Mycobacterium tuberculosis proteasomal ATPase Mpa has a ß-grasp domain that hinders docking with the proteasome core protease.

Mycobacterium tuberculosis (Mtb) has a proteasome system that is essential for its ability to cause lethal infections in mice. A key component of the system is the proteasomal adenosine triphosphatase (ATPase) Mpa, which captures, unfolds, and translocates protein substrates into the Mtb proteasome core particle for degradation. Here, we report the crystal structures of near full-length hexameric Mtb Mpa in apo and ADP-bound forms. Surprisingly, the structures revealed a ubiquitin-like ß-grasp domain that precedes the proteasome-activating carboxyl terminus. This domain, which was only found in bacterial proteasomal ATPases, buries the carboxyl terminus of each protomer in the central channel of the hexamer and hinders the interaction of Mpa with the proteasome core protease. Thus, our work reveals the structure of a bacterial proteasomal ATPase in the hexameric form, and the structure finally explains why Mpa is unable to stimulate robust protein degradation in vitro in the absence of other, yet-to-be-identified co-factors.

Zunyimycins B and C, New Chloroanthrabenzoxocinones Antibiotics against Methicillin-Resistant Staphylococcus aureus and Enterococci from Streptomyces sp. FJS31-2.

This study performed an optimization of the fermentation conditions to activate the expression of the zunyimycin family biosynthesis genes of the zunyimycin-producing streptomycetes strain Streptomyces sp. FJS31-2. Bioassay-guided isolation and purification by varied chromatographic methods yielded two new compounds of the zunyimycin derivatives, namely, 31-2-7 and 31-2-8, accompanied with three known anthrabenzoxocinones family members of zunyimycin A, BE24566B, and chloroanthrabenzoxocinone. Their structures were elucidated by NMR, HRESIMS, IR, UV, and CD. Results showed that these two compounds were structurally similar to the previously reported compound zunyimycin A but differed in positions and number of chlorine atom substitution. The two novel compounds were called zunyimycins B and C. Antibacterial activity assay indicated that zunyimycin C showed a good inhibitory effect on the methicillin-resistant Staphylococcus aureus and Enterococci.

Structural insights into the unique single-stranded DNA-binding mode of Helicobacter pylori DprA.

Natural transformation (NT) in bacteria is a complex process, including binding, uptake, transport and recombination of exogenous DNA into the chromosome, consequently generating genetic diversity and driving evolution. DNA processing protein A (DprA), which is distributed among virtually all bacterial species, is involved in binding to the internalized single-stranded DNA (ssDNA) and promoting the loading of RecA on ssDNA during NTs. Here we present the structures of DNA_processg_A (DprA) domain of the Helicobacter pylori DprA (HpDprA) and its complex with an ssDNA at 2.20 and 1.80 Å resolutions, respectively. The complex structure revealed for the first time how the conserved DprA domain binds to ssDNA. Based on structural comparisons and binding assays, a unique ssDNA-binding mode is proposed: the dimer of HpDprA binds to ssDNA through two small, positively charged binding pockets of the DprA domains with classical Rossmann folds and the key residue Arg52 is re-oriented to 'open' the pocket in order to accommodate one of the bases of ssDNA, thus enabling HpDprA to grasp substrate with high affinity. This mode is consistent with the oligomeric composition of the complex as shown by electrophoretic mobility-shift assays and static light scattering measurements, but differs from the direct polymeric complex of Streptococcus pneumoniae DprA-ssDNA.

Optimizing the robustness of higher-low order coupled networks.

