The phospholipase A effector PlaA from Legionella pneumophila: expression, purification and crystallization
Legionella pneumophila encodes an extracellular secreted phospholipase A named PlaA that is translocated by the type II secretion system. It plays an essential role in maintaining the integrity of Legionella-containing vacuoles in L. pneumophila pathogenesis. Here, it is shown that PlaA has a main lysophospholipase activity to hydrolyze fatty-acyl groups in lysophospholipids. Although it has a very low phospholipase A activity to catalyze the hydrolysis of fatty-acyl groups in phospholipids, PlaA can bind phospholipids such as 1,2-dipalmitoylphosphatidylcholine with a dissociation constant of 11.1 µ M. Sequence-alignm...
Source: Acta Crystallographica Section F - March 1, 2020 Category: Biochemistry Authors: Qu, X. Song, X. Zhang, N. Ma, J. Ge, H. Tags: phospholipase A Legionella pneumophila effector proteins research communications Source Type: research
Structure determination of the human TRPV1 ankyrin-repeat domain under nonreducing conditions
TRPV1, a member of the transient receptor potential (TRP) channels family, has been found to be involved in redox sensing. The crystal structure of the human TRPV1 ankyrin-repeat domain (TRPV1-ARD) was determined at 4.5 Å resolution under nonreducing conditions. This is the first report of the crystal structure of a ligand-free form of TRPV1-ARD and in particular of the human homologue. The structure showed a unique conformation in finger loop 3 near Cys258, which is most likely to be involved in inter-subunit disulfide-bond formation. Also, in human TRPV1-ARD it was possible for solvent to access Cys258. This structu...
Source: Acta Crystallographica Section F - March 1, 2020 Category: Biochemistry Authors: Tanaka, M. Hayakawa, K. Ogawa, N. Kurokawa, T. Kitanishi, K. Ite, K. Matsui, T. Mori, Y. Unno, M. Tags: TRPV1 akyrin-repeat domain human nonreducing conditions research communications Source Type: research
Engineering the Fab fragment of the anti-IgE omalizumab to prevent Fab crystallization and permit IgE-Fc complex crystallization
Immunoglobulin E (IgE) plays a central role in the allergic response, in which cross-linking of allergen by Fc ∊ RI-bound IgE triggers mast cell and basophil degranulation and the release of inflammatory mediators. The high-affinity interaction between IgE and Fc ∊ RI is a long-standing target for therapeutic intervention in allergic disease. Omalizumab is a clinically approved anti-IgE monoclonal antibody that binds to free IgE, also with high affinity, preventing its interaction with Fc ∊ RI. All attempts to crystallize the pre-formed complex between the omalizumab Fab and the Fc region of IgE (IgE-Fc), to understa...
Source: Acta Crystallographica Section F - March 1, 2020 Category: Biochemistry Authors: Mitropoulou, A.N. Ceska, T. Heads, J.T. Beavil, A.J. Henry, A.J. McDonnell, J.M. Sutton, B.J. Davies, A.M. Tags: omalizumab allergy Fab immunoglobulin E antibody protein engineering X-ray crystallography research communications Source Type: research
Structural and biochemical characterization of mitochondrial citrate synthase 4 from Arabidopsis thaliana
Citrate synthase (CS) catalyzes the conversion of oxaloacetate and acetyl coenzyme A into citrate and coenzyme A in the mitochondrial tricarboxylic acid (TCA) cycle. In plants, mitochondrial metabolism, including the TCA cycle, occurs in interaction with photosynthetic metabolism. The controlled regulation of several enzymes in the TCA cycle, such as CS, is important in plants. Here, the first crystal structure of a plant mitochondrial CS, CSY4 from Arabidopsis thaliana (AtCSY4), has been determined. Structural comparison of AtCSY4 with mitochondrial CSs revealed a high level of similarity. Inhibition analysis showed a sim...
Source: Acta Crystallographica Section F - February 29, 2020 Category: Biochemistry Authors: Nishio, K. Mizushima, T. Tags: citrate synthase mitochondria TCA cycle Arabidopsis thaliana X-ray crystallography research communications Source Type: research
The crystal structure of the catalytic domain of tau tubulin kinase 2 in complex with a small-molecule inhibitor
Tau proteins play an important role in the proper assembly and function of neurons. Hyperphosphorylation of tau by kinases such as tau tubulin kinase (TTBK) has been hypothesized to cause the aggregation of tau and the formation of neurofibrillary tangles (NFTs) that lead to the destabilization of microtubules, thereby contributing to neurodegenerative diseases such as Alzheimer's disease (AD). There are two TTBK isoforms with highly homologous catalytic sites but with distinct tissue distributions, tau phosphorylation patterns and loss-of-function effects. Inhibition of TTBK1 reduces the levels of NFT formation involved i...
