Bacteriophage N4 large terminase: expression, purification and X-ray crystallographic analysis
Genome packaging is a critical step in the assembly of dsDNA bacteriophages and is carried out by a powerful molecular motor known as the large terminase. To date, wild-type structures of only two large terminase proteins are available, and more structural information is needed to understand the genome-packaging mechanism. Towards this goal, the large and small terminase proteins from bacteriophage N4, which infects the Escherichia coli K12 strain, have been cloned, expressed and purified. The purified putative large terminase protein hydrolyzes ATP, and this is enhanced in the presence of the small terminase. The large te...
Source: Acta Crystallographica Section F - March 22, 2018 Category: Biochemistry Authors: Wangchuk, J. Prakash, P. Bhaumik, P. Kondabagil, K. Tags: bacteriophage N4 genome packaging dsDNA virus large terminase small terminase research communications Source Type: research
Crystal structure of pyrimidine-nucleoside phosphorylase from Bacillus subtilis in complex with imidazole and sulfate
Pyrimidine-nucleoside phosphorylase catalyzes the phosphorolytic cleavage of thymidine and uridine with equal activity. Investigation of this protein is essential for anticancer drug design. Here, the structure of this protein from Bacillus subtilis in complex with imidazole and sulfate is reported at 1.9 Å resolution, which is an improvement on the previously reported structure at 2.6 Å resolution. The localization and position of imidazole in the nucleoside-binding site reflects the possible binding of ligands that possess an imidazole ring. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - March 22, 2018 Category: Biochemistry Authors: Balaev, V.V. Prokofev, I.I. Gabdoulkhakov, A.G. Betzel, C. Lashkov, A.A. Tags: X-ray analysis protein crystallography nucleoside phosphorylases nucleosides imidazole pyrimidine-nucleoside phosphorylase Bacillus subtilis research communications Source Type: research
Crystal structure of chorismate mutase from Burkholderia phymatum
The bacterium Burkholderia phymatum is a promiscuous symbiotic nitrogen-fixating bacterium that belongs to one of the largest groups of Betaproteobacteria. Other Burkholderia species are known to cause disease in plants and animals, and some are potential agents for biological warfare. Structural genomics efforts include characterizing the structures of enzymes from pathways that can be targeted for drug development. As part of these efforts, chorismate mutase from B. phymatum was produced and crystallized, and a 1.95 Å resolution structure is reported. This enzyme shares less than 33% sequence identity with othe...
Source: Acta Crystallographica Section F - March 22, 2018 Category: Biochemistry Authors: Asojo, O.A. Subramanian, S. Abendroth, J. Exley, I. Lorimer, D.D. Edwards, T.E. Myler, P.J. Tags: chorismate mutase Burkholderia phymatum structural genomics Seattle Structural Genomics Center for Infectious Disease isomerase shikimate pathway research communications Source Type: research
Crystal structure of an inferred ancestral bacterial pyruvate decarboxylase
Pyruvate decarboxylase (PDC; EC 18.104.22.168) is a key enzyme in homofermentative metabolism where ethanol is the major product. PDCs are thiamine pyrophosphate- and Mg2+ ion-dependent enzymes that catalyse the non-oxidative decarboxylation of pyruvate to acetaldehyde and carbon dioxide. As this enzyme class is rare in bacteria, current knowledge of bacterial PDCs is extremely limited. One approach to further the understanding of bacterial PDCs is to exploit the diversity provided by evolution. Ancestral sequence reconstruction (ASR) is a method of computational molecular evolution to infer extinct ancestral protein sequences, ...
Source: Acta Crystallographica Section F - February 26, 2018 Category: Biochemistry Authors: Buddrus, L. Andrews, E.S.V. Leak, D.J. Danson, M.J. Arcus, V.L. Crennell, S.J. Tags: ancestral sequence reconstruction pyruvate decarboxylase lyases crystal structure TPP-dependent enzymes research communications Source Type: research
Crystal structure of the Kelch domain of human NS1-binding protein at 1.98 Å resolution
NS1-binding protein (NS1-BP), which belongs to the Kelch protein superfamily, was first identified as a novel human 70 kDa protein that interacts with NS1 of Influenza A virus. It is involved in many cell functions, including pre-mRNA splicing, the ERK signalling pathway, the aryl hydrocarbon receptor (AHR) pathway, F-actin organization and protein ubiquitylation. However, the structure of NS1-BP is still unknown, which may impede functional studies. Here, the structure of the C-terminal Kelch domain of NS1-BP (NS1-BP-C; residues 330 – 642) was determined at 1.98 Å resolution. The Kelch domain adopts a ...
Source: Acta Crystallographica Section F - February 26, 2018 Category: Biochemistry Authors: Guo, L. Liu, Y. Tags: NS1-binding protein Kelch domain ERK signalling pathway pre-mRNA splicing aryl hydrocarbon receptor (AHR) pathway F-actin organization protein ubiquitylation X-ray crystallography research communications Source Type: research
Crystal structure of the MSMEG_4306 gene product from Mycobacterium smegmatis
The MSMEG_4306 gene from Mycobacterium smegmatis encodes a protein of unknown function with 242 amino-acid residues that contains a conserved zinc-ribbon domain at its C-terminus. Here, the crystal structure of MSMEG_4306 determined by the single-wavelength anomalous dispersion method using just one zinc ion co-purified with the protein is reported. The crystal structure of MSMEG_4306 shows a coiled-coil helix domain in the N-terminal region and a zinc-ribbon domain in the C-terminal region. A structural similarity search against the Protein Data Bank using MSMEG_4306 as a query revealed two similar structures, namely CT39...
Source: Acta Crystallographica Section F - February 26, 2018 Category: Biochemistry Authors: Kumar, A. Karthikeyan, S. Tags: MSMEG_4306 Mycobacterium smegmatis X-ray crystallography zinc SAD Rv2229c zinc ribbon coiled-coil helix research communications Source Type: research
Expression, purification and crystallization of phosphoribosyl transferase from a mycobacteriophage
Tuberculosis (TB) continues to remain a leading cause of death globally. Of particular concern is the emergence and rise in incidence of multidrug-resistant and extremely drug-resistant cases of TB. To counter this threat, it is important to explore alternative therapies, including phage therapy. Phage BTCU-1 specifically infects Mycobacterium spp. and kills the majority of them. Intriguingly, many proteins from the phage do not share high amino-acid sequence identity with proteins from species other than phages. Here, the expression, purification and crystallization of one such protein, a putative phosphoribosyl transfera...
