Structural insights into the specific interaction between Geobacillus stearothermophilus tryptophanyl-tRNA synthetase and antimicrobial Chuangxinmycin.

The potential antimicrobial compound Chuangxinmycin (CXM) targets the tryptophanyl-tRNA synthetase (TrpRS) of both Gram-negative and Gram-positive bacteria. However, the specific steric recognition mode and interaction mechanism between CXM and TrpRS is unclear. Here, we studied this interaction using recombinant GsTrpRS from Geobacillus stearothermophilus by X-ray crystallography and molecular dynamics (MD) simulations. The crystal structure of the recombinant GsTrpRS in complex with CXM was experimentally determined to a resolution at 2.06 Å. After analysis using a complex-structure probe, MD simulations, and site-directed mutation verification through isothermal titration calorimetry, the interaction between CXM and GsTrpRS was determined to involve the key residues M129, D132, I133, and V141 of GsTrpRS. We further evaluated binding affinities between GsTrpRS WT/mutants and CXM; GsTrpRS was found to bind CXM through hydrogen bonds with D132 and hydrophobic interactions between the lipophilic tricyclic ring of CXM and M129, I133, and V141 in the substrate-binding pockets. This study elucidates the precise interaction mechanism between CXM and its target GsTrpRS at the molecular level and provides a theoretical foundation and guidance for the screening and rational design of more effective CXM analogs against both Gram-negative and Gram-positive bacteria.

Expression, purification and X-ray crystal diffraction analysis of alcohol dehydrogenase 1 from Artemisia annua L.

Alcohol dehydrogenase 1 identified from Artemisia annua (AaADH1) is a 40 kDa protein that predominately expressed in young leaves and buds, and catalyzes dehydrogenation of artemisinic alcohol to artemisinic aldehyde in artemisinin biosynthetic pathway. In this study, AaADH1 encoding gene was subcloned into vector pET-21a(+) and expressed in Escherichia coli. BL21(DE3), and purified by Co2+ affinity chromatography. Anion exchange chromatography was performed until the protein purity reached more than 90%. Crystallization of AaADH1 was conducted for further investigation of the molecular mechanism of catalysis, and hanging-drop vapour diffusion method was used in experiments. The results showed that the apo AaADH1 crystal diffracted to 2.95 Å resolution, and belongs to space group P1, with unit-cell parameters, a = 77.53 Å, b = 78.49 Å, c = 102.44 Å, α = 71.88°, ß = 74.02°, γ = 59.97°. The crystallization condition consists of 0.1 M Bis-Tris pH 6.0, 13% (w/v) PEG 8000 and 5% (v/v) glycerol.

The Complex Structure of Protein AaLpxC from Aquifex aeolicus with ACHN-975 Molecule Suggests an Inhibitory Mechanism at Atomic-Level against Gram-Negative Bacteria.

New drugs with novel antibacterial targets for Gram-negative bacterial pathogens are desperately needed. The protein LpxC is a vital enzyme for the biosynthesis of lipid A, an outer membrane component of Gram-negative bacterial pathogens. The ACHN-975 molecule has high enzymatic inhibitory capacity against the infectious diseases, which are caused by multidrug-resistant bacteria, but clinical research was halted because of its inflammatory response in previous studies. In this work, the structure of the recombinant UDP-3-O-(R-3-hydroxymyristol)-N-acetylglucosamine deacetylase from Aquifex aeolicus in complex with ACHN-975 was determined to a resolution at 1.21 Å. According to the solved complex structure, ACHN-975 was docked into the AaLpxC's active site, which occupied the site of AaLpxC substrate. Hydroxamate group of ACHN-975 forms five-valenced coordination with resides His74, His226, Asp230, and the long chain part of ACHN-975 containing the rigid alkynyl groups docked in further to interact with the hydrophobic area of AaLpxC. We employed isothermal titration calorimetry for the measurement of affinity between AaLpxC mutants and ACHN-975, and the results manifest the key residues (His74, Thr179, Tyr212, His226, Asp230 and His253) for interaction. The determined AaLpxC crystal structure in complex with ACHN-975 is expected to serve as a guidance and basis for the design and optimization of molecular structures of ACHN-975 analogues to develop novel drug candidates against Gram-negative bacteria.

Classification of ovarian cancer associated with BRCA1 mutations, immune checkpoints, and tumor microenvironment based on immunogenomic profiling.