Enhancing the robustness of complex networks is of great practical significance as it ensures the stable operation of infrastructure systems. We measure its robustness by examining the size of the largest connected component of the network after initial attacks. However, traditional research on network robustness enhancement has mainly focused on low-order networks, with little attention given to higher-order networks, particularly higher-low order coupling networks(the largest connected component of the network must exist in both higher-order and low-order networks). To address this issue, this paper proposes robust optimization methods for higher-low order coupled networks based on the greedy algorithm and the simulated annealing algorithm. By comparison, we found that the simulated annealing algorithm performs better. The proposed method optimizes the topology of the low-order network and the higher-order network by randomly reconnecting the edges, thereby enhancing the robustness of the higher-order and low-order coupled network. The experiments were conducted on multiple real networks to evaluate the change in the robustness coefficient before and after network optimization. The results demonstrate that the proposed method can effectively improve the robustness of both low-order and higher-order networks, ultimately enhancing the robustness of higher-low order coupled networks.

Structures of aminophenol dioxygenase in complex with intermediate, product and inhibitor.

Dioxygen activation by nonhaem Fe(II) enzymes containing the 2-His-1-carboxylate facial triad has been extensively studied in recent years. Here, crystal structures of 2-aminophenol 1,6-dioxygenase, an enzyme that represents a minor group of extradiol dioxygenases and that catalyses the ring opening of 2-aminophenol, in complex with the lactone intermediate (4Z,6Z)-3-iminooxepin-2(3H)-one and the product 2-aminomuconic 6-semialdehyde and in complex with the suicide inhibitor 4-nitrocatechol are reported. The Fe-ligand binding schemes observed in these structures revealed some common geometrical characteristics that are shared by the published structures of extradiol dioxygenases, suggesting that enzymes that catalyse the oxidation of noncatecholic compounds are very likely to utilize a similar strategy for dioxygen activation and the fission of aromatic rings as the canonical mechanism. The Fe-ligation arrangement, however, is strikingly enantiomeric to that of all other 2-His-1-carboxylate enzymes apart from protocatechuate 4,5-dioxygenase. This structural variance leads to the generation of an uncommon O(-)-Fe(2+)-O(-) species prior to O(2) binding, which probably forms the structural basis on which APD distinguishes its specific substrate and inhibitor, which share an analogous molecular structure.

Crystal structures of a Populus tomentosa 4-coumarate:CoA ligase shed light on its enzymatic mechanisms.

4-Coumaric acid:CoA ligase (4CL) is the central enzyme of the plant-specific phenylpropanoid pathway. It catalyzes the synthesis of hydroxycinnamate-CoA thioesters, the precursors of lignin and other important phenylpropanoids, in two-step reactions involving the formation of hydroxycinnamate-AMP anhydride and then the nucleophilic substitution of AMP by CoA. In this study, we determined the crystal structures of Populus tomentosa 4CL1 in the unmodified (apo) form and in forms complexed with AMP and adenosine 5'-(3-(4-hydroxyphenyl)propyl)phosphate (APP), an intermediate analog, at 2.4, 2.5, and 1.9 Å resolution, respectively. 4CL1 consists of two globular domains connected by a flexible linker region. The larger N-domain contains a substrate binding pocket, while the C-domain contains catalytic residues. Upon binding of APP, the C-domain rotates 81° relative to the N-domain. The crystal structure of 4CL1-APP reveals its substrate binding pocket. We identified residues essential for catalytic activities (Lys-438, Gln-443, and Lys-523) and substrate binding (Tyr-236, Gly-306, Gly-331, Pro-337, and Val-338) based on their crystal structures and by means of mutagenesis and enzymatic activity studies. We also demonstrated that the size of the binding pocket is the most important factor in determining the substrate specificities of 4CL1. These findings shed light on the enzymatic mechanisms of 4CLs and provide a solid foundation for the bioengineering of these enzymes.

Expression, crystallization and preliminary crystallographic study of GluB from Corynebacterium glutamicum.

