The mitochondrial respiratory Complex II or succinate:ubiquinone oxidoreductase (SQR) is an integral membrane protein complex in both the tricarboxylic acid cycle and aerobic respiration. Here we report the first crystal structure of Complex II from porcine heart at 2.4 A resolution and its complex structure with inhibitors 3-nitropropionate and 2-thenoyltrifluoroacetone (TTFA) at 3.5 A resolution. Complex II is comprised of two hydrophilic proteins, flavoprotein (Fp) and iron-sulfur protein (Ip), and two transmembrane proteins (CybL and CybS), as well as prosthetic groups required for electron transfer from succinate to ubiquinone. The structure correlates the protein environments around prosthetic groups with their unique midpoint redox potentials. Two ubiquinone binding sites are discussed and elucidated by TTFA binding. The Complex II structure provides a bona fide model for study of the mitochondrial respiratory system and human mitochondrial diseases related to mutations in this complex.

Coronavirus replication and transcription machinery involves multiple virus-encoded nonstructural proteins (nsp). We report the crystal structure of the hexadecameric nsp7-nsp8 supercomplex from the severe acute respiratory syndrome coronavirus at 2.4-angstroms resolution. nsp8 has a novel 'golf-club' fold with two conformations. The supercomplex is a unique hollow, cylinder-like structure assembled from eight copies of nsp8 and held tightly together by eight copies of nsp7. With an internal diameter of approximately 30 angstroms, the central channel has dimensions and positive electrostatic properties favorable for nucleic acid binding, implying that its role is to confer processivity on RNA-dependent RNA polymerase.

The unique coronavirus transcription/replication machinery comprised of multiple virus-encoded nonstructural proteins (nsp) plays a vital role during initial and intermediate phases of the viral life cycle. The crystal structure of mouse hepatitis virus strain A59 (MHV-A59) nsp15 is reported at 2.15-Å resolution. nsp15 is an XendoU endoribonuclease and is the first one from this family to have its structure unveiled. The MHV-A59 nsp15 monomer structure has a novel protein fold. Two nsp15 trimers form a back-to-back hexamer that is believed to be the functional unit. The structure reveals the catalytic site including the highly conserved residues His262, His277, and Lys317, which is supported by mutagenesis analysis. Gel filtration and enzyme activity assays confirmed that the hexamer is the active form for nsp15 and demonstrate the specificity of nsp15 for uridylate. The high sequence conservation of nsp15 in coronaviruses, including that of severe acute respiratory syndrome, suggests that this protein may provide a new target for the design of antiviral therapeutics.Mouse hepatitis virus (MHV) belongs to group II of the genus Coronavirus (CoV), together with bovine coronavirus, human coronavirus strain OC43, and the recently identified severe acute respiratory syndrome coronavirus (SARS-CoV) (2). The two strains of MHV that have been extensively studied to date are A59 (MHV-A59) and JHM (MHV-JHM or MHV-4). These coronavirus strains cause a variety of diseases in susceptible mice, such as enteritis, hepatitis, and panencephalitis, with acute and chronic demyelination that is histologically similar to multiple sclerosis in humans (13,14). Much of our knowledge concerning the replication mechanism of coronaviruses has been acquired from the use of MHV as a model for pathogenesis, docking and entry, receptor usage, transcription, replication, polymerase function, assembly, and release (31). MHV-A59 in particular is used extensively as a model to study the role of the immune system in virus-induced ventral nervous system demyelination. To date, the pathology of demyelination is still not clear; thus, further insights into the replication and transcription of the model are required to better use this model for further study of demyelination, replication, and transcription of other coronaviruses.MHV-A59 is a positive-strand RNA coronavirus with a genome of 31 kb in length, of which about 22 kb is encompassed by the replicase gene containing two large overlapping open reading frames (ORF), termed ORF1a and ORF1b. The replicase gene expresses two large polyproteins, pp1a (495 kDa) and pp1ab (803 kDa), where expression of pp1ab involves a Ϫ1 ribosomal frameshift just upstream of the ORF1a translation termination codon (31). Neither pp1a nor pp1ab is detected intact in MHV-infected cells, since they are cotranslationally and posttranslationally processed by three proteases (two papain-like proteases, PLP1 and PLP2, and a main protease, M pro ) into at least 14 mature nonstructural proteins (1,23,29). These nonstructural p...