BACKGROUND: Ovarian cancer is a highly fatal gynecological malignancy and new, more effective treatments are needed. Immunotherapy is gaining attention from researchers worldwide, although it has not proven to be consistently effective in the treatment of ovarian cancer. We studied the immune landscape of ovarian cancer patients to improve the efficacy of immunotherapy as a treatment option. METHODS: We obtained expression profiles, somatic mutation data, and clinical information from The Cancer Genome Atlas. Ovarian cancer was classified based on 29 immune-associated gene sets, which represented different immune cell types, functions, and pathways. Single-sample gene set enrichment (ssGSEA) was used to quantify the activity or enrichment levels of the gene sets in ovarian cancer, and the unsupervised machine learning method was used sort the classifications. Our classifications were validated using Gene Expression Omnibus datasets. RESULTS: We divided ovarian cancer into three subtypes according to the ssGSEA score: subtype 1 (low immunity), subtype 2 (median immunity), and subtype 3 (high immunity). Most tumor-infiltrating immune cells and immune checkpoint molecules were upgraded in subtype 3 compared with those in the other subtypes. The tumor mutation burden (TMB) was not significantly different among the three subtypes. However, patients with BRCA1 mutations were consistently detected in subtype 3. Furthermore, most immune signature pathways were hyperactivated in subtype 3, including T and B cell receptor signaling pathways, PD-L1 expression and PD-1 checkpoint pathway the NF-κB signaling pathway, Th17 cell differentiation and interleukin-17 signaling pathways, and the TNF signaling pathway. CONCLUSION: Ovarian cancer subtypes that are based on immune biosignatures may contribute to the development of novel therapeutic treatment strategies for ovarian cancer.

Kindlin­2 suppresses cervical cancer cell migration through AKT/mTOR­mediated autophagy induction.

Kindlin­2 plays a carcinogenic or tumor­suppressor role in various tumors. However, its role in cervical cancer remains unclear. In the present study, kindlin­2 expression was first analyzed using public expression data and clinical specimens. It was revealed that kindlin­2 was downregulated in cervical cancer tissues, and low expression of kindlin­2 was associated with poor disease­free survival. In addition, kindlin­2 was overexpressed and knocked down in two cell lines to study its effect in cervical cancer cells. The results revealed that kindlin­2 promoted cell autophagy and inactivated AKT/mTOR signaling. Rescue experiments indicated that the regulation of autophagy by kindlin­2 was dependent on the AKT/mTOR signaling pathway. Furthermore, it was revealed that kindlin­2 inhibited cell migration, and autophagy was required for this process. Collectively, these findings revealed the role and mechanism of kindlin­2 in the autophagy and migration of cervical cancer cells.

[Simple three-variable screening tool for identification of patients with obstructive sleep apnea-hypopnea syndrome in middle-aged male snorers from Guangxi region: a multi-center study].

OBJECTIVE: To evaluate the correlation between three variables of neck circumference, body mass index (BMI) and Epworth sleepiness scale (ESS) and obstructive sleep apnea-hypopnea syndrome (OSAHS) and the value of three-variable screening tool among snorers in Guangxi region. METHODS: From May 2009 to June 2014, 2 955 middle-aged consecutive patients with snoring were recruited from four hospitals of Guangxi Province. All subjects underwent polysomnography (PSG) and physical examinations including neck circumference, BMI and ESS. The diffrences of neck circumference, BMI and ESS were compared between OSAHS and non-OSAHS groups; the correlation between apnea-hypopnea index (AHI) and neck circumference, BMI and ESS were analyzed. The optimal cutoff point of three variables was selected by SPSS's Optimal Binning methods and screening score was assigned according to the Logistic regression coefficient. The recommended cutoff points were neck circumference ≥ 38.5 cm (3 points), BMI ≥ 25.7 kg/m(2) (5 points), ESS ≥ 9 points (1 point) for males and neck circumference ≥ 34.5 cm (5 points), BMI ≥ 24.5 kg/m(2) (2 points), ESS ≥ 7 points (1 points) for females. The best integral value was obtained through the receiver operating characteristic (ROC) curve. RESULTS: A total of 2 803 subjects fulfilled the study criteria. There were 2 366 males and 437 females. The average values of neck circumference, BMI and ESS were (38.7 ± 3.5) cm, (27.3 ± 3.6) kg/m(2) and (8.6 ± 5.4) score respectively. Both males and females had a positive association between AHI and neck circumference, BMI and ESS (r = 0.389, 0.485, 0.293, 0.386, 0.439 and 0.291 respectively, all P < 0.001). For males, at an integral value ≥ 6, the sensitivity was 71.4% in the diagnosis of OSAHS, specificity 65.5% and positive predictive value 91.1%. For females, at an integral value ≥ 3, the sensitivity was 73.3% in the diagnosis of OSAHS, specificity 63.6% and positive predictive value 66.3%. CONCLUSIONS: In middle-aged snorers in Guangxi, neck circumference, BMI and ESS are correlated with OSAHS. And a positive association exists between AHI and neck circumference, BMI and ESS. Simple three-variable screening tool is highly valuable for diagnosing OSAHS, especially among male snorers.