GluB is a substrate-binding protein (SBP) which participates in the uptake of glutamic acid in Corynebacterium glutamicum, a Gram-positive bacterium. It is part of an ATP-binding cassette (ABC) transporter system. Together with the transmembrane proteins GluC and GluD and the cytoplasmic protein GluA, which couples the hydrolysis of ATP to the translocation of glutamate, they form a highly active glutamate-uptake system. As part of efforts to study the amino-acid metabolism, especially the metabolism of glutamic acid by C. glutamicum, a bacterium that is widely used in the industrial production of glutamic acid, the GluB protein was expressed, purified and crystallized, an X-ray diffraction data set was collected to a resolution of 1.9 Å and preliminary crystallographic analysis was performed. The crystal belonged to space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 82.50, c = 72.69 Å.

Non-Coding RNAs Regulate Spinal Cord Injury-Related Neuropathic Pain via Neuroinflammation.

Secondary chronic neuropathic pain (NP) in addition to sensory, motor, or autonomic dysfunction can significantly reduce quality of life after spinal cord injury (SCI). The mechanisms of SCI-related NP have been studied in clinical trials and with the use of experimental models. However, in developing new treatment strategies for SCI patients, NP poses new challenges. The inflammatory response following SCI promotes the development of NP. Previous studies suggest that reducing neuroinflammation following SCI can improve NP-related behaviors. Intensive studies of the roles of non-coding RNAs in SCI have discovered that ncRNAs bind target mRNA, act between activated glia, neuronal cells, or other immunocytes, regulate gene expression, inhibit inflammation, and influence the prognosis of NP.

Crystal structure of HugZ, a novel heme oxygenase from Helicobacter pylori.

The crystal structure of a heme oxygenase (HO) HugZ from Helicobacter pylori complexed with heme has been solved and refined at 1.8 Å resolution. HugZ is part of the iron acquisition mechanism of H. pylori, a major pathogen of human gastroenteric diseases. It is required for the adaptive colonization of H. pylori in hosts. Here, we report that HugZ is distinct from all other characterized HOs. It exists as a dimer in solution and in crystals, and the dimer adopts a split-barrel fold that is often found in FMN-binding proteins but has not been observed in hemoproteins. The heme is located at the intermonomer interface and is bound by both monomers. The heme iron is coordinated by the side chain of His(245) and an azide molecule when it is present in crystallization conditions. Experiments show that Arg(166), which is involved in azide binding, is essential for HugZ enzymatic activity, whereas His(245), surprisingly, is not, implying that HugZ has an enzymatic mechanism distinct from other HOs. The placement of the azide corroborates the observed γ-meso specificity for the heme degradation reaction, in contrast to most known HOs that have α-meso specificity. We demonstrate through sequence and structural comparisons that HugZ belongs to a new heme-binding protein family with a split-barrel fold. Members of this family are widespread in pathogenic bacteria and may play important roles in the iron acquisition of these bacteria.

Crystal structure of Campylobacter jejuni ChuZ: a split-barrel family heme oxygenase with a novel heme-binding mode.

The heme oxygenase ChuZ is part of the iron acquisition mechanism of Campylobacter jejuni, a major pathogen causing enteritis in humans. ChuZ is required for C. jejuni to use heme as the sole iron source. The crystal structure of ChuZ was resolved at 2.5Å, and it was revealed to be a homodimer with a split-barrel fold. One heme-binding site was at the dimer interface and another novel heme-binding site was found on the protein surface. Heme was bound in this site by four histidine side-chains through hydrophobic interactions. Based on stoichiometry studies and comparisons with other proteins, the possibility that similar heme-binding site exists in homologous proteins and its possible functions are discussed. The structural and mutagenesis analyses reported here establish ChuZ and ChuZ homologs as a new bacterial heme oxygenase family apart from the canonical and IsdG/I families. Our studies provide insight into the enzymatic mechanisms and structure-function relationship of ChuZ.

Crystallization and preliminary crystallographic studies of Campylobacter jejuni ChuZ, a member of a novel haem oxygenase family.