Source: Acta Crystallographica Section F - February 26, 2018 Category: Biochemistry Authors: Li, L. Lee, E. Shaw, N. Tags: mycobacteriophages tuberculosis X-ray diffraction phosphoribosyl transferases phage BTCU-1 research communications Source Type: research
A novel inhibitor stabilizes the inactive conformation of MAPK-interacting kinase 1
Mitogen-activated protein kinase (MAPK)-interacting kinases 1 (Mnk1) and 2 (Mnk2) modulate translation initiation through the phosphorylation of eukaryotic translation initiation factor 4E, which promotes tumorigenesis. However, Mnk1 and Mnk2 are dispensable in normal cells, suggesting that the inhibition of Mnk1 and Mnk2 could be effective in cancer therapy. To provide a structural basis for Mnk1 inhibition, a novel Mnk1 inhibitor was discovered and the crystal structure of Mnk1 in complex with this inhibitor was determined. The crystal structure revealed that the inhibitor binds to the autoinhibited state of Mnk1, stabil...
Source: Acta Crystallographica Section F - February 26, 2018 Category: Biochemistry Authors: Matsui, Y. Yasumatsu, I. Yoshida, K. Iimura, S. Ikeno, Y. Nawano, T. Fukano, H. Ubukata, O. Hanzawa, H. Tanzawa, F. Isoyama, T. Tags: kinase kinase inhibitors inactive conformations transferases Mnk1 MAPK-interacting kinase 1 DS12881479 research communications Source Type: research
Enhancement of the thermostability of mouse claudin-3 on complex formation with the carboxyl-terminal region of Clostridium perfringens enterotoxin improves crystal quality
Tight junctions regulate substance permeation through intercellular spaces as a physical barrier or a paracellular pathway, and play an important role in maintaining the internal environment. Claudins, which are tetraspan-transmembrane proteins, are pivotal components of tight junctions. In mammals 27 claudin subtypes have been identified, each of which interacts with specific subtypes. Although the crystal structures of several subtypes have been determined, the molecular mechanisms underlying subtype specificity remain unclear. Here, mouse claudin-3 (mCldn3) was crystallized in complex with the C-terminal region of Clost...
Source: Acta Crystallographica Section F - February 26, 2018 Category: Biochemistry Authors: Nakamura, S. Fujiyoshi, Y. Irie, K. Tags: tight junctions mouse claudin-3 Clostridium perfringens enterotoxins crystallization thermostability research communications Source Type: research
Cloning, purification and structure determination of the HIV integrase-binding domain of lens epithelium-derived growth factor
Lens epithelium-derived growth factor (LEDGF)/p75 is the dominant binding partner of HIV-1 integrase in human cells. The crystal structure of the HIV integrase-binding domain (IBD) of LEDGF has been determined in the absence of ligand. IBD was overexpressed in Escherichia coli, purified and crystallized by sitting-drop vapour diffusion. X-ray diffraction data were collected at Diamond Light Source to a resolution of 2.05 Å . The crystals belonged to space group P21, with eight polypeptide chains in the asymmetric unit arranged as an unusual octamer composed of four domain-swapped IBD dimers. IBD exists as a mixtu...
Source: Acta Crystallographica Section F - February 26, 2018 Category: Biochemistry Authors: Hannon, C. Cruz-Migoni, A. Platonova, O. Owen, R.L. Nettleship, J.E. Miller, A. Carr, S.B. Harris, G. Rabbitts, T.H. Phillips, S.E.V. Tags: HIV integrase-binding domain lens epithelium-derived growth factor human immunodeficiency virus domain swapping research communications Source Type: research
The first crystal structure of manganese superoxide dismutase from the genus Staphylococcus
A recombinant Staphylococcus equorum manganese superoxide dismutase (MnSOD) with an Asp13Arg substitution displays activity over a wide range of pH, at high temperature and in the presence of chaotropic agents, and retains 50% of its activity after irradiation with UVC for up to 45 min. Interestingly, Bacillus subtilis MnSOD does not have the same stability, despite having a closely similar primary structure and thus presumably also tertiary structure. Here, the crystal structure of S. equorum MnSOD at 1.4 Å resolution is reported that may explain these differences. The crystal belonged to space group P3221, ...
Source: Acta Crystallographica Section F - February 26, 2018 Category: Biochemistry Authors: Retnoningrum, D.S. Yoshida, H. Arumsari, S. Kamitori, S. Ismaya, W.T. Tags: manganese superoxide dismutase Staphylococcus equorum MnSOD structural comparison thermostable enzymes research communications Source Type: research
High-resolution structure of the Influenza A virus PB2cap binding domain illuminates the changes induced by ligand binding
In this report, the anatomic behavior of the influenza A virus PB2cap domain is established by solving the crystal structure of native influenza A virus PB2cap at 1.52 Å resolution. By comparing it with the ligand-bound structure, the dissociation and rotation of the ligand-binding domain in PB2cap from the C-terminal domain is identified. This domain movement is present in many PB2cap structures, suggesting its functional relevance for polymerase activity. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - February 26, 2018 Category: Biochemistry Authors: Constantinides, A. Gumpper, R. Severin, C. Luo, M. Tags: high resolution structures transcription polymerase basic protein 2 RNA polymerase cap-snatching domain movement Influenza A virus research communications Source Type: research
YopT domain of the PfhB2 toxin from Pasteurella multocida: protein expression, characterization, crystallization and crystallographic analysis
Pasteurella multocida causes respiratory-tract infections in a broad range of animals, as well as opportunistic infections in humans. P. multocida secretes a multidomain toxin called PfhB2, which contains a YopT-like cysteine protease domain at its C-terminus. The YopT domain of PfhB2 contains a well conserved Cys – His – Asp catalytic triad that defines YopT family members, and shares high sequence similarity with the prototype YopT from Yersinia sp. To date, only one crystal structure of a YopT family member has been reported; however, additional structural information is needed to help characterize the varie...