Crystal structure analysis of a recombinant predicted acetamidase/formamidase from the thermophile Thermoanaerobacter tengcongensis.

The structure of acetamidase/formamidase (Amds/Fmds) from the archaeon Thermoanaerobacter tengcongensis has been determined by X-ray diffraction analysis using MAD data in a crystal of space group P21, with unit-cell parameters a = 41.23 (3), b = 152.88 (6), c = 100.26 (7) Å, ß = 99.49 (3) ° and been refined to a crystallographic R-factor of 17.4% and R-free of 23.7%. It contains two dimers in one asymmetric unit, in which native Amds/Fmds (TE19) contains of the 32 kDa native protein. The final model consists of 4 monomer (299 amino acids residues with additional 2 expression tag amino acids residues), 5 Ca²âº, 4 Zn²âº and 853 water molecules. The monomer is composed by the following: an N-domain which is featuring by three-layers ß/ß/ß; a prominent excursion between N-terminal end of strand ß7 and ß11, which contains four-stranded antiparallel ß sheet; an C-domain which is formed by the last 82 amino acid residues with the feature of mixed α/ß structure. The protein contains ion-pair Ca²âº-Zn²âº. The portion of three-layer ß/ß/ß along with the loops provides four protein ligands to the tightly bound Ca²âº, three water molecules complete the coordination; and provides five protein ligands to the tightly bound Zn²âº, one water molecule complete the coordination.

Probing the effect of MODY mutations near the co-activator-binding pocket of HNF4α.

HNF4α (hepatocyte nuclear factor 4α) is a culprit gene product for a monogenic and dominantly inherited form of diabetes, referred to as MODY (maturity onset diabetes of the young). As a member of the NR (nuclear receptor) superfamily, HNF4α recruits transcriptional co-activators such as SRC-1α (steroid receptor co-activator-1α) and PGC-1α (peroxisome-proliferator-activated receptor γ co-activator-1α) through the LXXLL-binding motifs for its transactivation, and our recent crystal structures of the complex provided the molecular details and the mechanistic insights into these co-activator recruitments. Several mutations have been identified from the MODY patients and, among these, point mutations can be very instructive site-specific measures of protein function and structure. Thus, in the present study, we probed the functional effects of the two MODY point mutations (D206Y and M364R) found directly near the LXXLL motif-binding site by conducting a series of experiments on their structural integrity and specific functional roles such as overall transcription, ligand selectivity, target gene recognition and co-activator recruitment. While the D206Y mutation has a subtle effect, the M364R mutation significantly impaired the overall transactivation by HNF4α. These functional disruptions are mainly due to their reduced ability to recruit co-activators and lowered protein stability (only with M364R mutation), while their DNA-binding activities and ligand selectivities are preserved. These results confirmed our structural predictions and proved that MODY mutations are loss-of-function mutations leading to impaired ß-cell function. These findings should help target selective residues for correcting mutational defects or modulating the overall activity of HNF4α as a means of therapeutic intervention.

Multiple binding modes between HNF4alpha and the LXXLL motifs of PGC-1alpha lead to full activation.

