Structures of chloramphenicol acetyltransferase III and Escherichia coli β -ketoacylsynthase III co-crystallized with partially hydrolysed acetyl-oxa(dethia)CoA
Acetyl coenzyme A (acetyl-CoA) is a reactive metabolite that nonproductively hydrolyzes in a number of enzyme active sites in the crystallization time frame. In order to elucidate the enzyme – acetyl-CoA interactions leading to catalysis, acetyl-CoA substrate analogs are needed. One possible analog for use in structural studies is acetyl-oxa(dethia)CoA (AcOCoA), in which the thioester S atom of CoA is replaced by an O atom. Here, structures of chloramphenicol acetyltransferase III (CATIII) and Escherichia coli ketoacylsynthase III (FabH) from crystals grown in the presence of partially hydrolyzed AcOCoA and the respectiv...
Source: Acta Crystallographica Section F - February 23, 2023 Category: Biochemistry Authors: Benjamin, A.B. Stunkard, L.M. Ling, J. Nice, J.N. Lohman, J.R. Tags: ketoacyl synthases substrate analogs transferases chloramphenicol acetyltransferase III Escherichia coli ketoacylsynthase III acetyl-oxa(dethia)CoA research communications Source Type: research
Structural and biophysical characterization of the Borna disease virus 1 phosphoprotein
In this study, the structure of the oligomerization domain of the phosphoprotein determined by X-ray crystallography is reported. The structural results are complemented with biophysical characterization using circular dichroism, differential scanning calorimetry and small-angle X-ray scattering. The data reveal the phosphoprotein to assemble into a stable tetramer, with the regions outside the oligomerization domain remaining highly flexible. A helix-breaking motif is observed between the α -helices at the midpoint of the oligomerization domain that appears to be conserved across the Bornaviridae. These data provide info...
Source: Acta Crystallographica Section F - February 23, 2023 Category: Biochemistry Authors: Whitehead, J.D. Grimes, J.M. Keown, J.R. Tags: bornavirus phosphoproteins X-ray crystallography negative-strand RNA viruses replication research communications Source Type: research
Purification and structure of luminal domain C of human Niemann – Pick C1 protein
Niemann – Pick C1 protein (NPC1) is a membrane protein that primarily resides in late endosomes and lysosomes, and plays an important role in cholesterol homeostasis in the cell. The second luminal domain of NPC1 (NPC1-C) serves as the intracellular receptor for Ebola and Marburg viruses. Here, the recombinant production of nonglycosylated and glycosylated NPC1-C and a new crystal form of the nonglycosylated protein are reported. The crystals belonged to space group P21 and diffracted to 2.3 Å resolution. The structure is similar to other reported structures of NPC1-C, with differences observed in the protruding loo...
Source: Acta Crystallographica Section F - February 2, 2023 Category: Biochemistry Authors: Odongo, L. Zadrozny, K.K. Diehl, W.E. Luban, J. White, J.M. Ganser-Pornillos, B.K. Tamm, L.K. Pornillos, O. Tags: Niemann – Pick C1 protein Ebola virus cholesterol membrane proteins research communications Source Type: research
An unusual disulfide-linked dimerization in the fluorescent protein rsCherryRev1.4
rsCherryRev1.4 has been reported as one of the reversibly photoswitchable variants of mCherry, and is an improved version with a faster off-switching speed and lower switching fatigue at high light intensities than its precursor rsCherryRev. However, rsCherryRev1.4 still has some limitations such as a tendency to dimerize as well as complex photophysical properties. Here, the crystal structure of rsCherryRev1.4 was determined at a resolution of 2 Å and it was discovered that it forms a dimer that shows disulfide bonding between the protomers. Mutagenesis, gel electrophoresis and size-exclusion chromatography strongly ...
Source: Acta Crystallographica Section F - February 2, 2023 Category: Biochemistry Authors: Bui, T.Y.H. Dedecker, P. Van Meervelt, L. Tags: disulfide bonds oligomerization fluorescent proteins rsCherryRev1.4 crystal structure research communications Source Type: research
Structure of pyridoxal 5 ′ -phosphate-bound d-threonine aldolase from Chlamydomonas reinhardtii
This study produced and investigated the crystal structure of DTA from Chlamydomonas reinhardtii (CrDTA) at 1.85 Å resolution. To our knowledge, this is the first report on the crystal structure of eukaryotic DTA. Compared with the structure of bacterial DTA, CrDTA has a similar arrangement of active-site residues. On the other hand, we speculated that some non-conserved residues alter the affinity for substrates and inhibitors. The structure of CrDTA could provide insights into the structural framework for structure-guided protein engineering studies to modify reaction selectivity. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - February 1, 2023 Category: Biochemistry Authors: Hirato, Y. Goto, M. Mizobuchi, T. Muramatsu, H. Tanigawa, M. Nishimura, K. Tags: d-threonine aldolase Chlamydomonas reinhardtii pyridoxal 5 ′ -phosphate d-amino acids research communications Source Type: research
Room-temperature serial synchrotron crystallography of the human phosphatase PTP1B
Room-temperature X-ray crystallography provides unique insights into protein conformational heterogeneity, but obtaining sufficiently large protein crystals is a common hurdle. Serial synchrotron crystallography (SSX) helps to address this hurdle by allowing the use of many medium- to small-sized crystals. Here, a recently introduced serial sample-support chip system has been used to obtain the first SSX structure of a human phosphatase, specifically protein tyrosine phosphatase 1B (PTP1B) in the unliganded (apo) state. In previous apo room-temperature structures, the active site and allosteric sites adopted alternate conf...