The haem oxygenase ChuZ from Campylobacter jejuni, a major enteric pathogen in humans, is part of the iron-acquisition mechanism that is involved in bacterial survival and persistence in hosts. The ChuZ-haemin complex has been purified and crystallized and diffraction data have been collected to 2.4 Å resolution. The ChuZ-haemin complex crystals belonged to space group C222(1), with unit-cell parameters a = 106.474, b = 106.698, c = 52.464 Å, α = ß = γ = 90°. The asymmetric unit contained one ChuZ monomer, with a Matthews coefficient of 2.58 Å(3) Da(-1).

Crystal structure of DNA gyrase B' domain sheds lights on the mechanism for T-segment navigation.

DNA gyrase is an indispensible marvelous molecular machine in manipulating the DNA topology for the prokaryotes. In the 'two-gate' mechanism of DNA topoisomerase, T-segment navigation from N- to DNA-gate is a critical step, but the structural basis supporting this scheme is unclear. The crystal structure of DNA gyrase B' subfragment from Mycobacterium tuberculosis reveals an intrinsic homodimer. The two subunits, each consisting of a Tail and a Toprim domain, are tightly packed one another to form a 'crab-like' organization never observed previously from yeast topo II. Structural comparisons show two orientational alterations of the Tail domain, which may be dominated by a 43-residue peptide at the B' module C-terminus. A highly conserved pentapeptide mediates large-scale intrasubunit conformational change as a hinge point. Mutational studies highlight the significant roles of a negatively charge cluster on a groove at dimer interface. On the basis of structural analysis and mutation experiments, a sluice-like model for T-segment transport is proposed.

User capabilities in eyes-free spatial target acquisition in immersive virtual reality environments.

In immersive virtual reality (VR) environments, users rely on the vision channel to search for objects. Such eyes-engaged interactive techniques may significantly degrade the interaction efficiency and user experience, particularly when users have to turn their head frequently to search for a target object in the limited field of view (FOV) of a head-mounted display (HMD). In this study, we systematically investigated user capabilities in eyes-free spatial target acquisition considering different horizontal angles, vertical angles, distances from the user's body, and body sides. Our results show that high acquisition accuracy and low task load are achieved for target locations at front and middle horizontal angles as well as those at middle vertical angles. Meanwhile, a trade-off cannot be achieved between the acquisition accuracy and the task load for target locations at long distances from the user's body. In addition, the acquisition accuracy and task load for the target locations vary with the body side. Our research findings can provide a deeper understanding of user capability in eyes-free target acquisition and offer concrete design guidelines for appropriate target arrangement for eyes-free target acquisition in immersive VR environments.

Crystal structure of human programmed cell death 10 complexed with inositol-(1,3,4,5)-tetrakisphosphate: a novel adaptor protein involved in human cerebral cavernous malformation.

Programmed cell death 10 (PDCD10) is a novel adaptor protein involved in human cerebral cavernous malformation, a common vascular lesion mostly occurring in the central nervous system. By interacting with different signal proteins, PDCD10 could regulate various physiological processes in the cell. The crystal structure of human PDCD10 complexed with inositol-(1,3,4,5)-tetrakisphosphate has been determined at 2.3A resolution. The structure reveals an integrated dimer via a unique assembly that has never been observed before. Each PDCD10 monomer contains two independent domains: an N-terminal domain with a new fold involved in the tight dimer assembly and a C-terminal four-helix bundle domain that closely resembles the focal adhesion targeting domain of focal adhesion kinase. An eight-residue flexible linker connects the two domains, potentially conferring mobility onto the C-terminal domain, resulting in the conformational variability of PDCD10. A variable basic cleft on the top of the dimer interface binds to phosphatidylinositide and regulates the intracellular localization of PDCD10. Two potential sites, respectively located on the two domains, are critical for recruiting different binding partners, such as germinal center kinase III proteins and the focal adhesion protein paxillin.

Crystallization and preliminary crystallographic studies of a flavin-dependent thymidylate synthase from Helicobacter pylori.

