Molecular basis of Mg2+ permeation through the human mitochondrial Mrs2 channel.

Mitochondrial RNA splicing 2 (Mrs2), a eukaryotic CorA ortholog, enables Mg2+ to permeate the inner mitochondrial membrane and plays an important role in mitochondrial metabolic function. However, the mechanism by which Mrs2 permeates Mg2+ remains unclear. Here, we report four cryo-electron microscopy (cryo-EM) reconstructions of Homo sapiens Mrs2 (hMrs2) under various conditions. All of these hMrs2 structures form symmetrical pentamers with very similar pentamer and protomer conformations. A special structural feature of Cl--bound R-ring, which consists of five Arg332 residues, was found in the hMrs2 structure. Molecular dynamics simulations and mitochondrial Mg2+ uptake assays show that the R-ring may function as a charge repulsion barrier, and Cl- may function as a ferry to jointly gate Mg2+ permeation in hMrs2. In addition, the membrane potential is likely to be the driving force for Mg2+ permeation. Our results provide insights into the channel assembly and Mg2+ permeation of hMrs2.

Quantitative analysis of the effect of radiation on mitochondria structure using coherent diffraction imaging with a clustering algorithm.

Radiation damage and a low signal-to-noise ratio are the primary factors that limit spatial resolution in coherent diffraction imaging (CDI) of biomaterials using X-ray sources. Introduced here is a clustering algorithm named ConvRe based on deep learning, and it is applied to obtain accurate and consistent image reconstruction from noisy diffraction patterns of weakly scattering biomaterials. To investigate the impact of X-ray radiation on soft biomaterials, CDI experiments were performed on mitochondria from human embryonic kidney cells using synchrotron radiation. Benefiting from the new algorithm, structural changes in the mitochondria induced by X-ray radiation damage were quantitatively characterized and analysed at the nanoscale with different radiation doses. This work also provides a promising approach for improving the imaging quality of biomaterials with XFEL-based plane-wave CDI.

Cryo-EM structure of the human TACAN in a closed state.

TACAN is an ion channel-like protein that may be involved in sensing mechanical pain. Here, we present the cryo-electron microscopic structure of human TACAN (hTACAN). hTACAN forms a dimer in which each protomer consists of a transmembrane globular domain (TMD) containing six helices and an intracellular domain (ICD) containing two helices. Molecular dynamic simulations suggest that each protomer contains a putative ion conduction pore. A single-point mutation of the key residue Met207 greatly increases membrane pressure-activated currents. In addition, each hTACAN subunit binds one cholesterol molecule. Our data show the molecular assembly of hTACAN and suggest that wild-type hTACAN is in a closed state.

Cryo-EM structure of the heptameric calcium homeostasis modulator 1 channel.

Calcium homeostasis modulator 1 (CALHM1) is a voltage- and Ca2+-gated ATP channel that plays an important role in neuronal signaling. However, as the previously reported CALHM structures are all in the ATP-conducting state, the gating mechanism of ATP permeation is still elusive. Here, we report cryo-EM reconstructions of two Danio rerio CALHM1 heptamers with ordered or flexible long C-terminal helices at resolutions of 3.2 Å and 2.9 Å, respectively, and one D. rerio CALHM1 octamer with flexible long C-terminal helices at a resolution of 3.5 Å. Structural analysis shows that the heptameric CALHM1s are in an ATP-nonconducting state with a central pore diameter of approximately 6.6 Å. Compared with those inside the octameric CALHM1, the N-helix inside the heptameric CALHM1 is in the "down" position to avoid steric clashing with the adjacent TM1 helix. Molecular dynamics simulations show that as the N-helix moves from the "down" position to the "up" position, the pore size of ATP molecule permeation increases significantly. Our results provide important information for elucidating the mechanism of ATP molecule permeation in the CALHM1 channel.

Cryo-EM structure of AMP-PNP-bound human mitochondrial ATP-binding cassette transporter ABCB7.

ATP-binding cassette subfamily B member 7 (ABCB7) is localized in the inner membrane of mitochondria, playing a critical role in iron metabolism. Here, we determined the structure of the nonhydrolyzable ATP analog adenosine-5'-(ß-γ-imido) triphosphate (AMP-PNP) bound human ABCB7 at 3.3 Å by single-particle electron cryo-microscopy (cryo-EM). The AMP-PNP-bound human ABCB7 shows an inverted V-shaped homodimeric architecture with an inward-facing open conformation. One AMP-PNP molecule and Mg2+ were identified in each nucleotide-binding domain (NBD) of the hABCB7 monomer. Moreover, four disease-causing missense mutations of human ABCB7 have been mapped to the structure, creating a hotspot map for X-linked sideroblastic anemia and ataxia disease. Our results provide a structural basis for further understanding the transport mechanism of the mitochondrial ABC transporter.