The severe acute respiratory syndrome coronavirus (SARS-CoV) nonstructural proteins nsp1 to nsp16 have been implicated by genetic analysis in the assembly of a functional replication/transcription complex. We report the crystal structure of nsp10 from SARS-CoV at 2.1-Å resolution. The nsp10 structure has a novel fold, and 12 identical subunits assemble to form a unique spherical dodecameric architecture. Two zinc fingers have been identified from the nsp10 monomer structure with the sequence motifs C-(X) 2 -C-(X) 5 -H-(X) 6 -C and C-(X) 2 -C-(X) 7 -C-(X)-C. The nsp10 crystal structure is the first of a new class of zinc finger protein threedimensional structures to be revealed experimentally. The zinc finger sequence motifs are conserved among all three coronavirus antigenic groups, implicating an essential function for nsp10 in all coronaviruses. Based on the structure, we propose that nsp10 is a transcription factor for coronavirus replication/transcription.Coronaviruses (CoVs) are positive-stranded RNA viruses known to cause highly prevalent respiratory and enteric diseases in humans and animals. About two-thirds of their genomes encode the viral replicase that mediates viral RNA synthesis (19). The replicase gene is comprised of two open reading frames at the 5Ј end of the genome, termed ORF1a and ORF1b (26). The upstream ORF1a encodes polyprotein pp1a (450 to 500 kDa), while ORF1a and ORF1b together encode pp1ab (750 to 800 kDa). Expression of the ORF1b-encoded half of pp1ab requires a Ϫ1 ribosomal frameshift during translation. The polyproteins pp1a and pp1ab undergo extensive proteolytic processing by viral proteases to produce 16 functional subunits known as nonstructural proteins, which then assemble to form the replicase complex required for viral replication and transcription.Prior to the global severe acute respiratory syndrome (SARS) outbreak in 2003, scant attention was paid to coronaviruses by researchers, as this genus of viruses causes severe diseases predominantly in animals and only comparatively mild diseases in humans. In the wake of the SARS outbreak, greater attention has been focused on the replicase proteins with a view to understanding the replication/transcription machinery and to identify new therapeutic targets. To date, the threedimensional structures of a series of nsp proteins have been reported (1). nsp5, also called the main protease or 3C-like protease, was the first SARS protein structure determined in 2003 (24) and has since been the focus of concerted efforts for the design of antiviral inhibitors. Last year, a broad-spectrum inhibitor was reported with efficient in vitro inactivation of multiple coronavirus main proteases, potent antiviral activity, and extremely low cellular toxicity (23). The structure of nsp9 was determined in 2004, and it was found to be a singlestranded RNA binding protein (4, 17). More recently, the complex structure between nsp7 and nsp8 revealed a hexadecameric assembly that should constitute a processivity factor for nsp12, an RNA-dependent RNA pol...

Rabbit hemorrhagic disease, first described in China in 1984, causes hemorrhagic necrosis of the liver. Its etiological agent, rabbit hemorrhagic disease virus (RHDV), belongs to the Lagovirus genus in the family Caliciviridae. The detailed molecular structure of any lagovirus capsid has yet to be determined. Here, we report a cryo-electron microscopic (cryoEM) reconstruction of wild-type RHDV at 6.5 Å resolution and the crystal structures of the shell (S) and protruding (P) domains of its major capsid protein, VP60, each at 2.0 Å resolution. From these data we built a complete atomic model of the RHDV capsid. VP60 has a conserved S domain and a specific P2 sub-domain that differs from those found in other caliciviruses. As seen in the shell portion of the RHDV cryoEM map, which was resolved to ∼5.5 Å, the N-terminal arm domain of VP60 folds back onto its cognate S domain. Sequence alignments of VP60 from six groups of RHDV isolates revealed seven regions of high variation that could be mapped onto the surface of the P2 sub-domain and suggested three putative pockets might be responsible for binding to histo-blood group antigens. A flexible loop in one of these regions was shown to interact with rabbit tissue cells and contains an important epitope for anti-RHDV antibody production. Our study provides a reliable, pseudo-atomic model of a Lagovirus and suggests a new candidate for an efficient vaccine that can be used to protect rabbits from RHDV infection.

