Structural insights into the orthosteric inhibition of P2X receptors by non-ATP analog antagonists.

P2X receptors are extracellular ATP-gated ion channels that form homo- or heterotrimers and consist of seven subtypes. They are expressed in various tissues, including neuronal and nonneuronal cells, and play critical roles in physiological processes such as neurotransmission, inflammation, pain, and cancer. As a result, P2X receptors have attracted considerable interest as drug targets, and various competitive inhibitors have been developed. However, although several P2X receptor structures from different subtypes have been reported, the limited structural information of P2X receptors in complex with competitive antagonists hampers the understanding of orthosteric inhibition, hindering the further design and optimization of those antagonists for drug discovery. We determined the cryogenic electron microscopy (cryo-EM) structures of the mammalian P2X7 receptor in complex with two classical competitive antagonists of pyridoxal-5'-phosphate derivatives, pyridoxal-5'-phosphate-6-(2'-naphthylazo-6'-nitro-4',8'-disulfonate) (PPNDS) and pyridoxal phosphate-6-azophenyl-2',5'-disulfonic acid (PPADS), and performed structure-based mutational analysis by patch-clamp recording as well as molecular dynamics (MD) simulations. Our structures revealed the orthosteric site for PPADS/PPNDS, and structural comparison with the previously reported apo- and ATP-bound structures showed how PPADS/PPNDS binding inhibits the conformational changes associated with channel activation. In addition, structure-based mutational analysis identified key residues involved in the PPNDS sensitivity of P2X1 and P2X3, which are known to have higher affinity for PPADS/PPNDS than other P2X subtypes.

Structural insights into the allosteric inhibition of P2X4 receptors.

P2X receptors are ATP-activated cation channels, and the P2X4 subtype plays important roles in the immune system and the central nervous system, particularly in neuropathic pain. Therefore, P2X4 receptors are of increasing interest as potential drug targets. 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. 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.

Crystal structure of the catalytic ATP-binding domain of the PhoR sensor histidine kinase.

The two-component regulatory system (TCS) is a major regulatory system in bacteria that occurs in response to environmental changes and involves the sensor histidine kinase (HK) protein and response regulator (RR) protein. Among the TCSs, PhoR/PhoB is crucial for bacteria to adapt to changes in environmental phosphate concentrations. In addition, recent studies have shown that PhoR binding to the MgtC virulence factor activates phosphate transport for normal pathogenesis. In this work, we determined the crystal structure of the catalytic ATP binding domain of the PhoR sensor histidine kinase from Vibrio cholera, compared the structure with the known HK protein structures and discussed the potential binding interface with MgtC.

Recent Advances in the Structural Biology of Mg2+ Channels and Transporters.

Magnesium ions (Mg2+) are the most abundant divalent cations in living organisms and are essential for various physiological processes, including ATP utilization and the catalytic activity of numerous enzymes. Therefore, the homeostatic mechanisms associated with cellular Mg2+ are crucial for both eukaryotic and prokaryotic organisms and are thus strictly controlled by Mg2+ channels and transporters. Technological advances in structural biology, such as the expression screening of membrane proteins, in meso phase crystallization, and recent cryo-EM techniques, have enabled the structure determination of numerous Mg2+ channels and transporters. In this review article, we provide an overview of the families of Mg2+ channels and transporters (MgtE/SLC41, TRPM6/7, CorA/Mrs2, CorC/CNNM), and discuss the structural biology prospects based on the known structures of MgtE, TRPM7, CorA and CorC.

The long ß2,3-sheets encoded by redundant sequences play an integral role in the channel function of P2X7 receptors.

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 ß2- and ß3-sheets and their linker (loop ß2,3), 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 ß2,3-loop affects P2X7 surface expression but, more importantly, that this loop affects channel gating of P2X7. We propose that the longer ß2,3-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.

Recent progress in the structural biology of P2X receptors.

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.

Fluorescence-detection size-exclusion chromatography utilizing nanobody technology for expression screening of membrane proteins.

GFP fusion-based fluorescence-detection size-exclusion chromatography (FSEC) has been widely employed for membrane protein expression screening. 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 for FSEC analysis. FSEC-Nb enables the evaluation of the expression, monodispersity and thermostability of membrane proteins without the need for purification but does not require direct GFP fusion to targeted proteins. Our results show FSEC-Nb as a powerful tool for expression screening of membrane proteins for structural and functional studies.