Expression, purification and microscopic characterization of transmembrane BAX Inhibitor-1 motif containing protein 5.

Transmembrane bax inhibitor-1 motif containing protein 5 (TMBIM5) is located on the inner membrane of mitochondria and is widely expressed in tissues but less frequently in the intestine and thymus. TMBIM5 affects mitochondrial cristae organization and is associated with Parkinson's disease. Here, we present the first report about expression, purification and the 2D classification projections derived from negatively stained electron micrographs of recombinant H. sapiens TMBIM5 (hTMBIM5). The described methods and results will support further structural and functional study of hTMBIM5.

Structural Insights into Ca2+ Permeation through Orai Channels.

Orai channels belong to the calcium release-activated calcium (CRAC) channel family. Orai channels are responsible for the influx of extracellular Ca2+ that is triggered by Ca2+ depletion from the endoplasmic reticulum (ER); this function is essential for many types of non-excitable cells. Extensive structural and functional studies have advanced the knowledge of the molecular mechanism by which Orai channels are activated. However, the gating mechanism that allows Ca2+ permeation through Orai channels is less well explained. Here, we reviewed and summarized the existing structural studies of Orai channels. We detailed the structural features of Orai channels, described structural comparisons of their closed and open states, and finally proposed a "push-pull" model of Ca2+ permeation.

The BAG2 and BAG6 Genes Are Involved in Multiple Abiotic Stress Tolerances in Arabidopsis Thaliana.

The BAG proteins are a family of multi-functional co-chaperones. In plants, BAG proteins were found to play roles both in abiotic and biotic stress tolerance. However, the function of Arabidopsis BAG2 remains largely unknown, whereas BAG6 is required for plants' defense to pathogens, although it remains unknown whether BAG6 is involved in plants' tolerance to abiotic stresses. Here, we show that both BAG2 and BAG6 are expressed in various tissues and are upregulated by salt, mannitol, and heat treatments and by stress-related hormones including ABA, ethylene, and SA. Germination of bag2, bag6 and bag2 bag6 seeds is less sensitive to ABA compared to the wild type (WT), whereas BAG2 and BAG6 overexpression lines are hypersensitive to ABA. bag2, bag6, and bag2 bag6 plants show higher survival rates than WT in drought treatment but display lower survival rates in heat-stress treatment. Consistently, these mutants showed differential expression of several stress- and ABA-related genes such as RD29A, RD29B, NCED3 and ABI4 compared to the WT. Furthermore, these mutants exhibit lower levels of ROS after drought and ABA treatment but higher ROS accumulation after heat treatment than the WT. These results suggest that BAG2 and BAG6 are negatively involved in drought stress but play a positive role in heat stress in Arabidopsis.

Structural basis for activation and allosteric modulation of full-length calcium-sensing receptor.

Calcium-sensing receptor (CaSR) is a class C G protein-coupled receptor (GPCR) that plays an important role in calcium homeostasis and parathyroid hormone secretion. Here, we present multiple cryo-electron microscopy structures of full-length CaSR in distinct ligand-bound states. Ligands (Ca2+ and l-tryptophan) bind to the extracellular domain of CaSR and induce large-scale conformational changes, leading to the closure of two heptahelical transmembrane domains (7TMDs) for activation. The positive modulator (evocalcet) and the negative allosteric modulator (NPS-2143) occupy the similar binding pocket in 7TMD. The binding of NPS-2143 causes a considerable rearrangement of two 7TMDs, forming an inactivated TM6/TM6 interface. Moreover, a total of 305 disease-causing missense mutations of CaSR have been mapped to the structure in the active state, creating hotspot maps of five clinical endocrine disorders. Our results provide a structural framework for understanding the activation, allosteric modulation mechanism, and disease therapy for class C GPCRs.

Cryo-EM structure of human ABCB8 transporter in nucleotide binding state.

Human ATP-binding cassette transporter 8 of subfamily B (hABCB8) is an ABC transporter that located in the inner membrane of mitochondria. The ABCB8 is involved in the maturation of Fe-S and protects the heart from oxidative stress. Here, we present the cryo-EM structure of human ABCB8 binding with AMPPNP in inward-facing conformation with resolution of 4.1 Å. hABCB8 shows an open-inward conformation when ATP is bound. Unexpectedly, cholesterol molecules were identified in the transmembrane domain of hABCB8. Our results provide structural basis for the transport mechanism of the ABC transporter in mitochondria.

Mitochondrial heat-shock cognate protein 70 contributes to auxin-mediated embryo development.