Source: Acta Crystallographica Section F - February 26, 2018 Category: Biochemistry Authors: Kumar, S. Hedrick, V. Mattoo, S. Tags: PfhB2 YopT cysteine protease catalytic triad Pasteurella multocida AvrPphB research communications Source Type: research
A thermostable and alkaline GDSL-motif esterase from Bacillus sp. K91: crystallization and X-ray crystallographic analysis
The esterase Est8 from the thermophilic bacterium Bacillus sp. K91 belongs to the GDSL family and is active on a variety of acetylated compounds, including 7-aminocephalosporanic acid. In contrast to other esterases of the GDSL family, the catalytic residues Asp182 and His185 were more pivotal for the catalytic activity of Est8 than the Ser11 residue. To better understand the biochemical and enzymatic properties of Est8, recombinant Est8 protein was purified and crystallized. Crystals of Est8 were obtained by the hanging-drop vapour-diffusion method using 2.0 M ammonium sulfate, 5%(v/v) 2-propanol as the crystallizatio...
Source: Acta Crystallographica Section F - January 26, 2018 Category: Biochemistry Authors: Ding, J. Zhu, H. Ye, Y. Li, J. Han, N. Wu, Q. Huang, Z. Meng, Z. Tags: Est8 crystallization X-ray crystallographic analysis GDSL-motif esterase research communications Source Type: research
Xylanase B from Clostridium cellulovorans 743B: overexpression, purification, crystallization and X-ray diffraction analysis
In this study, XynB was cloned, overexpressed, purified and crystallized. XynB was crystallized using the hanging-drop vapour-diffusion method in the presence of 0.2 M sodium acetate trihydrate, 0.1 M Tris – HCl pH 8.5, 32%(w/v) PEG 4000 at 293 K. X-ray diffraction analysis revealed that the crystal diffracted to 1.95 Å resolution and belonged to space group P212121, with unit-cell parameters a = 74.28, b = 77.55, c = 88.20 Å , α = β = γ = 90 ° . The data-evaluation statistics revealed high quality of the collected data, thereby establishing a solid b...
Source: Acta Crystallographica Section F - January 26, 2018 Category: Biochemistry Authors: Nakajima, D. Nagano, A. Shibata, T. Tanaka, R. Kuroda, K. Ueda, M. Miyake, H. Tags: xylanases xylanase B Clostridium cellulovorans cellulosome CBM family 22 glycoside hydrolase family 10 research communications Source Type: research
Structural characterization of Porphyromonas gingivalis enoyl-ACP reductase II (FabK)
Enoyl-acyl carrier protein (ACP) reductase II (FabK) is a critical rate-limiting enzyme in the bacterial type II fatty-acid synthesis (FAS II) pathway. FAS II pathway enzymes are markedly disparate from their mammalian analogs in the FAS I pathway in both structure and mechanism. Enzymes involved in bacterial fatty-acid synthesis represent viable drug targets for Gram-negative pathogens, and historical precedent exists for targeting them in the treatment of diseases of the oral cavity. The Gram-negative organism Porphyromonas gingivalis represents a key causative agent of the costly and highly prevalent disease known as ch...
Source: Acta Crystallographica Section F - January 26, 2018 Category: Biochemistry Authors: Hevener, K.E. Santarsiero, B.D. Lee, H. Jones, J.A. Boci, T. Johnson, M.E. Mehboob, S. Tags: Porphyromonas gingivalis periodontal disease enoyl-ACP reductase II FabK crystal structure research communications Source Type: research
Ribokinase from Leishmania donovani: purification, characterization and X-ray crystallographic analysis
Leishmania is an auxotrophic protozoan parasite which acquires d-ribose by transporting it from the host cell and also by the hydrolysis of nucleosides. The enzyme ribokinase (RK) catalyzes the first step of ribose metabolism by phosphorylating d-ribose using ATP to produce d-ribose-5-phosphate. To understand its structure and function, the gene encoding RK from L. donovani was cloned, expressed and purified using affinity and size-exclusion chromatography. Circular-dichroism spectroscopy of the purified protein showed comparatively more α -helix in the secondary-structure content, and thermal unfolding revealed the ...
Source: Acta Crystallographica Section F - January 26, 2018 Category: Biochemistry Authors: Gatreddi, S. Are, S. Qureshi, I.A. Tags: Leishmania donovani d-ribose metabolism ribokinase crystallization thermal stability research communications Source Type: research
Structure of bovine cytochrome c oxidase in the ligand-free reduced state at neutral pH
Cytochrome c oxidase (CcO), the terminal oxidase in cellular respiration, couples proton pumping to O2 reduction. Mammalian CcO resides in the inner mitochondrial membrane. Previously, a model of H-pathway proton pumping was proposed based on various CcO crystal structures. However, all previously determined structures were solved using crystals obtained at pH 5.7, which differs from the environmental pH of CcO in the inner membrane. The structures of fully oxidized and ligand-free reduced CcO at pH 7.3 have now been determined. Structural comparison between the oxidized and reduced states revealed that the structural alte...
Source: Acta Crystallographica Section F - January 26, 2018 Category: Biochemistry Authors: Luo, F. Shinzawa-Itoh, K. Hagimoto, K. Shimada, A. Shimada, S. Yamashita, E. Yoshikawa, S. Tsukihara, T. Tags: cytochrome c oxidase membrane-protein complexes X-ray structure neutral pH research communications Source Type: research
X-ray crystallographic and high-speed AFM studies of peroxiredoxin 1 from Chlamydomonas reinhardtii
In this study, high-speed atomic force microscopy (HS-AFM) images of CrPRX1 and an X-ray crystallographic analysis have enabled examination of the oligomeric state of CrPRX1. Diffraction data from a crystal of the Cys174Ser mutant of CrPRX1 indicate the existence of noncrystallographic fivefold symmetry. HS-AFM images of CrPRX1 further show that CrPRX1 particles form rings with pentagonal rotational symmetry. On the basis of these findings, the oligomeric state of CrPRX1 is discussed and it is concluded that this PRX exists in a ring-shaped decameric form comprising a pentamer of dimers. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - January 26, 2018 Category: Biochemistry Authors: Charoenwattanasatien, R. Tanaka, H. Zinzius, K. Hochmal, A.K. Mutoh, R. Yamamoto, D. Hippler, M. Kurisu, G. Tags: photosynthesis 2-Cys peroxiredoxin oligomeric state peroxiredoxin 1 atomic force microscopy Chlamydomonas reinhardtii research communications Source Type: research
Human CCL5 trimer: expression, purification and initial crystallographic studies
The chemokine CCL5 is considered to be a potential therapeutic target because of its ability to recruit immune cells to inflammatory sites. CCL5 aggregates under physiological conditions, and high-order oligomer formation is considered to be significant for cell migration, immune-cell activation and HIV cell entry. The structure of the high-order oligomer is unknown and the mechanism by which the oligomer is derived has yet to be established. Here, a CCL5 mutant (CCL5-E66S) which is deficient in oligomer formation was mixed with native CCL5 to prepare a protein trimer. At an optimized ratio the trimeric CCL5 crystallized, ...