Hepatocyte nuclear factor 4alpha (HNF4alpha) is a novel nuclear receptor that participates in a hierarchical network of transcription factors regulating the development and physiology of such vital organs as the liver, pancreas, and kidney. Among the various transcriptional coregulators with which HNF4alpha interacts, peroxisome proliferation-activated receptor gamma (PPARgamma) coactivator 1alpha (PGC-1alpha) represents a novel coactivator whose activation is unusually robust and whose binding mode appears to be distinct from that of canonical coactivators such as NCoA/SRC/p160 family members. To elucidate the potentially unique molecular mechanism of PGC-1alpha recruitment, we have determined the crystal structure of HNF4alpha in complex with a fragment of PGC-1alpha containing all three of its LXXLL motifs. Despite the presence of all three LXXLL motifs available for interactions, only one is bound at the canonical binding site, with no additional contacts observed between the two proteins. However, a close inspection of the electron density map indicates that the bound LXXLL motif is not a selected one but an averaged structure of more than one LXXLL motif. Further biochemical and functional studies show that the individual LXXLL motifs can bind but drive only minimal transactivation. Only when more than one LXXLL motif is involved can significant transcriptional activity be measured, and full activation requires all three LXXLL motifs. These findings led us to propose a model wherein each LXXLL motif has an additive effect, and the multiple binding modes by HNF4alpha toward the LXXLL motifs of PGC-1alpha could account for the apparent robust activation by providing a flexible mechanism for combinatorial recruitment of additional coactivators and mediators.

Structural basis of natural promoter recognition by a unique nuclear receptor, HNF4alpha. Diabetes gene product.

HNF4alpha (hepatocyte nuclear factor 4alpha) plays an essential role in the development and function of vertebrate organs, including hepatocytes and pancreatic beta-cells by regulating expression of multiple genes involved in organ development, nutrient transport, and diverse metabolic pathways. As such, HNF4alpha is a culprit gene product for a monogenic and dominantly inherited form of diabetes, known as maturity onset diabetes of the young (MODY). As a unique member of the nuclear receptor superfamily, HNF4alpha recognizes target genes containing two hexanucleotide direct repeat DNA-response elements separated by one base pair (DR1) by exclusively forming a cooperative homodimer. We describe here the 2.0 angstroms crystal structure of human HNF4alpha DNA binding domain in complex with a high affinity promoter element of another MODY gene, HNF1alpha, which reveals the molecular basis of unique target gene selection/recognition, DNA binding cooperativity, and dysfunction caused by diabetes-causing mutations. The predicted effects of MODY mutations have been tested by a set of biochemical and functional studies, which show that, in contrast to other MODY gene products, the subtle disruption of HNF4alpha molecular function can cause significant effects in afflicted MODY patients.

Expression, purification, crystallization and preliminary crystallographic study of a potential metal-dependent hydrolase with cyclase activity from Thermoanaerobacter tengcongensis.

The putative metal-dependent hydrolase gene TTE1006 from Thermoanaerobacter tengcongensis strain MB4T (T = type strain; Genbank accession No. AE008691) was heterologously expressed in Escherichia coli. The 205-amino-acid gene product was purified and crystallized. The crystal used for data collection belongs to space group P2(1), with unit-cell parameters a = 85.2, b = 62.1, c = 172.4 A, beta = 104.2 degrees. Using a synchrotron-radiation source, the resolution limit of the data reached 1.87 A. Eight molecules were estimated to be present in the asymmetric unit, with a solvent content of 48%. Structure determination is ongoing using the multiple-wavelength anomalous diffraction (MAD) method and also the molecular-replacement (MR) method.

Crystallization and preliminary crystallographic study of a recombinant predicted acetamidase/formamidase from the thermophile Thermoanaerobacter tengcongensis.

No crystal structures are yet available for homologues of a predicted acetamidase/formamidase (Amds/Fmds) from the archaeon Thermoanaerobacter tengcongensis. The Amds/Fmds gene was cloned and expressed as a soluble protein in Escherichia coli. Native Amds/Fmds and its SeMet-substituted form were purified and crystallized by vapour diffusion in hanging drops at 296 K. The native crystals, which were grown in PEG 8000, belong to the monoclinic space group P2(1), with unit-cell parameters a = 41.23 (3), b = 152.88 (6), c = 100.26 (7) A, beta = 99.49 (3) degrees. The diffraction data were collected to 2.00 A resolution using synchrotron radiation. Based on a predicted solvent content of 50%, a Matthews coefficient of 2.44 A3 Da(-1) and two main peaks in the self-rotation function, the asymmetric unit is predicted to contain two dimers of the 32 kDa native protein. MAD data were collected for the SeMet protein, but the corresponding crystals display different unit-cell parameters and appear to contain four dimers in the asymmetric unit.