A FRET-based screening method to detect potential inhibitors of the binding of CNNM3 to PRL2.

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.

Molecular mechanisms of human P2X3 receptor channel activation and modulation by divalent cation bound ATP.

P2X3 receptor channels expressed in sensory neurons are activated by extracellular ATP and serve important roles in nociception and sensory hypersensitization, making them attractive therapeutic targets. Although several P2X3 structures are known, it is unclear how physiologically abundant Ca2+-ATP and Mg2+-ATP activate the receptor, or how divalent cations regulate channel function. We used structural, computational and functional approaches to show that a crucial acidic chamber near the nucleotide-binding pocket in human P2X3 receptors accommodates divalent ions in two distinct modes in the absence and presence of nucleotide. The unusual engagement between the receptor, divalent ion and the γ-phosphate of ATP enables channel activation by ATP-divalent complex, cooperatively stabilizes the nucleotide on the receptor to slow ATP unbinding and recovery from desensitization, a key mechanism for limiting channel activity. These findings reveal how P2X3 receptors recognize and are activated by divalent-bound ATP, aiding future physiological investigations and drug development.

Structural insights into the competitive inhibition of the ATP-gated P2X receptor channel.

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.

ATP-dependent modulation of MgtE in Mg2+ homeostasis.

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.

Conductance of P2X4 purinergic receptor is determined by conformational equilibrium in the transmembrane region.

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,ß-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,ß-methylene ATP-bound states. Our NMR analyses revealed that, in the α,ß-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 (<100 s(-1)), suggesting that the small population of the open conformation causes the partial activation in this state. Our NMR analyses also revealed that the transmembrane region adopts the open conformation in the state bound to the inhibitor trinitrophenyl-ATP, and thus the antagonism is due to the closure of ion pathways, except for the pore in the transmembrane region: i.e., the lateral cation access in the extracellular region.

Structural Insights into Divalent Cation Modulations of ATP-Gated P2X Receptor Channels.

P2X 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 Zn(2+) 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 Zn(2+) 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 Mg(2+). Overall, our work provides structural insights into the divalent cation modulations of P2X receptors.

Structural basis for dynamic mechanism of proton-coupled symport by the peptide transporter POT.

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 ß-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.

Structural basis for the drug extrusion mechanism by a MATE multidrug transporter.

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 membrane. MATE transporters confer multidrug resistance to bacterial pathogens and cancer cells, 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 medicine. 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 Å 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.

Molecular mechanism of ATP binding and ion channel activation in P2X receptors.

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 ß-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.

Structural basis of novel interactions between the small-GTPase and GDI-like domains in prokaryotic FeoB iron transporter.

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.

Crystallization and preliminary X-ray diffraction analysis of the truncated cytosolic domain of the iron transporter FeoB.

FeoB-family proteins are widely distributed in bacteria and archaea and are involved in high-affinity Fe(2+) uptake through the plasma membrane. FeoB consists of an N-terminal cytosolic region followed by a C-terminal transmembrane region. The cytosolic region contains small GTPase and GDP dissociation inhibitor-like domains, which serve a regulatory function. The truncated cytosolic region of the iron transporter FeoB from Thermotoga maritima was overexpressed, purified and crystallized. Four native or SeMet crystal forms in a nucleotide-free state or in complex with either GDP or GMPPNP diffracted to resolutions of between 1.5 and 2.1 A.

Crystal structure of the MgtE Mg2+ transporter.

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.

Crystallization and preliminary X-ray diffraction analysis of the cytosolic domain of the Mg2+ transporter MgtE.

The MgtE family of Mg(2+) transporters is ubiquitously conserved in all domains of life. The cytosolic domains of the MgtE Mg(2+) transporters include a cystathionine-beta-synthase (CBS) domain which is known to play a regulatory function in transporter proteins. The cytosolic domain of MgtE from Thermus thermophilus was overexpressed, purified and crystallized in the presence and absence of Mg(2+). The crystals formed in the presence of Mg(2+) diffracted X-rays to 2.3 A resolution using synchrotron radiation, belong to space group P6(5)22 with unit-cell parameters a = b = 57.7, c = 317.6 A and are expected to contain one molecule in the asymmetric unit. The crystals formed in the absence of Mg(2+) diffracted X-rays to 3.5 A resolution using synchrotron radiation, belong to space group P2(1)2(1)2(1) with unit-cell parameters a = 77.0, b = 100.3, c = 100.3 A and are expected to contain two molecules in the asymmetric unit.