Source: Acta Crystallographica Section F - January 26, 2018 Category: Biochemistry Authors: Chen, Y.-C. Li, K.-M. Zarivach, R. Sun, Y.-J. Sue, S.-C. Tags: human CCL5 trimer RANTES chemokines research communications Source Type: research
The Pex4p – Pex22p complex from Hansenula polymorpha: biophysical analysis, crystallization and X-ray diffraction characterization
This article reports the crystallization of Pex4p and of the Pex4p – Pex22p complex from the yeast Hansenula polymorpha, and data collection from their crystals to 2.0 and 2.85 Å resolution, respectively. The resulting structures are likely to provide important insights to understand the molecular mechanism of the Pex4p – Pex22p complex and its role in peroxisome biogenesis. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - January 26, 2018 Category: Biochemistry Authors: Ali, A.M. Atmaj, J. Adawy, A. Lunev, S. Van Oosterwijk, N. Yan, S.R. Williams, C. Groves, M.R. Tags: Pex4 Pex22 ubiquitin-conjugating enzyme peroxisome import Hansenula polymorpha research communications Source Type: research
Protein Crystallography – Methods and Protocols. Edited by A. Wlodawer, Z. Dauter and M. Jaskolski. Humana Press, 2017. Pp XIII + 672. Hardcover. Price GBP 100, USD 159. ISBN 9781493969982.
(Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - December 23, 2017 Category: Biochemistry Authors: Liebschner, D. Tags: book reviews macromolecular crystallography protein science methods and protocols Source Type: research
Structure of the mouse acidic amino acid decarboxylase GADL1
Pyridoxal 5 ′ -phosphate (PLP) is a ubiquitous cofactor in various enzyme classes, including PLP-dependent decarboxylases. A recently discovered member of this class is glutamic acid decarboxylase-like protein 1 (GADL1), which lacks the activity to decarboxylate glutamate to γ -aminobutyrate, despite its homology to glutamic acid decarboxylase. Among the acidic amino acid decarboxylases, GADL1 is most similar to cysteine sulfinic acid decarboxylase (CSAD), but the physiological function of GADL1 is unclear, although its expression pattern and activity suggest a role in neurotransmitter and neuroprotectant metab...
Source: Acta Crystallographica Section F - December 23, 2017 Category: Biochemistry Authors: Raasakka, A. Mahootchi, E. Winge, I. Luan, W. Kursula, P. Haavik, J. Tags: decarboxylases pyridoxal phosphate catalysis conformational change research communications Source Type: research
Crystal structures of the archaeal RNase P protein Rpp38 in complex with RNA fragments containing a K-turn motif
In this study, the crystal structure of PhoRpp38 in complex with the K-turn in P12.2 was improved to 2.1 Å resolution and the structure of PhoRpp38 in complex with the K-turn in P12.1 was also determined at a resolution of 3.1 Å . Both structures revealed that Lys35, Asn38 and Glu39 in PhoRpp38 interact with characteristic G · A and A · G pairs in the K-turn, while Thr37, Asp59, Lys84, Glu94, Ala96 and Ala98 in PhoRpp38 interact with the three-nucleotide bulge in the K-turn. Moreover, an extended stem-loop containing P10 – P12.2 in complex with PhoRpp38, as well as PhoRpp21 and PhoRp...
Source: Acta Crystallographica Section F - December 23, 2017 Category: Biochemistry Authors: Oshima, K. Gao, X. Hayashi, S. Ueda, T. Nakashima, T. Kimura, M. Tags: archaea K-turn L7Ae family Pyrococcus horikoshii ribonuclease P research communications Source Type: research
Solution NMR structures of oxidized and reduced Ehrlichia chaffeensis thioredoxin: NMR-invisible structure owing to backbone dynamics
Thioredoxins are small ubiquitous proteins that participate in a diverse variety of redox reactions via the reversible oxidation of two cysteine thiol groups in a structurally conserved active site. Here, the NMR solution structures of a reduced and oxidized thioredoxin from Ehrlichia chaffeensis (Ec-Trx, ECH_0218), the etiological agent responsible for human monocytic ehrlichiosis, are described. The overall topology of the calculated structures is similar in both redox states and is similar to those of other thioredoxins: a five-stranded, mixed β -sheet ( β 1 – β 3 – β 2 – β 4 &...
Source: Acta Crystallographica Section F - December 23, 2017 Category: Biochemistry Authors: Buchko, G.W. Hewitt, S.N. Van Voorhis, W.C. Myler, P.J. Tags: human monocytic ehrlichiosis infectious diseases thioredoxin reactive oxygen species structural biology molecular dynamics Ehrlichia chaffeensis NMR research communications Source Type: research
Structural characterization of a novel monotreme-specific protein with antimicrobial activity from the milk of the platypus
Monotreme lactation protein (MLP) is a recently identified protein with antimicrobial activity. It is present in the milk of monotremes and is unique to this lineage. To characterize MLP and to gain insight into the potential role of this protein in the evolution of lactation, the crystal structure of duck-billed platypus (Ornithorhynchus anatinus) MLP was determined at 1.82 Å resolution. This is the first structure to be reported for this novel, mammalian antibacterial protein. MLP was expressed as a FLAG epitope-tagged protein in mammalian cells and crystallized readily, with at least three space groups being o...