Source: Acta Crystallographica Section F - December 20, 2022 Category: Biochemistry Authors: Sharma, S. Ebrahim, A. Keedy, D.A. Tags: X-ray crystallography phosphatases apo protein tyrosine phosphatase 1B allostery room-temperature serial crystallography research communications Source Type: research
Crystal structure of the C-terminal domain of the plant-specific microtubule-associated protein Spiral2
In this study, the crystal structure of the conserved C-terminal domain of Spiral2 was determined using the single-wavelength anomalous dispersion method. Refinement of the model to a resolution of 2.2 Å revealed a helix – turn – helix fold with seven α -helices. The protein crystallized as a dimer, but SEC-MALS analysis showed the protein to be monomeric. A structural homology search revealed that the protein has similarity to the C-terminal domain of the katanin regulatory subunit p80. The structure presented here suggests that the C-terminal domain of Spiral2 represents a new class of microtubule dynamics modu...
Source: Acta Crystallographica Section F - December 20, 2022 Category: Biochemistry Authors: Ohno, M. Higuchi, Y. Hayashi, I. Tags: microtubules Spiral2 plants Physcomitrella patens katanin research communications Source Type: research
The structure of a tautomerase superfamily member linked to the type VI secretion system of Acinetobacter baumannii
Bacteria exploit specialized secretion systems to assist in competition for resources, in collaboration and in communication. Here, a protocol for the recombinant production, purification and crystallization of a protein linked to the Acinetobacter baumannii type VI secretion system is provided. A high-resolution structure of this trimeric protein is reported, revealing the characteristic dual β – α – β subunit fold typical of longer subunit members of the tautomerase superfamily. The protein does not appear to be toxic to bacteria or yeast under the conditions tested. The possible biological role of this protein is...
Source: Acta Crystallographica Section F - December 8, 2022 Category: Biochemistry Authors: Pankov, G. Mol Avelar, G. Buchanan, G. Coulthurst, S.J. Hunter, W.N. Tags: Acinetobacter baumannii 4-oxalocrotonate tautomerase tautomerase superfamily type VI secretion system research communications Source Type: research
Joint X-ray/neutron structure of Lentinus similis AA9_A at room temperature
Lytic polysaccharide monooxygenases (LPMOs) are copper metalloenzymes which cleave polysaccharides oxidatively and are important in pathogen biology, carbon cycling and biotechnology. The Lentinus similis family AA9 isoform A (LsAA9_A) has been extensively studied as a model system because its activity towards smaller soluble saccharide substrates has allowed detailed structural characterization of its interaction with a variety of substrates by X-ray crystallography at high resolution. Here, the joint X-ray/neutron room-temperature crystallographic structure of carbohydrate-free LsAA9_A in the copper(II) resting state ref...
Source: Acta Crystallographica Section F - December 8, 2022 Category: Biochemistry Authors: Tandrup, T. Lo Leggio, L. Meilleur, F. Tags: lytic polysaccharide monooxygenases Lentinus similis AA9_A copper metalloenzymes protonation states neutron crystallography research communications Source Type: research
The structure of His-tagged Geobacillus stearothermophilus purine nucleoside phosphorylase reveals a `spanner in the works'
The 1.72 Å resolution structure of purine nucleoside phosphorylase from Geobacillus stearothermophilus, a thermostable protein of potential interest for the biocatalytic synthesis of antiviral nucleoside compounds, is reported. The structure of the N-terminally His-tagged enzyme is a hexamer, as is typical of bacterial homologues, with a trimer-of-dimers arrangement. Unexpectedly, several residues of the recombinant tobacco etch virus protease (rTEV) cleavage site from the N-terminal tag are located in the active site of the neighbouring subunit in the dimer. Key to this interaction is a tyrosine residue, which sits w...