Source: Acta Crystallographica Section F - February 29, 2020 Category: Biochemistry Authors: Marcotte, D.J. Spilker, K.A. Wen, D. Hesson, T. Patterson, T.A. Kumar, P.R. Chodaparambil, J.V. Tags: TTBK2 kinase domain tau proteins Alzheimer's disease research communications Source Type: research
High-resolution crystal structures of Escherichia coli FtsZ bound to GDP and GTP
Bacterial cytokinesis is mediated by the Z-ring, which is formed by the prokaryotic tubulin homolog FtsZ. Recent data indicate that the Z-ring is composed of small patches of FtsZ protofilaments that travel around the bacterial cell by treadmilling. Treadmilling involves a switch from a relaxed (R) state, favored for monomers, to a tense (T) conformation, which is favored upon association into filaments. The R conformation has been observed in numerous monomeric FtsZ crystal structures and the T conformation in Staphylococcus aureus FtsZ crystallized as assembled filaments. However, while Escherichia coli has served as a m...
Source: Acta Crystallographica Section F - February 4, 2020 Category: Biochemistry Authors: Schumacher, M.A. Ohashi, T. Corbin, L. Erickson, H.P. Tags: FtsZ cytokinesis treadmilling protofilaments Z-ring research communications Source Type: research
Crystal structures of the cell-division protein FtsZ from Klebsiella pneumoniae and Escherichia coli
In this study, crystal structures of the enzymatic domains of FtsZ from Klebsiella pneumoniae (KpFtsZ) and Escherichia coli (EcFtsZ) were determined at 1.75 and 2.50 Å resolution, respectively. Both FtsZs form straight protofilaments in the crystals, and the two structures adopted relaxed (R) conformations. The T3 loop, which is involved in GTP/GDP binding and FtsZ assembly/disassembly, adopted a unique open conformation in KpFtsZ, while the T3 loop of EcFtsZ was partially disordered. The crystal structure of EcFtsZ can explain the results from previous functional analyses using EcFtsZ mutants. (Source: Acta Crysta...
Source: Acta Crystallographica Section F - February 4, 2020 Category: Biochemistry Authors: Yoshizawa, T. Fujita, J. Terakado, H. Ozawa, M. Kuroda, N. Tanaka, S. Uehara, R. Matsumura, H. Tags: cell division FtsZ Escherichia coli Klebsiella pneumoniae research communications Source Type: research
An unexpected co-crystal structure of the calpain PEF(S) domain with Hfq reveals a potential chaperone function of Hfq
Calpain is a Ca2+-activated, heterodimeric cysteine protease consisting of a large catalytic subunit and a small regulatory subunit. Dysregulation of this enzyme is involved in a range of pathological conditions such as cancer, Alzheimer's disease and rheumatoid arthritis, and thus calpain I is a drug target with potential therapeutic applications. Difficulty in the production of this enzyme has hindered structural and functional investigations in the past, although heterodimeric calpain I can be generated by Escherichia coli expression in low yield. Here, an unexpected structure discovered during crystallization trials of...
Source: Acta Crystallographica Section F - February 4, 2020 Category: Biochemistry Authors: Cresser-Brown, J. Rizkallah, P. Jin, Y. Roth, C. Miller, D.J. Allemann, R.K. Tags: calpain expression Hfq chaperones cysteine protease research communications Source Type: research
The thermodynamic profile and molecular interactions of a C(9)-cytisine derivative-binding acetylcholine-binding protein from Aplysia californica
Cytisine, a natural product with high affinity for clinically relevant nicotinic acetylcholine receptors (nAChRs), is used as a smoking-cessation agent. The compound displays an excellent clinical profile and hence there is an interest in derivatives that may be further improved or find use in the treatment of other conditions. Here, the binding of a cytisine derivative modified by the addition of a 3-(hydroxypropyl) moiety (ligand 4) to Aplysia californica acetylcholine-binding protein (AcAChBP), a surrogate for nAChR orthosteric binding sites, was investigated. Isothermal titration calorimetry revealed that the favorable...
Source: Acta Crystallographica Section F - February 2, 2020 Category: Biochemistry Authors: Davis, S. Rego Campello, H. Gallagher, T. Hunter, W.N. Tags: acetylcholine-binding protein crystal structure cytisine ligand-gated ion channel nicotine research communications Source Type: research
Conformational flexibility within the small domain of human serine racemase
Serine racemase (SR) is a pyridoxal 5 ′ -phosphate (PLP)-containing enzyme that converts l-serine to d-serine, an endogenous co-agonist for the N-methyl-d-aspartate receptor (NMDAR) subtype of glutamate ion channels. SR regulates d-serine levels by the reversible racemization of l-serine to d-serine, as well as the catabolism of serine by α , β -elimination to produce pyruvate. The modulation of SR activity is therefore an attractive therapeutic approach to disorders associated with abnormal glutamatergic signalling since it allows an indirect modulation of NMDAR function. In the present study, a 1.89 Å resolution...