Source: Acta Crystallographica Section F - December 23, 2017 Category: Biochemistry Authors: Newman, J. Sharp, J.A. Enjapoori, A.K. Bentley, J. Nicholas, K.R. Adams, T.E. Peat, T.S. Tags: novel folds SAD phasing monotremes platypus antibacterial monotreme lactation protein research communications Source Type: research
Production, biophysical characterization and initial crystallization studies of the N- and C-terminal domains of DsbD, an essential enzyme in Neisseria meningitidis
The membrane protein DsbD is a reductase that acts as an electron hub, translocating reducing equivalents from cytoplasmic thioredoxin to a number of periplasmic substrates involved in oxidative protein folding, cytochrome c maturation and oxidative stress defence. DsbD is a multi-domain protein consisting of a transmembrane domain (t-DsbD) flanked by two periplasmic domains (n-DsbD and c-DsbD). Previous studies have shown that DsbD is required for the survival of the obligate human pathogen Neisseria meningitidis. To help understand the structural and functional aspects of N. meningitidis DsbD, the two periplasmic domains...
Source: Acta Crystallographica Section F - December 23, 2017 Category: Biochemistry Authors: Smith, R.P. Whitten, A.E. Paxman, J.J. Kahler, C.M. Scanlon, M.J. Heras, B. Tags: disulfide catalysis DsbD Neisseria meningitidis membrane proteins research communications Source Type: research
Structure of aspartate β -semialdehyde dehydrogenase from Francisella tularensis
Aspartate β -semialdehyde dehydrogenase (ASADH) is an enzyme involved in the diaminopimelate pathway of lysine biosynthesis. It is essential for the viability of many pathogenic bacteria and therefore has been the subject of considerable research for the generation of novel antibiotic compounds. This manuscript describes the first structure of ASADH from Francisella tularensis, the causative agent of tularemia and a potential bioterrorism agent. The structure was determined at 2.45 Å resolution and has a similar biological assembly to other bacterial homologs. ASADH is known to be dimeric in bacteria and hav...
Source: Acta Crystallographica Section F - December 15, 2017 Category: Biochemistry Authors: Mank, N.J. Pote, S. Majorek, K.A. Arnette, A.K. Klapper, V.G. Hurlburt, B.K. Chruszcz, M. Tags: amino-acid biosynthesis lysine biosynthesis aspartate β -semialdehyde dehydrogenase oxidoreductases Francisella tularensis tularemia research communications Source Type: research
Crystal structure of cytoplasmic acetoacetyl-CoA thiolase from Saccharomyces cerevisiae
Thiolases are vital enzymes which participate in both degradative and biosynthetic pathways. Biosynthetic thiolases catalyze carbon – carbon bond formation by a Claisen condensation reaction. The cytoplasmic acetoacetyl-CoA thiolase from Saccharomyces cerevisiae, ERG10, catalyses carbon – carbon bond formation in the mevalonate pathway. The structure of a S. cerevisiae biosynthetic thiolase has not previously been reported. Here, crystal structures of apo ERG10 and its Cys91Ala variant were solved at resolutions of 2.2 and 1.95 Å , respectively. The structure determined shows that ERG10 shares the cha...
Source: Acta Crystallographica Section F - December 15, 2017 Category: Biochemistry Authors: Zhou, P. Zhu, Z. Hidayatullah Khan, M. Zheng, P. Teng, M. Niu, L Tags: acetoacetyl-CoA thiolase Claisen condensation mevalonate pathway X-ray crystallography Saccharomyces cerevisiae research communications Source Type: research
Structure and stability of the Human respiratory syncytial virus M2 – 1 RNA-binding core domain reveals a compact and cooperative folding unit
Human syncytial respiratory virus is a nonsegmented negative-strand RNA virus with serious implications for respiratory disease in infants, and has recently been reclassified into a new family, Pneumoviridae. One of the main reasons for this classification is the unique presence of a transcriptional antiterminator, called M2 – 1. The puzzling mechanism of action of M2 – 1, which is a rarity among antiterminators in viruses and is part of the RNA polymerase complex, relies on dissecting the structure and function of this multidomain tetramer. The RNA-binding activity is located in a monomeric globular `core' dom...
Source: Acta Crystallographica Section F - December 15, 2017 Category: Biochemistry Authors: Molina, I.G. Josts, I. Almeida Hernandez, Y. Esperante, S. Salgueiro, M. Garcia Alai, M.M. de Prat-Gay, G. Tidow, H. Tags: viral proteins crystal structure small-angle X-ray scattering protein folding Human syncytial respiratory virus research communications Source Type: research
The novel thermostable cellulose-degrading enzyme DtCel5H from Dictyoglomus thermophilum: crystallization and X-ray crystallographic analysis
Cellulose-based products constitute the great majority of municipal waste, and applications of cellulases in the conversion of waste biomass to biofuels will be a key technology in future biorefineries. Currently, multi-enzymatic pre-treatment of biomass is a crucial step in making carbohydrates more accessible for subsequent fermentation. Using bioinformatics analysis, endo- β -(1,4)-glucanase from Dictyoglomus thermophilum (DtCel5H) was identified as a new member of glycosyl hydrolase family 5. The gene encoding DtCel5H was cloned and the recombinant protein was overexpressed for crystallization and biophysical stud...
Source: Acta Crystallographica Section F - December 14, 2017 Category: Biochemistry Authors: Squeglia, F. Berisio, R. Ruggiero, A. Tags: crystal structure endoglucanase Dictyoglomus thermophilum thermostability biomass renewable energy research communications Source Type: research
Crystal structure of SAM-dependent methyltransferase from Pyrococcus horikoshii
Methyltransferases (MTs) are enzymes involved in methylation that are needed to perform cellular processes such as biosynthesis, metabolism, gene expression, protein trafficking and signal transduction. The cofactor S-adenosyl-l-methionine (SAM) is used for catalysis by SAM-dependent methyltransferases (SAM-MTs). The crystal structure of Pyrococcus horikoshii SAM-MT was determined to a resolution of 2.1 Å using X-ray diffraction. The monomeric structure consists of a Rossmann-like fold (domain I) and a substrate-binding domain (domain II). The cofactor (SAM) molecule binds at the interface between adjacent subuni...