The ThyX enzymes that have recently been identified in various bacteria, including some important human pathogens such as Helicobacter pylori and Mycobacterium tuberculosis, are flavin-dependent thymidylate synthases that function in the place of classic thymidylate synthase enzymes in the biosynthesis of dTMP, which is one of the building blocks of DNA. They are promising targets for the development of novel antibiotics because they utilize catalytic mechanisms that are distinct from those of the classic thymidylate synthases found in most organisms, including humans. In this study, H. pylori ThyX was purified and crystallized in complex with flavin adenine dinucleotide (FAD) and a diffraction data set was collected to 2.5 A resolution. The crystals belonged to space group C2, with unit-cell parameters a = 221.92, b = 49.43, c = 143.02 A, beta = 98.84 degrees .

Crystallization and preliminary crystallographic studies of Helicobacter pylori HugZ, a novel haem oxygenase.

The haem oxygenase HugZ is part of the iron-acquisition mechanism that is essential for the adaptive colonization of Helicobacter pylori, a major pathogen of gastroenteric diseases. The HugZ-haemin complex has been purified and crystallized and diffraction data sets have been collected to 1.8 A resolution. The HugZ-haemin complex crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 88.40, b = 139.37, c = 152.97 A.

Crystallization and preliminary crystallographic studies of Mycobacterium tuberculosis DNA gyrase B C-terminal domain, part of the enzyme reaction core.

DNA gyrase subunit B C-terminal domain (GyrB-CTD) is a functional module of DNA gyrase which participates in forming the core of DNA gyrase and plays critical roles in G-segment binding and T-segment loading and passage. Here, the purification, crystallization and preliminary X-ray crystallographic studies of GyrB-CTD from Mycobacterium tuberculosis H37Rv are reported. Diffraction data were collected from crystals of native GyrB-CTD and its selenomethionine derivative to resolutions of 2.8 and 3.0 A, respectively. These crystals belonged to space group P2(1)2(1)2(1) with similar unit-cell parameters. The native protein crystals had unit-cell parameters a = 52.831, b = 52.763, c = 192.579 A.

Crystal structure of Mabinlin II: a novel structural type of sweet proteins and the main structural basis for its sweetness.

The crystal structure of a sweet protein Mabinlin II (Mab II) isolated from the mature seeds of Capparis masaikai Levl. grown in Southern China has been determined at 1.7A resolution by the SIRAS method. The Mab II 3D structure features in an "all alpha" fold mode consisting of A- and B-chains crosslinked by four disulfide bridges, which is distinct from all known sweet protein structures. The Mabinlin II molecule shows an amphiphilic surface, a cationic face (Face A) and a neutral face (Face B). A unique structural motif consisting of B54-B64 was found in Face B, which adopts a special sequence, NL-P-NI-C-NI-P-NI, featuring four [Asn-Leu/Ile] units connected by three conformational-constrained residues, thus is called the [NL/I] tetralet motif. The experiments for testing the possible interactions of separated A-chain and B-chain and the native Mabinlin II to the sweet-taste receptor were performed through the calcium imaging experiments with the HEK293E cells coexpressed hT1R2/T1R3. The result shows that hT1R2/T1R3 responds to both the integrated Mabinlin II and the individual B-chain in the same scale, but not to A-chain. The sweetness evaluation further identified that the separated B-chain can elicit the sweetness alone, but A-chain does not. All data in combination revealed that the sweet protein Mabinlin II can interact with the sweet-taste receptor hT1R2/T1R3 to elicit its sweet taste, and the B-chain with a unique [NL/I] tetralet motif is the essential structural element for the interaction with sweet-taste receptor to elicit the sweetness, while the A-chain may play a role in gaining a long aftertaste for the integrate Mabinlin II. The findings reported in this paper will be advantage for understanding the diversity of sweet proteins and engineering research for development of a unique sweetener for the food and agriculture based on the Mabinlin II structure as a native model.

Research on Root Responses to Pb and Zn Combined Stress of Carex putuoshan.

