Data of radiation damage on selenomethionine-substituted single-domain substrate-binding protein.

Radiation damage is an inherent issue in X-ray crystallography. It not only damages macromolecular crystals, which lowers the quality of the diffraction intensity, but results in inaccurate structural information. Among the various types of radiation damage, little is known regarding the damage to selenomethionine, an amino acid contained in some proteins. Recently, radiation damage to the selenomethionine-substituted single-domain substrate-binding domain from Rhodothermus marinus (SeMet-RmSBP) was investigated. Global and specific radiation damage from four datasets collected by repeatedly exposing a single RmSBP-SeMet crystal to X-rays were analyzed. The results indicated that the B-factor value of the selenium atom in selenomethionine was significantly increased compared with other atoms. To date, no images of radiation damage have been reported for selenomethionine-substituted proteins. Therefore, these data may be used to study radiation damage in macromolecular crystallography. This study provides insight into radiation damage associated with selenomethionine.

Structural Basis of the Inhibition of L-Methionine γ-Lyase from Fusobacterium nucleatum.

Fusobacterium nucleatum is a lesion-associated obligate anaerobic pathogen of destructive periodontal disease; it is also implicated in the progression and severity of colorectal cancer. Four genes (FN0625, FN1055, FN1220, and FN1419) of F. nucleatum are involved in producing hydrogen sulfide (H2S), which plays an essential role against oxidative stress. The molecular functions of Fn1419 are known, but their mechanisms remain unclear. We determined the crystal structure of Fn1419 at 2.5 Å, showing the unique conformation of the PLP-binding site when compared with L-methionine γ-lyase (MGL) proteins. Inhibitor screening for Fn1419 with L-cysteine showed that two natural compounds, gallic acid and dihydromyricetin, selectively inhibit the H2S production of Fn1419. The chemicals of gallic acid, dihydromyricetin, and its analogs containing trihydroxybenzene, were potentially responsible for the enzyme-inhibiting activity on Fn1419. Molecular docking and mutational analyses suggested that Gly112, Pro159, Val337, and Arg373 are involved in gallic acid binding and positioned close to the substrate and pyridoxal-5'-phosphate-binding site. Gallic acid has little effect on the other H2S-producing enzymes (Fn1220 and Fn1055). Overall, we proposed a molecular mechanism underlying the action of Fn1419 from F. nucleatum and found a new lead compound for inhibitor development.

Regulation of BRCA1 stability through the tandem UBX domains of isoleucyl-tRNA synthetase 1.

Aminoacyl-tRNA synthetases (ARSs) have evolved to acquire various additional domains. These domains allow ARSs to communicate with other cellular proteins in order to promote non-translational functions. Vertebrate cytoplasmic isoleucyl-tRNA synthetases (IARS1s) have an uncharacterized unique domain, UNE-I. Here, we present the crystal structure of the chicken IARS1 UNE-I complexed with glutamyl-tRNA synthetase 1 (EARS1). UNE-I consists of tandem ubiquitin regulatory X (UBX) domains that interact with a distinct hairpin loop on EARS1 and protect its neighboring proteins in the multi-synthetase complex from degradation. Phosphomimetic mutation of the two serine residues in the hairpin loop releases IARS1 from the complex. IARS1 interacts with BRCA1 in the nucleus, regulates its stability by inhibiting ubiquitylation via the UBX domains, and controls DNA repair function.

Crystal structure of human brain-type fatty acid-binding protein FABP7 complexed with palmitic acid.