Source: Acta Crystallographica Section F - November 28, 2022 Category: Biochemistry Authors: Given, F.M. Moran, F. Johns, A.S. Titterington, J.A. Allison, T.M. Crittenden, D.L. Johnston, J.M. Tags: purine nucleoside phosphorylase Geobacillus stearothermophilus His tags affinity tags research communications Source Type: research
Crystal structure of Sphingobacterium multivorum serine palmitoyltransferase complexed with tris(hydroxymethyl)aminomethane
Serine palmitoyltransferase (SPT) catalyses the first reaction in sphingolipid biosynthesis: the decarboxylative condensation of l-serine (l-Ser) and palmitoyl-CoA to form 3-ketodihydrosphingosine. SPT from Sphingobacterium multivorum has been isolated and its crystal structure in complex with l-Ser has been determined at 2.3 Å resolution (PDB entry 3a2b). However, the quality of the crystal was not good enough to judge the conformation of the cofactor molecule and the orientations of the side chains of the amino-acid residues in the enzyme active site. The crystal quality was improved by revision of the purification ...
Source: Acta Crystallographica Section F - November 28, 2022 Category: Biochemistry Authors: Ikushiro, H. Takahashi, A. Murakami, T. Katayama, A. Sawai, T. Goto, H. Miyahara, I. Kamiya, N. Yano, T. Tags: serine palmitoyltransferase PLP-dependent enzymes sphingolipids X-ray crystallography research communications Source Type: research
Relating protein crystal structure to ligand-binding thermodynamics
This article focuses on saccharide binding to lectins as a theme, yet after 25 years or so it is still a work in progress to connect 3D structure to binding energies. Whilst this study involved one type of protein (lectins) and one class of ligand (monosaccharides), i.e. it was specific, it was of general importance, as measured for instance by its wide impact. The impetus for writing this update now, as a Scientific Comment, is that a breakthrough in neutron crystal structure determinations of saccharide-bound lectins has been achieved. It is suggested here that this new research from neutron protein crystallography could...
Source: Acta Crystallographica Section F - November 28, 2022 Category: Biochemistry Authors: Helliwell, J.R. Tags: protein ligand binding thermodynamics and structure lectins saccharides scientific comment Source Type: research
Ibuprofen: a weak inhibitor of carbonic anhydrase II
This study discusses the potential development of CA inhibitors utilizing the carboxylic acid moiety. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - October 14, 2022 Category: Biochemistry Authors: Combs, J. Andring, J. McKenna, R. Tags: carbonic anhydrases carboxylic acid-based inhibitors ibuprofen X-ray crystallography kinetics research communications Source Type: research
Crystals of SctV from different species reveal variable symmetry for the cytosolic domain of the type III secretion system export gate
Type III secretion systems (T3SSs) are proteinaceous devices employed by Gram-negative bacteria to directly transport proteins into a host cell. Substrate recognition and secretion are strictly regulated by the export apparatus of the so-called injectisome. The export gate SctV engages chaperone-bound substrates of the T3SS in its nonameric cytoplasmic domain. Here, the purification and crystallization of the cytoplasmic domains of SctV from Photorhabdus luminescens (LscVC) and Aeromonas hydrophila (AscVC) are reported. Self-rotation functions revealed that LscVC forms oligomers with either eightfold or ninefold symmetry i...
Source: Acta Crystallographica Section F - October 14, 2022 Category: Biochemistry Authors: Gilzer, D. Baum, E. Lieske, N. Kowal, J.L. Niemann, H.H. Tags: type III secretion systems export-gate protein SctV Photorhabdus luminescens Aeromonas hydrophila cyclic oligomers low-resolution crystallography molecular replacement oligomerization self-rotation function research communications Source Type: research
Crystal structure of the capsular polysaccharide-synthesis enzyme CapG from Staphylococcus aureus
Bacterial capsular polysaccharides provide protection against environmental stress and immune evasion from the host immune system, and are therefore considered to be attractive therapeutic targets for the development of anti-infectious reagents. Here, we focused on CapG, one of the key enzymes in the synthesis pathway of capsular polysaccharides type 5 (CP5) from the opportunistic pathogen Staphylococcus aureus. SaCapG catalyses the 2-epimerization of UDP-N-acetyl-d-talosamine (UDP-TalNAc) to UDP-N-acetyl-d-fucosamine (UDP-FucNAc), which is one of the nucleotide-activated precursors for the synthesis of the trisaccharide r...
Source: Acta Crystallographica Section F - October 14, 2022 Category: Biochemistry Authors: Tien, N. Ho, C.-Y. Lai, S.-J. Lin, Y.-C. Yang, C.-S. Wang, Y.-C. Huang, W.-C. Chen, Y. Chang, J.-J. Tags: crystal structure Staphylococcus aureus 2-epimerases capsular polysaccharides CapG research communications Source Type: research