Source: Acta Crystallographica Section F - November 24, 2017 Category: Biochemistry Authors: Pampa, K.J. Madan Kumar, S. Hema, M.K. Kumara, K. Naveen, S. Kunishima, N. Lokanath, N.K. Tags: methyltransferases Rossmann-like fold SAM-binding residues Pyrococcus horikoshii research communications Source Type: research
The Pseudomonas syringae pv. actinidiae chemoreceptor protein F (PscF) periplasmic sensor domain: cloning, purification and X-ray crystallographic analysis
Nitrate- and nitrite-sensing (NIT) domains are found associated with a wide variety of bacterial receptors, including chemoreceptors. However, the structure of a chemoreceptor-associated NIT domain has not yet been characterized. Recently, a chemoreceptor named PscF was identified from the plant pathogen Pseudomonas syringae pv. actinidiae that is predicted to contain a periplasmic NIT domain. The PscF sensor domain (PscF-SD; residues 42 – 332) was cloned into an appropriate expression vector, recombinantly produced in Escherichia coli BL21-Gold(DE3) cells and purified via immobilized metal-affinity and size-exclusio...
Source: Acta Crystallographica Section F - November 24, 2017 Category: Biochemistry Authors: Oulavallickal, T. Brewster, J.L. McKellar, J.L.O. Fairhurst, M.J. Tenci, N.A. Gerth, M.L. Tags: chemotaxis NIT domain chemoreceptor ligand-binding domain sensor domain Pseudomonas syringae research communications Source Type: research
DNA-binding domain of myelin-gene regulatory factor: purification, crystallization and X-ray analysis. Corrigendum
An extra affiliation is added for the authors of the article by Wu et al. [(2017), Acta Cryst. F73, 393 – 397]. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - November 21, 2017 Category: Biochemistry Authors: Wu, W. Zhen, X. Shi, N. Tags: myelin-gene regulation MRF DNA-binding domain trimeric transcription factor X-ray crystallography corrigendum addenda and errata Source Type: research
Crystal structure of the second fibronectin type III (FN3) domain from human collagen α 1 type XX
Collagen α 1 type XX, which contains fibronectin type III (FN3) repeats involving six FN3 domains (referred to as the FN#1 – FN#6 domains), is an unusual member of the fibril-associated collagens with interrupted triple helices (FACIT) subfamily of collagens. The results of standard protein BLAST suggest that the FN3 repeats might contribute to collagen α 1 type XX acting as a cytokine receptor. To date, solution NMR structures of the FN#3, FN#4 and FN#6 domains have been determined. To obtain further structural evidence to understand the relationship between the structure and function of the FN3 repeats ...
Source: Acta Crystallographica Section F - November 21, 2017 Category: Biochemistry Authors: Zhao, J. Ren, J. Wang, N. Cheng, Z. Yang, R. Lin, G. Guo, Y. Cai, D. Xie, Y. Zhao, X. Tags: crystal structure fibronectin type III domain FN3 domain collagen α 1 type XX research communications Source Type: research
Meditope – Fab interaction: threading the hole
Meditope, a cyclic 12-residue peptide, binds to a unique binding side between the light and heavy chains of the cetuximab Fab. In an effort to improve the affinity of the interaction, it was sought to extend the side chain of Arg8 in the meditope, a residue that is accessible from the other side of the meditope binding site, in order to increase the number of interactions. These modifications included an n-butyl and n-octyl extension as well as hydroxyl, amine and carboxyl substitutions. The atomic structures of the complexes and the binding kinetics for each modified meditope indicated that each extension threaded through...
Source: Acta Crystallographica Section F - November 18, 2017 Category: Biochemistry Authors: Bzymek, K.P. Ma, Y. Avery, K.N. Horne, D.A. Williams, J.C. Tags: meditope monoclonal antibodies X-ray crystallography surface plasmon resonance research communications Source Type: research
Crystal structure of an anti-idiotype variable lymphocyte receptor
Variable lymphocyte receptors (VLRs), the leucine-rich repeat (LRR)-based antigen receptors of jawless fish, have great utility in a wide variety of biochemical and biological applications, similar to classical Ig-based antibodies. VLR-based reagents may be particularly useful when traditional antibodies are not available. An anti-idiotype lamprey VLR, VLR39, has previously been identified that recognizes the heavy-chain CDR3 of the B-cell receptor (BCR) of a leukemic clone from a patient with chronic lymphocytic leukemia (CLL). VLR39 was used successfully to track the re-emergence of this clone in the patient following ch...
Source: Acta Crystallographica Section F - November 14, 2017 Category: Biochemistry Authors: Collins, B.C. Nakahara, H. Acharya, S. Cooper, M.D. Herrin, B.R. Wilson, I.A. Tags: structural biology crystallography immunology antibodies B-cell receptors anti-idiotype leucine-rich repeats variable lymphocyte receptors research communications Source Type: research
Swit_4259, an acetoacetate decarboxylase-like enzyme from Sphingomonas wittichii RW1
The Gram-negative bacterium Sphingomonas wittichii RW1 is notable for its ability to metabolize a variety of aromatic hydrocarbons. Not surprisingly, the S. wittichii genome contains a number of putative aromatic hydrocarbon-degrading gene clusters. One of these includes an enzyme of unknown function, Swit_4259, which belongs to the acetoacetate decarboxylase-like superfamily (ADCSF). Here, it is reported that Swit_4259 is a small (28.8 kDa) tetrameric ADCSF enzyme that, unlike the prototypical members of the superfamily, does not have acetoacetate decarboxylase activity. Structural characterization shows that the tert...