The brain-type fatty acid-binding protein FABP7, which is expressed in astrocytes and neural progenitors, is a member of the intracellular lipid-binding protein family. This protein is not only involved in various cellular functions such as metabolism, inflammation and energy homeostasis, but also in diseases such as cognitive disorders and tumors. Structures of unsaturated fatty acids, such as oleic acid (OA) and docosahexaenoic acid (DHA), bound to FABP7 have been elucidated; however, structures of saturated fatty acids bound to FABP7 remain unknown. To better understand fatty acid recognition, here the crystal structure of human brain-type fatty acid-binding protein FABP7 complexed with palmitic acid (PA), a saturated fatty acid, is reported at a resolution of 1.6 Å. The PA bound to the fatty acid-binding pocket of FABP7 assumed a U-shaped conformation. The carboxylate moiety of PA interacted with Tyr129, Arg127 and, via a water bridge, with Arg107 and Thr54, whereas its aliphatic chain was stabilized by hydrophobic interactions with Met21, Leu24, Thr30, Thr37, Pro39, Phe58 and Asp77. Structural comparison showed that PA, OA and DHA exhibited unique binding conformations in the fatty acid-binding pocket, stabilized by distinct amino-acid interactions. The binding of PA to FABP7 exhibits a unique binding conformation when compared with other human FABPs (FABP3-FABP5 and FABP8) expressed in other tissues. Based on the crystal and fatty acid structures, it was suggested that PA, which prefers a linear form in nature, required a greater conformational change in its aliphatic chain to bind to the fatty acid-binding pocket in a U-shaped conformation, compared with the cis configurations of OA or DHA. This, together with the length of the aliphatic chain, was considered to be one of the factors determining the binding affinity of PA to FABP7. These results provide a better understanding of fatty acid recognition by FABP7 and expand the knowledge of the binding of PA to FABPs.

Crystal Structure of Aeromonas hydrophila Cytoplasmic 5'-Methylthioadenosine/S-Adenosylhomocysteine Nucleosidase.

5'-Methylthioadenosine/S-adenosyl-l-homocysteine (MTA/SAH) nucleosidase (MTAN) is an important enzyme in a number of critical biological processes. Mammals do not express MtaN, making this enzyme an attractive antibacterial drug target. In pathogen Aeromonas hydrophila, two MtnN subfamily genes (MtaN-1 and MtaN-2) play important roles in the periplasm and cytosol, respectively. We previously reported structural and functional analyses of MtaN-1, but little is known regarding MtaN-2 due to the lack of a crystal structure. Here, we determined the crystal structure of cytosolic A. hydrophila MtaN-2 in complex with adenine (ADE), which is a cleavage product of adenosine. AhMtaN-1 and AhMtaN-2 exhibit a high degree of similarity in the α-ß-α sandwich fold of the core structural motif. However, there is a structural difference in the nonconserved extended loop between ß7 and α3 that is associated with the channel depth of the substrate-binding pocket and dimerization. The ADE molecules in the substrate-binding pockets of AhMtaN-1 and AhMtaN-2 are stabilized with π-π stacking by Trp199 and Phe152, respectively, and the hydrophobic residues surrounding the ribose-binding sites differ. A structural comparison of AhMtaN-2 with other MtaN proteins showed that MtnN subfamily proteins exhibit a unique substrate-binding surface and dimerization interface.

Multifarious injection chamber for molecular structure study (MICOSS) system: development and application for serial femtosecond crystallography at Pohang Accelerator Laboratory X-ray Free-Electron Laser.

The multifarious injection chamber for molecular structure study (MICOSS) experimental system has been developed at the Pohang Accelerator Laboratory X-ray Free-Electron Laser for conducting serial femtosecond crystallography. This system comprises several instruments such as a dedicated sample chamber, sample injectors, sample environment diagnostic system and detector stage for convenient distance manipulation. Serial femtosecond crystallography experiments of lysozyme crystals have been conducted successfully. The diffraction peaks have reached to ∼1.8 Šresolution at the photon energy of 9.785 keV.

Focusing X-ray free-electron laser pulses using Kirkpatrick-Baez mirrors at the NCI hutch of the PAL-XFEL.

The Pohang Accelerator Laboratory X-ray Free-Electron Laser (PAL-XFEL) is a recently commissioned X-ray free-electron laser (XFEL) facility that provides intense ultrashort X-ray pulses based on the self-amplified spontaneous emission process. The nano-crystallography and coherent imaging (NCI) hutch with forward-scattering geometry is located at the hard X-ray beamline of the PAL-XFEL and provides opportunities to perform serial femtosecond crystallography and coherent X-ray diffraction imaging. To produce intense high-density XFEL pulses at the interaction positions between the X-rays and various samples, a microfocusing Kirkpatrick-Baez (KB) mirror system that includes an ultra-precision manipulator has been developed. In this paper, the design of a KB mirror system that focuses the hard XFEL beam onto a fixed sample point of the NCI hutch, which is positioned along the hard XFEL beamline, is described. The focusing system produces a two-dimensional focusing beam at approximately 2 µm scale across the 2-11 keV photon energy range. XFEL pulses of 9.7 keV energy were successfully focused onto an area of size 1.94 µm × 2.08 µm FWHM.

