structures of MgtE combined with biochemical analyses that reveal the molecular basis for selectivity.

Proton-dependent oligopeptide transporters (POTs) are major facilitator superfamily (MFS) proteins that mediate the uptake of peptides and peptide-like molecules, using the inwardly directed H + gradient across the membrane. The human POT family transporter peptide transporter 1 is present in the brush border membrane of the small intestine and is involved in the uptake of nutrient peptides and drug molecules such as β-lactam antibiotics. Although previous studies have provided insight into the overall structure of the POT family transporters, the question of how transport is coupled to both peptide and H + binding remains unanswered. Here we report the high-resolution crystal structures of a bacterial POT family transporter, including its complex with a dipeptide analog, alafosfalin. These structures revealed the key mechanistic and functional roles for a conserved glutamate residue (Glu310) in the peptide binding site. Integrated structural, biochemical, and computational analyses suggested a mechanism for H + -coupled peptide symport in which protonated Glu310 first binds the carboxyl group of the peptide substrate. The deprotonation of Glu310 in the inward open state triggers the release of the bound peptide toward the intracellular space and salt bridge formation between Glu310 and Arg43 to induce the state transition to the occluded conformation.

ATP-gated P2X receptors and acid-sensing ion channels are cation-selective, trimeric ligand-gated ion channels unrelated in amino acid sequence. Nevertheless, initial crystal structures of the P2X4 receptor and acid-sensing ion channel 1a in resting/closed and in non conductive/desensitized conformations, respectively, revealed common elements of architecture. Recent structures of both channels have revealed the ion channels in open conformations. Here we focus on common elements of architecture, conformational change and ion permeation, emphasizing general principles of structure and mechanism in P2X receptors and in acid-sensing ion channels and showing how these two sequence-disparate families of ligand-gated ion channel harbor unexpected similarities when viewed through a structural lens.

MgtE is a prokaryotic Mg(2+) transporter that controls cellular Mg(2+) concentrations. We previously reported crystal structures of the cytoplasmic region of MgtE, consisting of 2 domains, that is, N and CBS, in the Mg(2+)-free and Mg(2+)-bound forms. The Mg(2+)-binding sites lay at the interface of the 2 domains, making the Mg(2+)-bound form compact and globular. In the Mg(2+)-free structure, however, the domains are far apart, and the Mg(2+)-binding sites are destroyed. Therefore, it is unclear how Mg(2+)-free MgtE changes its conformation to accommodate Mg(2+) ions. Here, we used paramagnetic relaxation enhancement (PRE) to characterize the relative orientation of the N and CBS domains in the absence of Mg(2+) in solution. When the residues on the surface of the CBS domain were labeled with nitroxide tags, significant PRE effects were observed for the residues in the N domain. No single structure satisfied the PRE profiles, suggesting that the N and CBS domains are not fixed in a particular orientation in solution. We then conducted ensemble simulated annealing calculations in order to obtain the atomic probability density and visualize the spatial distribution of the N domain in solution. The results indicate that the N domain tends to occupy the space near its position in the Mg(2+)-bound crystal structure, facilitating efficient capture of Mg(2+) with increased intracellular Mg(2+) concentration, which is necessary to close the gate.