Source: Acta Crystallographica Section F - November 14, 2017 Category: Biochemistry Authors: Mydy, L.S. Mashhadi, Z. Knight, T.W. Fenske, T. Hagemann, T. Hoppe, R.W. Han, L. Miller, T.R. Schwabacher, A.W. Silvaggi, N.R. Tags: acetoacetate decarboxylase-like enzyme aldolase biodegradation dehydratase enzyme structure enzyme mechanism enzyme catalysis X-ray crystallography research communications Source Type: research
Structure of the Bacillus anthracis dTDP-l-rhamnose-biosynthetic enzyme dTDP-4-dehydrorhamnose 3,5-epimerase (RfbC)
The exosporium layer of Bacillus anthracis spores is rich in l-rhamnose, a common bacterial cell-wall component, which often contributes to the virulence of pathogens by increasing their adherence and immune evasion. The biosynthetic pathway used to form the activated l-rhamnose donor dTDP-l-rhamnose consists of four enzymes (RfbA, RfbB, RfbC and RfbD) and is an attractive drug target because there are no homologs in mammals. It was found that co-purifying and screening RfbC (dTDP-6-deoxy-d-xylo-4-hexulose 3,5-epimerase) from B. anthracis in the presence of the other three B. anthracis enzymes of the biosynthetic pathway y...
Source: Acta Crystallographica Section F - November 10, 2017 Category: Biochemistry Authors: Shornikov, A. Tran, H. Macias, J. Halavaty, A.S. Minasov, G. Anderson, W.F. Kuhn, M.L. Tags: RfbC dTDP-4-dehydrorhamnose 3,5-epimerase Bacillus anthracis Anthrax research communications Source Type: research
A low-cost method for visible fluorescence imaging
A wide variety of crystallization solutions are screened to establish conditions that promote the growth of a diffraction-quality crystal. Screening these conditions requires the assessment of many crystallization plates for the presence of crystals. Automated systems for screening and imaging are very expensive. A simple approach to imaging trace fluorescently labeled protein crystals in crystallization plates has been devised, and can be implemented at a cost as low as $50. The proteins β -lactoglobulin B, trypsin and purified concanavalin A (ConA) were trace fluorescently labeled using three different fluorescent p...
Source: Acta Crystallographica Section F - November 10, 2017 Category: Biochemistry Authors: Tarver, C.L. Pusey, M. Tags: fluorescence trace fluorescent labeling low-cost imaging protein crystallization research communications Source Type: research
Crystal structure of a pyridoxal 5 ′ -phosphate-dependent aspartate racemase derived from the bivalve mollusc Scapharca broughtonii
Aspartate racemase (AspR) is a pyridoxal 5 ′ -phosphate (PLP)-dependent enzyme that is responsible for d-aspartate biosynthesis in vivo. To the best of our knowledge, this is the first study to report an X-ray crystal structure of a PLP-dependent AspR, which was resolved at 1.90 Å resolution. The AspR derived from the bivalve mollusc Scapharca broughtonii (SbAspR) is a type II PLP-dependent enzyme that is similar to serine racemase (SR) in that SbAspR catalyzes both racemization and dehydration. Structural comparison of SbAspR and SR shows a similar arrangement of the active-site residues and nucleotide-bin...
Source: Acta Crystallographica Section F - November 6, 2017 Category: Biochemistry Authors: Mizobuchi, T. Nonaka, R. Yoshimura, M. Abe, K. Takahashi, S. Kera, Y. Goto, M. Tags: pyridoxal 5 ′ -phosphate aspartate racemase crystal structure Scapharca broughtonii serine racemase research communications Source Type: research
Structure of the Bacillus anthracis dTDP-l-rhamnose-biosynthetic enzyme dTDP-4-dehydrorhamnose reductase (RfbD)
Bacillus anthracis is the causative agent of the deadly disease Anthrax. Its use in bioterrorism and its ability to re-emerge have brought renewed interest in this organism. B. anthracis is a Gram-positive bacterium that adds l-rhamnose to its cell-wall polysaccharides using the activated donor dTDP- β -l-rhamnose. The enzymes involved in the biosynthesis of the activated donor are absent in humans, which make them ideal targets for therapeutic development to combat pathogens. Here, the 2.65 Å resolution crystal structure of the fourth enzyme in the dTDP- β -l-rhamnose-biosynthetic pathway from B. anthr...
Source: Acta Crystallographica Section F - November 6, 2017 Category: Biochemistry Authors: Law, A. Stergioulis, A. Halavaty, A.S. Minasov, G. Anderson, W.F. Kuhn, M.L. Tags: RfbD dTDP-4-dehydrorhamnose reductase Bacillus anthracis Anthrax research communications Source Type: research
Biochemical and structural studies of mutants indicate concerted movement of the dimer interface and ligand-binding region of Mycobacterium tuberculosis pantothenate kinase
Two point mutants and the corresponding double mutant of Mycobacterium tuberculosis pantothenate kinase have been prepared and biochemically and structurally characterized. The mutants were designed to weaken the affinity of the enzyme for the feedback inhibitor CoA. The mutants exhibit reduced activity, which can be explained in terms of their structures. The crystals of the mutants are not isomorphous to any of the previously analysed crystals of the wild-type enzyme or its complexes. The mycobacterial enzyme and its homologous Escherichia coli enzyme exhibit structural differences in their nucleotide complexes in the di...
Source: Acta Crystallographica Section F - October 30, 2017 Category: Biochemistry Authors: Paul, A. Kumar, P. Surolia, A. Vijayan, M. Tags: homodimers CoA biosynthesis nucleotide binding concerted movement structural transformation pantothenate kinase Mycobacterium tuberculosis research communications Source Type: research
Pteridine glycosyltransferase from Chlorobium tepidum: crystallization and X-ray analysis
The pteridine glycosyltransferase (PGT) found in Chlorobium tepidum (CtPGT) catalyzes the conversion of l-threo-tetrahydrobiopterin to 1-O-(l-threo-biopterin-2 ′ -yl)- β -N-acetylglucosamine using UDP-N-acetylglucosamine. The gene for CtPGT was cloned, and selenomethionine-derivatized protein was overexpressed and purified using various chromatographic techniques. The protein was crystallized by the hanging-drop vapour-diffusion method using 0.24 M triammonium citrate pH 7.0, 14%(w/v) PEG 3350 as a reservoir solution. Multiple-wavelength anomalous diffraction data were collected to 2.15 Å resolutio...