Structures of CRISPR Cas3 offer mechanistic insights into Cascade-activated DNA unwinding and degradation.

CRISPR drives prokaryotic adaptation to invasive nucleic acids such as phages and plasmids, using an RNA-mediated interference mechanism. Interference in type I CRISPR-Cas systems requires a targeting Cascade complex and a degradation machine, Cas3, which contains both nuclease and helicase activities. Here we report the crystal structures of Thermobifida fusca Cas3 bound to single-stranded (ss) DNA substrate and show that it is an obligate 3'-to-5' ssDNase that preferentially accepts substrate directly from the helicase moiety. Conserved residues in the HD-type nuclease coordinate two irons for ssDNA cleavage. We demonstrate ATP coordination and conformational flexibility of the SF2-type helicase domain. Cas3 is specifically guided toward Cascade-bound target DNA by a PAM sequence, through physical interactions with both the nontarget substrate strand and the CasA protein. The sequence of recognition events ensures well-controlled DNA targeting and degradation of foreign DNA by Cascade and Cas3.

Crystal structure of an EfPDF complex with Met-Ala-Ser based on crystallographic packing.

PDF (peptide deformylase) plays a critical role in the production of mature proteins by removing the N-formyl polypeptide of nascent proteins in the prokaryote cell system. This protein is essential for bacterial growth, making it an attractive target for the design of new antibiotics. Accordingly, PDF has been evaluated as a drug target; however, architectural mechanism studies of PDF have not yet fully elucidated its molecular function. We recently reported the crystal structure of PDF produced by Enterococcus faecium [K.H. Nam, J.I. Ham, A. Priyadarshi, E.E. Kim, N. Chung, K.Y. Hwang, "Insight into the antibacterial drug design and architectural mechanism of peptide recognition from the E. faecium peptide deformylase structure", Proteins 74 (2009) 261-265]. Here, we present the crystal structure of the EfPDF complex with MAS (Met-Ser-Ala), thereby not only delineating the architectural mechanism for the recognition of mimic-peptides by N-terminal cleaved expression peptide, but also suggesting possible targets for rational design of antibacterial drugs. In addition to their implications for drug design, these structural studies will facilitate elucidation of the architectural mechanism responsible for the peptide recognition of PDF.

Thyroglobulin measurement in fine-needle aspirate washouts: the criteria for neck node dissection for patients with thyroid cancer.

BACKGROUND: Several studies report that detection of thyroglobulin (Tg) in fine-needle aspiration (FNA) biopsy washout fluid from lymph nodes identifies recurrences/metastases of differentiated papillary thyroid cancer (DPTC) in the neck with higher sensitivity and specificity than fine-needle aspiration cytology (FNAC). However, the diagnostic FNA-Tg cutoff values have not yet been established. OBJECTIVE: To determine an appropriate diagnostic threshold value for Tg levels in FNA washout fluid in patients with neck node metastases or recurrences of DPTC. DESIGN: We performed ultrasound (US)-guided FNAC and measured Tg levels in FNA washout fluid (FNA-Tg). Final diagnoses were confirmed by histological examination of excised specimens or by follow-up examinations for at least 24 months. PATIENTS: A total of 168 ultrasonographically detected lymph nodes from 168 patients with DPTC were included. MEASUREMENTS: In comparison with FNAC, we evaluated diagnostic sensitivity, specificity and accuracy of metastasis detection according to several predetermined threshold levels: 1, 10, 100 ng/ml, mean+2SD of node-negative patients, and FNA-Tg/serum-Tg > 1. RESULTS: The diagnostic sensitivity was lowest at 77.3% for FNAC alone. The lower FNA-Tg threshold levels showed higher diagnostic sensitivity whereas the higher FNA-Tg showed higher specificity. In addition to the FNAC results, FNA-Tg levels showed 95.0% sensitivity, 81.6% specificity, 92.6% in positive predictive value (PPV) and 87.0% in negative predictive value (NPV) with the threshold of FNA-Tg level at 10 ng/ml, serum-Tg or mean+2SD of FNA-Tg measured in node-negative patients. The diagnostic accuracy of FNA-Tg was higher in neck node recurrences after thyroid surgery than in the metastasis of patients waiting for surgery. CONCLUSION: FNA-Tg measurement as well as FNAC should be performed either before or after surgery to evaluate neck node metastases or recurrences in patients with differentiated thyroid carcinomas. We recommend that the threshold values for FNA-Tg levels should be > 10 ng/ml if the serum-Tg level or the mean+2SD in node-negative patients is not available for reference.