Pb hyper-accumulated Carex putuoshan was taken as experimental material and subjected to combined stress of Pb and Zn. The differential expression of proteins in their roots were analyzed by Proteomic Approach. The protein that was directly involved in the cellular defense under the Pb and Zn combined stress was separated, and expression of those genes was analyzed with Carex Evergold as control. The results were obtained by MALDI-TOF/MS analysis. After applying Pb and Zn combined stress, the expression of 9 protein spots (including 7 different proteins, 2 identical proteins, 1 unknown protein) in Carex putuoshan root was found to be significantly up-regulated. Five proteins were obtained from the 9 proteins related to carbohydrate metabolism, including malate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, frutose-1,6-bisphosphate aldolase, enolase, and 6- phosphogluconate dehydrogenase. Two proteins were related to protein biosynthesis, including isoflavone reductase and phytochelatin synthase (PCS). From these proteins, the most important protein is PCS, which is a key enzyme in the synthesis of phytochelatins (PCs) and plays an important role in chelation. It is directly involved in cellular defense under Pb and Zn stress. After Pb and Zn combined stress, the CpPCS in Carex putuoshan was cloned. The full length of cDNA is 1461 bps, and it encodes 486 amino acids with molecular weight of 53.86 kD and pI value of 6.12. Two typical phytochelatin synthase subfamily domains constitute CpPCS protein, which includes three adjacent Cys-Cys elements in the C-terminal region. Phylogenetic analysis of PCS proteins from different species showed that it had the closest relationship with the Oryza sativa and Triticum aestivum. Real-time quantitative PCR analysis indicated that CpPCS and CePCS (Carex Evergold) genes were expressed in the root. The CpPCS and CePCS genes were up-regulated by Pb and Zn treatments. The expression of CpPCS was higher than that of CePCS under the same condition. The study found that CpPCS expression was increased by Pb and Zn stress in the Carex putuoshan enrichment process of Pb, which lead to high expression of PCS protein. CpPCS improved the accumulation ability and resistance of Carex putuoshan to heavy metals with the expression level of glucose metabolism related proteins increasing after Pb and Zn stress.

[Efficacy and safety of azosemide in patients with edema and ascites].

OBJECTIVE: To assess the efficacy and safety of azosemide in patients with edema and ascites. METHODS: A multicentral, randomized, double-blind, controlled clinical trial was applied. All 223 patients (cardiac edema 92, hepatogenic edema 63, renal edema 68) were randomized to azoesmide and furosemide group, and all patients were treated for 2 weeks. Patients with cardiac or renal edema took azosemide (30 mg/d) or furosemide (20 mg/d); patients with hepatogenic edema took azosemide (60 mg/d) or furosemide (40 mg/d). The dosage were adjusted to azosemide 60 mg/d (cardiac, renal edema), 90 mg (hepatogeic edema); or furosemide 40 mg/d (cardiac, renal edema), 60 mg (hepatogeic edema), if diuretic effects were not obtained at the end of third day. RESULTS: At the end of the study, the weight changes were (2.87+/-3.10) kg and (2.81 +/-2.84) kg; the total effective rate of edema lessen was 89.19% and 89.81%; the total effective rate of heart function improvement was 64.44% and 66.66%; the 24 h urine output increased (321.85 +/-669.52) ml and (273.80 +/-645.72) ml for azosemide and furosemide, respectively. The total effective rate of ascites lessen (tested by B-ultrasound) was 89.28% and 86.66%; abdominal girth decreased (5.20 +/-3.58) cm and (5.03 +/-3.74) cm for azosemide and furosemide, respectively. The adverse event rate was 23.01% in azosemide group and 21.01% in furosemide group; the main adverse effects were hypokalemia, hyperuricemia, hypertriglyceridemia and thirsty. CONCLUSION: Azosemide could effectively lessen edema, improve heart function and decrease ascitesûit is well tolerated and is particularly useful for the diuretic treatment.