Source: Acta Crystallographica Section F - October 30, 2017 Category: Biochemistry Authors: Killivalavan, A. Park, Y.S. Lee, K.H. Tags: pteridine glycosyltransferase Chlorobium tepidum tetrahydrobiopterin UDP-N-acetylglucosamine l-threo-tetrahydrobiopterin research communications Source Type: research
Structure of the Bacillus anthracis dTDP-l-rhamnose-biosynthetic enzyme glucose-1-phosphate thymidylyltransferase (RfbA)
In this study, the three-dimensional structure of the first protein of this pathway, glucose-1-phosphate thymidylyltransferase (RfbA), from Bacillus anthracis was determined. In other organisms this enzyme is referred to as RmlA. RfbA was co-crystallized with the products of the enzymatic reaction, dTDP- α -d-glucose and pyrophosphate, and its structure was determined at 2.3 Å resolution. This is the first reported thymidylyltransferase structure from a Gram-positive bacterium. RfbA shares overall structural characteristics with known RmlA homologs. However, RfbA exhibits a shorter sequence at its C-terminu...
Source: Acta Crystallographica Section F - October 30, 2017 Category: Biochemistry Authors: Baumgartner, J. Lee, J. Halavaty, A.S. Minasov, G. Anderson, W.F. Kuhn, M.L. Tags: RfbA glucose-1-phosphate thymidylyltransferase Bacillus anthracis Anthrax research communications Source Type: research
Structural studies of domain movement in active-site mutants of porphobilinogen deaminase from Bacillus megaterium
The enzyme porphobilinogen deaminase (PBGD) is one of the key enzymes in tetrapyrrole biosynthesis. It catalyses the formation of a linear tetrapyrrole from four molecules of the substrate porphobilinogen (PBG). It has a dipyrromethane cofactor (DPM) in the active site which is covalently linked to a conserved cysteine residue through a thioether bridge. The substrate molecules are linked to the cofactor in a stepwise head-to-tail manner during the reaction, which is catalysed by a conserved aspartate residue: Asp82 in the B. megaterium enzyme. Three mutations have been made affecting Asp82 (D82A, D82E and D82N) and their ...
Source: Acta Crystallographica Section F - October 30, 2017 Category: Biochemistry Authors: Guo, J. Erskine, P. Coker, A.R. Wood, S.P. Cooper, J.B. Tags: protein crystallography structural biology porphobilinogen deaminase Bacillus megaterium tetrapyrrole biosynthesis research communications Source Type: research
Periplasmic form of dipeptidyl aminopeptidase IV from Pseudoxanthomonas mexicana WO24: purification, kinetic characterization, crystallization and X-ray crystallographic analysis
Dipeptidyl aminopeptidase IV (DAP IV or DPP IV) from Pseudoxanthomonas mexicana WO24 (PmDAP IV) preferentially cleaves substrate peptides with Pro or Ala at the P1 position [NH2-P2-P1(Pro/Ala)-P1 ′ -P2 ′ … ]. For crystallographic studies, the periplasmic form of PmDAP IV was overproduced in Escherichia coli, purified and crystallized in complex with the tripeptide Lys-Pro-Tyr using the hanging-drop vapour-diffusion method. Kinetic parameters of the purified enzyme against a synthetic substrate were also determined. X-ray diffraction data to 1.90 Å resolution were collected from a triclinic crys...
Source: Acta Crystallographica Section F - October 23, 2017 Category: Biochemistry Authors: Roppongi, S. Tateoka, C. Fujimoto, M. Iizuka, I. Morisawa, S. Nakamura, A. Honma, N. Suzuki, Y. Shida, Y. Ogasawara, W. Tanaka, N. Sakamoto, Y. Nonaka, T. Tags: DAP dipeptidyl aminopeptidase DPP4 DPP IV Pseudoxanthomonas mexicana research communications Source Type: research
Crystallization and X-ray diffraction analysis of native and selenomethionine-substituted PhyH-DI from Bacillus sp. HJB17
Phytases are phosphatases that hydrolyze phytates to less phosphorylated myo-inositol derivatives and inorganic phosphate. β -Propeller phytases, which are very diverse phytases with improved thermostability that are active at neutral and alkaline pH and have absolute substrate specificity, are ideal substitutes for other commercial phytases. PhyH-DI, a β -propeller phytase from Bacillus sp. HJB17, was found to act synergistically with other single-domain phytases and can increase their efficiency in the hydrolysis of phytate. Crystals of native and selenomethionine-substituted PhyH-DI were obtained using the vap...
Source: Acta Crystallographica Section F - October 23, 2017 Category: Biochemistry Authors: Lu, F. Zhang, B. Liu, Y. Song, Y. Guo, G. Feng, D. Huang, H. Yang, P. Gao, W. Guo, S. Yao, B. Tags: β -propeller phytases crystallization X-ray diffraction selenomethionine-substituted protein research communications Source Type: research
Structure of the staphylococcal enterotoxin B vaccine candidate S19 showing eliminated superantigen activity
Four mutations (N23A, Y90A, R110A and F177A) were introduced into S19, a vaccine candidate for staphylococcal enterotoxin B (SEB), resulting in a lower binding affinity towards the T-cell receptor beta chain (TCB) and reducing its superantigen activity. The structure of S19 was solved and was superposed on the native or complex structure of SEB. In the superposition model, mutations that were introduced seemed to reduce the number of hydrogen bonds at the SEB – TCB interface. S19 also displayed an unexpected structural change around the flexible-loop region owing to the Y90A mutation. This local structural change pro...
Source: Acta Crystallographica Section F - October 20, 2017 Category: Biochemistry Authors: Jeong, W.H. Song, D.H. Hur, G.H. Jeong, S.T. Tags: SEB staphylococcal enterotoxin B T-cell receptor beta chain recombinant protein vaccines bacterial superantigens research communications Source Type: research
The N253F mutant structure of trehalose synthase from Deinococcus radiodurans reveals an open active-site topology
In this study, the apo structure of the DrTS N253F mutant displays a new open conformation with an empty active site. Analysis of these structures suggests that substrate binding induces a domain rotation to close the active site. Such a substrate-induced domain rotation has also been observed in some other GH13 enzymes. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - October 20, 2017 Category: Biochemistry Authors: Chow, S.-Y. Wang, Y.-L. Hsieh, Y.-C. Lee, G.-C. Liaw, S.-H. Tags: trehalose synthase glycoside hydrolase family 13 enzyme mechanism conformational change domain rotation X-ray crystallography research communications Source Type: research