Structural studies of human brain-type creatine kinase complexed with the ADP-Mg2+-NO3- -creatine transition-state analogue complex.

Creatine kinase is a member of the phosphagen kinase family, which catalyzes the reversible phosphoryl transfer reaction that occurs between ATP and creatine to produce ADP and phosphocreatine. Here, three structural aspects of human-brain-type-creatine-kinase (hBB-CK) were identified by X-ray crystallography: the ligand-free-form at 2.2A; the ADP-Mg2+, nitrate, and creatine complex (transition-state-analogue complex; TSAC); and the ADP-Mg2+-complex at 2.0A. The structures of ligand-bound hBB-CK revealed two different monomeric states in a single homodimer. One monomer is a closed form, either bound to TSAC or the ADP-Mg2+-complex, and the second monomer is an unliganded open form. These structural studies provide a detailed mechanism indicating that the binding of ADP-Mg2+ alone may trigger conformational changes in hBB-CK that were not observed with muscle-type-CK.

Papillary thyroid carcinoma with thyroid-associated orbitopathy in a euthyroid state.

PURPOSE: Thyroid cancers with concurrent thyroid-associated orbitopathy (TAO) are extremely rare. The present study reports 5 unusual cases of papillary thyroid microcarcinoma in patients presenting with TAO in an euthyroid state. METHODS: We retrospectively reviewed the records of 5 patients (4 female, 1 male) with no known history of thyroid disease, who initially presented with TAO and were subsequently found to have thyroid cancer. TAO was diagnosed by a combination of computed tomography and clinical symptoms and signs. All patients underwent routine serologic assessment of thyroid hormones and thyroid-related autoantibodies including thyrotropin-receptor antibodies, as well as imaging studies such as neck ultrasonography or thyroid scintiscan with I. RESULTS: Patients presented with asymmetric proptosis (cases 1-3), ocular motility restriction (cases 1-3), and eyelid retraction (cases 1-5). None of the patients showed clinical signs of hyperthyroidism, and all returned normal thyroid function test data. Thyroid nodules were detected by neck imaging; subsequent fine-needle aspiration biopsies were performed with the cytologic result of papillary carcinoma. In no cases did ophthalmic symptoms resolve following cancer treatment (e.g., thyroidectomy and radioactive iodine therapy). All patients were positive for thyroid-binding inhibiting immunoglobulin in low titers. CONCLUSIONS: Although coincident occurrence is rare, patients presenting with TAO should be carefully evaluated for the possible presence of papillary microcarcinoma, which can develop in a setting of systemic autoimmunity without inducing hyperthyroidism.

Crystallization and preliminary X-ray crystallographic analysis of the tRNA-specific adenosine deaminase from Streptococcus pyogenes.

The tRNA-specific adenosine deaminase from the pathogenic bacteria Streptococcus pyogenes (spTAD) has been overexpressed in Escherichia coli and crystallized in the presence of Zn2+ ion at 295 K using ammonium sulfate as a precipitant. Flash-cooled crystals of spTAD diffracted to 2.0 A using 30%(v/v) glycerol as a cryoprotectant. X-ray diffraction data have been collected to 2.0 A using synchrotron radiation. The crystal belongs to the tetragonal space group P4(2)2(1)2, with unit-cell parameters a = b = 81.042, c = 81.270 A. The asymmetric unit contains one subunit of spTAD, with a corresponding crystal volume per protein weight (VM) of 3.3 A3 Da(-1) and a solvent content of 62.7%.