In Arabidopsis thaliana, mitochondrial-localized heat-shock cognate protein 70-1 (mtHSC70-1) plays an important role in vegetativegrowth. However, whether mtHSC70-1 affects reproductive growth remains unknown. Here, we found that the mtHSC70-1 gene was expressed in the provascular cells of the embryo proper from the early heart stage onward during embryogenesis. Phenotypic analyses of mthsc70-1 mutants revealed that mtHSC70 deficiency leads to defective embryo development and that this effect is mediated by auxin. In addition to a dwarf phenotype, the mthsc70-1 mutant displayed defects in flower morphology, anther development, and embryogenesis. At early developmental stages, the mthsc70-1 embryos exhibited abnormal cell divisions in both embryo proper and suspensor cells. From heart stage onward, they displayed an abnormal shape such as with no or very small cotyledon protrusions, had aberrant number of cotyledons, or were twisted. These embryo defects were associated with reduced or ectopic expression of auxin responsive reporter DR5rev:GFP. Consistently, the expression of auxin biosynthesis and polar auxin transport genes were markedly altered in mthsc70-1. On the other hand, mitochondrial retrograde regulation (MRR) was enhanced in mthsc70-1. Treatment of wild-type plants with an inhibitor that activates mitochondrial retrograde signaling reduced the expression level of auxin biosynthesis and polar auxin transport genes and induced phenotypes similar to those of mthsc70-1. Taken together, our data reveal that loss of function of mtHSC70-1 induces MRR, which inhibits auxin biosynthesis and polar auxin transport, leading to abnormal auxin gradients and defective embryo development.

Expression, purification and microscopic characterization of human ATP-binding cassette sub-family B member 7 protein.

The ATP-binding cassette sub-family B member 7 (ABCB7) is a membrane transport protein located on the inner membrane of mitochondria, which could be involved in the transport of heme from the mitochondria to the cytosol. ABCB7 also plays a central role in the maturation of cytosolic iron-sulfur (Fe/S) cluster-containing proteins, and mutations can cause a series of mitochondrial defects. X-linked sideroblastic anemia and ataxia (XLSA-A) is a rare cause of early onset ataxia, which may be overlooked due to the usually mild asymptomatic anemia. The genetic defect has been identified as a mutation in the ABCB7 gene at Xq12-q13. Here, we report the expression, purification and the 2D projections derived from negatively stained electron micrographs of recombinant H. sapiens ABCB7 (hABCB7), paving the way from an atomic structure determination of ABCB7.

Receptor-targeting nanomaterials alleviate binge drinking-induced neurodegeneration as artificial neurotrophins.

The distinguished properties of nanomaterials promote us to explore whether their intrinsic activities would be beneficial to disease treatment. Furthermore, understanding the molecular mechanism is thereby crucial for biomedical applications. Here, we investigate the therapeutic effects of single-walled carbon nanotubes (SWNTs) in a rat model of binge alcohol-induced neurodegeneration. With selection from four types of SWNT structures, bundled SWNTs (bSWNTs) facilitated the recovery of learning and memory via enhancing neuroprotection and neuroregeneration. We screened the potential target for bSWNTs, and found that bSWNTs have the abilities to directly interact with neurotrophic receptors, especially tropomyosin-related kinase B (TrkB). Moreover, similar to the actions of endogenous neurotrophins, bSWNTs could trigger the dimerization and phosphorylation of TrkB, while these conformational changes resulted in activating their downstream signals involved in neuroprotection and neuroregeneration. With relatively clear mechanisms, these "artificial neurotrophins" provide a proof-of-concept example as an efficiently therapeutic strategy for the treatment of neurodegenerative diseases.

Cryo-EM structure of the calcium homeostasis modulator 1 channel.

Calcium homeostasis modulator 1 (CALHM1) is a voltage-gated ATP release channel that plays an important role in neural gustatory signaling and the pathogenesis of Alzheimer's disease. Here, we present a cryo-electron microscopy structure of full-length Ca2+-free CALHM1 from Danio rerio at an overall resolution of 3.1 Å. Our structure reveals an octameric architecture with a wide pore diameter of ~20 Å, presumably representing the active conformation. The overall structure is substantially different from that of the isoform CALHM2, which forms both undecameric hemichannels and gap junctions. The N-terminal small helix folds back to the pore and forms an antiparallel interaction with transmembrane helix 1. Structural analysis revealed that the extracellular loop 1 region within the dimer interface may contribute to oligomeric assembly. A positive potential belt inside the pore was identified that may modulate ion permeation. Our structure offers insights into the assembly and gating mechanism of the CALHM1 channel.

Crystal Structures of the C-Glycosyltransferase UGT708C1 from Buckwheat Provide Insights into the Mechanism of C-Glycosylation.