Source: Acta Crystallographica Section F - February 2, 2020 Category: Biochemistry Authors: Koulouris, C.R. Bax, B.D. Atack, J.R. Roe, S.M. Tags: serine racemase d-serine NMDA receptors drug design domain structure subdomain ligand-induced reorientation research communications Source Type: research
Structure of the AAVhu.37 capsid by cryoelectron microscopy
Adeno-associated viruses (AAVs) are used as in vivo gene-delivery vectors in gene-therapy products and have been heavily investigated for numerous indications. Over 100 naturally occurring AAV serotypes and variants have been isolated from primate samples. Many reports have described unique properties of these variants (for instance, differences in potency, target cell or evasion of the immune response), despite high amino-acid sequence conservation. AAVhu.37 is of interest for clinical applications owing to its proficient transduction of the liver and central nervous system. The sequence identity of the AAVhu.37 VP1 to th...
Source: Acta Crystallographica Section F - February 2, 2020 Category: Biochemistry Authors: Kaelber, J.T. Yost, S.A. Webber, K.A. Firlar, E. Liu, Y. Danos, O. Mercer, A.C. Tags: adeno-associated viruses gene therapy gene-delivery vectors capsid cryo-EM structure AAVhu.37 research communications Source Type: research
The structure of MP-4 from Mucuna pruriens at 2.22 Å resolution
The structure of the MP-4 protein was previously determined at a resolution of 2.8 Å . Owing to the unavailability of gene-sequence information at the time, the side-chain assignment was carried out on the basis of a partial sequence available through Edman degradation, sequence homology to orthologs and electron density. The structure of MP-4 has now been determined at a higher resolution (2.22 Å ) in another space group and all of the structural inferences that were presented in the previous report of the structure were validated. In addition, the present data allowed an improved assignment of side chains and e...
Source: Acta Crystallographica Section F - January 31, 2020 Category: Biochemistry Authors: Jain, A. Kumar, A. Shikhi, M. Kumar, A. Nair, D.T. Salunke, D.M. Tags: Mucuna pruriens MP-4 high resolution plant proteins protease inhibitors research communications Source Type: research
Cocktailed fragment screening by X-ray crystallography of the antibacterial target undecaprenyl pyrophosphate synthase from Acinetobacter baumannii
Direct soaking of protein crystals with small-molecule fragments grouped into complementary clusters is a useful technique when assessing the potential of a new crystal system to support structure-guided drug discovery. It provides a robustness check prior to any extensive crystal screening, a double check for assay binding cutoffs and structural data for binding pockets that may or may not be picked out in assay measurements. The structural output from this technique for three novel fragment molecules identified to bind to the antibacterial target Acinetobacter baumannii undecaprenyl pyrophosphate synthase are reported, a...
Source: Acta Crystallographica Section F - December 31, 2019 Category: Biochemistry Authors: Thorpe, J.H. Wall, I.D. Sinnamon, R.H. Taylor, A.N. Stavenger, R.A. Tags: undecaprenyl pyrophosphate synthase fragment screening Acinetobacter baumannii research communications Source Type: research
The Escherichia coli RnlA – RnlB toxin – antitoxin complex: production, characterization and crystallization
The Escherichia coli rnlAB operon encodes a toxin – antitoxin module that is involved in protection against infection by bacteriophage T4. The full-length RnlA – RnlB toxin – antitoxin complex as well as the toxin RnlA were purified to homogeneity and crystallized. When the affinity tag is placed on RnlA, RnlB is largely lost during purification and the resulting crystals exclusively comprise RnlA. A homogeneous preparation of RnlA – RnlB containing stoichiometric amounts of both proteins could only be obtained using a His tag placed C-terminal to RnlB. Native mass spectrometry and SAXS indicate a 1:1 stoichiometry...
Source: Acta Crystallographica Section F - December 31, 2019 Category: Biochemistry Authors: Garcia Rodriguez, G. Talavera Perez, A. Konijnenberg, A. Sobott, F. Michiels, J. Loris, R. Tags: toxin – antitoxin bacterial stress response macromolecular complex Escherichia coli research communications Source Type: research
The RING domain of mitochondrial E3 ubiquitin ligase 1 and its complex with Ube2D2: crystallization and X-ray diffraction
Mitochondrial E3 ubiquitin ligase 1 (MUL1) is located in the mitochondrial outer membrane and regulates various biological processes, including apoptosis, cell growth, mitophagy and mitochondrial dynamics. The C-terminal region of MUL1 faces the cytoplasm and contains the RING domain (MUL1-RING) where the Ub~E2 thioester binds. Unlike most RING-type E3 enzymes, MUL1-RING alone does not have an additional region that recruits a substrate protein, yet is still able to ubiquitylate the substrate, the p53 protein. Nevertheless, the exact mechanism of the ubiquitylation of p53 by MUL1-RING has not yet been elucidated. In order ...
Source: Acta Crystallographica Section F - December 31, 2019 Category: Biochemistry Authors: Lee, S.-O. Lee, C.-K. Ryu, K.-S. Chi, S.-W. Tags: mitochondrial E3 ubiquitin ligase 1 MUL1 RING domain MUL1-RING Ube2D2 ubiquitylation crystallization research communications Source Type: research