Ferroptosis, triggered by discoordination of iron, thiols and lipids, leads to accumulation of 15-hydroperoxy-arachidonoyl-PE (15-HpETE-PE) generated by complexes of 15-lipoxygenase (15-LOX) and a scaffold protein, PEBP1. As Ca 2+ -independent phospholipase PLA 2 (iPLA 2 β, PLA2G6/PNPLA9 gene), can preferentially hydrolyze peroxidized phospholipids, it may eliminate ferroptotic 15-HpETE-PE death signal. Here we demonstrate that by hydrolyzing 15-HpETE-PE, iPLA 2 β averts ferroptosis whereas its genetic or pharmacological inactivation sensitizes cells to ferroptosis. Given that PLA2G6/PNPLA9 mutations relate to neurodegeneration, we examined fibroblasts from a patient with a Parkinson’s disease (PD)-associated mutation fPD R747W and found selectively decreased 15-HpETE-PE hydrolyzing activity, 15-HpETE-PE accumulation and elevated sensitivity to ferroptosis. CRISPR-CAS9-engineered PNPLA9 R748W/R748W mice exhibited progressive parkinsonian motor deficits and 15-HpETE-PE accumulation. Elevated 15-HpETE-PE levels were also detected in midbrains of rotenone-infused parkinsonian rats and α-synuclein mutant SNCA-A53T mice with decreased iPLA 2 β expression and PD-relevant phenotype. Thus, iPLA 2 β is a new ferroptosis regulator and its mutations may be implicated in PD pathogenesis.

The voltage-gated proton channel Hv1 has a voltage sensor domain but lacks a pore domain. Although the C-terminal domain of Hv1 is known to be responsible for dimeric architecture of the channel, its role and structure are not known. We report that the full-length Hv1 is mainly localized in intracellular compartment membranes rather than the plasma membrane. Truncation of either the N or C terminus alone or both together revealed that the N-terminal deletion did not alter localization, but deletion of the C terminus either alone or together with the N terminus resulted in expression throughout the cell. These results indicate that the C terminus is essential for Hv1 localization but not the N terminus. In the 2.0 Å structure of the C-terminal domain, the two monomers form a dimer via a parallel ␣-helical coiled-coil, in which one chloride ion binds with the N atom of Arg 264. A pH-dependent structural change of the protein has been observed, but it remains a dimer irrespective of pH value.Voltage-gated proton channel (Hv channel) currents were observed first in snail neurons (1) and later found in many mammalian cells, such as alveolar epithelium cells of the lung, microglia of the brain, skeletal muscle, and many blood cells, including macrophages, neutrophils, and eosinophils (2-5). The Hv channels in mammalian phagocytes were originally proposed to be responsible for the proton-transporting pathway, which regulates intracellular pH during oxygen consumption associated with phagocytosis, called "respiratory burst" (5, 6). Hv channels are activated by depolarization and intracellular acidification, whose activities maintain intracellular pH neutral to keep reactive oxygen species generation (7, 8). Hv channels not only regulate pH in cytoplasm but could also provide protons in the phagosome, a closed membrane compartment for killing and digestion of a pathogen (5). Hv channels are extremely selective for H ϩ , with no detectable permeability to other cations (2, 9, 10). The voltage activation relationship of Hv channels depends strongly on both the intracellular pH (pH i ) and extracellular pH (pH o ). Increasing pH o or lowering pH i promotes H ϩ channel opening by shifting the activation threshold to more negative potentials (4, 5). Furthermore, Hv currents are known to be inhibited by submillimolar concentrations of Zn 2ϩ and Cd 2ϩ and other divalent cations (11). Recently, the human and mouse voltage-gated proton channels, called Hv1 and mVSOP, respectively, were identified using bioinformatics searches based on known cation channels (Hv1) and the voltage sensor domain of Ciona intestinalis VSP (mVSOP) (12, 13). Hv1/mVSOP currents are activated under depolarizing voltage, sensitive to the membrane pH gradient, H ϩ -selective, and Zn 2ϩ -sensitive. Hv1/mVSOP is a predicted 273/269 amino acids in length and contains three predicted domains: N-terminal acid and proline-rich domain, transmembrane voltage sensor domain, and C-terminal domain. Voltagegated K ϩ channels are composed of four subunits, each of which...

SUMMARY Thermosomes are group II chaperonins responsible for protein refolding in an ATP-dependent manner. Little is known regarding the conformational changes of thermosomes during their functional cycle due to lack of high-resolution structure in open state. Here we report the first complete crystal structure of thermosome (rATcpnβ) in open state from Acidianus tengchongensis. There is a ~30° rotation of the apical and lid domains compared to the previous closed structure. Besides, the structure reveals a conspicuous hydrophobic patch in the lid domain and residues locating in this patch are conserved across species. Both the closed and open forms of rATcpnβ were also reconstructed by electron microscopy (EM). Structural fitting revealed the detailed conformational change from open to closed state. Structural comparison as well as protease K digestion indicated only ATP binding without hydrolysis does not induce chamber closure of thermosome.