C-Glycosyltransferases (CGTs) catalyze the formation of C-glycosidic bonds for the biosynthesis of C-glycosides, but the underlying mechanism is unclear. This process improves the solubility and bioavailability of specialized metabolites, which play important roles in plant growth and development and represent rich resources for drug discovery. Here, we performed functional and structural studies of the CGT UGT708C1 from buckwheat (Fagopyrum esculentum). Enzymatic analysis showed that UGT708C1 is capable of utilizing both UDP-galactose and UDP-glucose as sugar donors. Our structural studies of UGT708C1 complexed with UDP-glucose and UDP identified the key roles of Asp382, Gln383, Thr151, and Thr150 in recognizing the sugar moiety of the donor substrate and Phe130, Tyr102, and Phe198 in binding and stabilizing the acceptor. A systematic site-directed mutagenesis study confirmed the important roles of these residues. Further structural analysis combined with molecular dynamics simulations revealed that phloretin binds to the acceptor binding pocket in a bent state with a precise spatial disposition and complementarity. These findings provide insights into a catalytic mechanism for CGTs.

Molecular architecture of the acetohydroxyacid synthase holoenzyme.

The acetohydroxyacid synthase (AHAS) holoenzyme catalyzes the first step of branch-chain amino acid biosynthesis and is essential for plants and bacteria. It consists of a regulatory subunit (RSU) and a catalytic subunit (CSU). The allosteric mechanism of the AHAS holoenzyme has remained elusive for decades. Here, we determined the crystal structure of the AHAS holoenzyme, revealing the association between the RSU and CSU in an A2B2 mode. Structural analysis in combination with mutational studies demonstrated that the RSU dimer forms extensive interactions with the CSU dimer, in which a conserved salt bridge between R32 and D120 may act as a trigger to open the activation loop of the CSU, resulting in the activation of the CSU by the RSU. Our study reveals the activation mechanism of the AHAS holoenzyme.

Expression, purification and oligomerization of the S-adenosylmethionine transporter.

The S-adenosylmethionine carrier (SAMC) is a membrane transport protein located on the inner membrane of mitochondria that catalyzes the import of S-adenosylmethionine (SAM) into the mitochondrial matrix. SAMC mutations can cause a series of mitochondrial defects, including those affecting RNA stability, protein modification, mitochondrial translation and biosynthesis. Here, we describe the expression, purification and oligomerization of SAMC. The SAMC genes from three species were cloned into a eukaryotic expression vector with a GFP tag, and confocal microscopy analysis showed that these SAMCs were localized to mitochondria. A BacMam expression system was used for the expression of D. rerio SAMC with a FLAG tag. A size-exclusion chromatography analysis showed that SAMC may form a hexamer. A negative-staining electron microscopy analysis showed that SAMC formed tiny uniform particles and also confirmed the oligomerization of SAMC.

Crystal structure and biochemical characterization of Striga hermonthica HYPO-SENSITIVE TO LIGHT 8 (ShHTL8) in strigolactone signaling pathway.

Striga is a parasitic weed that disperses easily, and its seeds can persist in the soil for many years, presenting long-term threats to food security. If SLs stimulate the seed germination of root parasitic weeds before planting, weeds will wither due to no host. Therefore, it is necessary to determine the mechanism of strigolactone (SL) signaling in Striga to reduce the impacts of this parasitic weed. Striga has eleven different kinds of HYPO-SENSITIVE to LIGHT (ShHTL) hydrolases. Different ShHTL hydrolases exhibit distinct responses to SLs, despite these ShHTLs exhibiting more than 60% sequence identity. Currently, structural information is available for only five ShHTL proteins, and more structural information is needed to design Striga germination stimulants or inhibitors. In this paper, we report the crystal structure of ShHTL8, which is determined at a resolution of 1.4 Å. Scanning fluorimetry and HPLC assays indicate that L125, M147, M154 and I194 are important binding sites, and of which L125 may act as a key holder involved in the catalytic reaction. Additionally, the corresponding residue, Y124 of ShHTL1 and F135 of ShHTL2 also play a significant role in the substrate recognition.

Molecular understanding of calcium permeation through the open Orai channel.

The Orai channel is characterized by voltage independence, low conductance, and high Ca2+ selectivity and plays an important role in Ca2+ influx through the plasma membrane (PM). How the channel is activated and promotes Ca2+ permeation is not well understood. Here, we report the crystal structure and cryo-electron microscopy (cryo-EM) reconstruction of a Drosophila melanogaster Orai (dOrai) mutant (P288L) channel that is constitutively active according to electrophysiology. The open state of the Orai channel showed a hexameric assembly in which 6 transmembrane 1 (TM1) helices in the center form the ion-conducting pore, and 6 TM4 helices in the periphery form extended long helices. Orai channel activation requires conformational transduction from TM4 to TM1 and eventually causes the basic section of TM1 to twist outward. The wider pore on the cytosolic side aggregates anions to increase the potential gradient across the membrane and thus facilitate Ca2+ permeation. The open-state structure of the Orai channel offers insights into channel assembly, channel activation, and Ca2+ permeation.