The A component (SmhA) of a tripartite pore-forming toxin from Serratia marcescens: expression, purification and crystallographic analysis
Tripartite α -pore-forming toxins are constructed of three proteins (A, B and C) and are found in many bacterial pathogens. While structures of the B and C components from Gram-negative bacteria have been described, the structure of the A component of a Gram-negative α -pore-forming toxin has so far proved elusive. SmhA, the A component from the opportunistic human pathogen Serratia marcescens, has been cloned, overexpressed and purified. Crystals were grown of selenomethionine-derivatized protein and anomalous data were collected. Phases were calculated and an initial electron-density map was produced. (Source: Acta Cry...
Source: Acta Crystallographica Section F - November 25, 2020 Category: Biochemistry Authors: Churchill-Angus, A.M. Sedelnikova, S.E. Schofield, T.H.B. Baker, P.J. Tags: pore-forming toxin crystallization ClyA family Serratia research communications Source Type: research
Structural characterization of a nonhydrolyzing UDP-GlcNAc 2-epimerase from Neisseria meningitidis serogroup A
Bacterial nonhydrolyzing UDP-N-acetylglucosamine 2-epimerases catalyze the reversible interconversion of UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylmannosamine (UDP-ManNAc). UDP-ManNAc is an important intermediate in the biosynthesis of certain cell-surface polysaccharides, including those in some pathogenic bacteria, such as Neisseria meningitidis and Streptococcus pneumoniae. Many of these epimerases are allosterically regulated by UDP-GlcNAc, which binds adjacent to the active site and is required to initiate UDP-ManNAc epimerization. Here, two crystal structures of UDP-N-acetylglucosamine 2-epimerase from Nei...
Source: Acta Crystallographica Section F - October 29, 2020 Category: Biochemistry Authors: Hurlburt, N.K. Guan, J. Ong, H. Yu, H. Chen, X. Fisher, A.J. Tags: UDP-GlcNAc UDP-ManNAc epimerases epimerization Rossmann fold X-ray crystallography Neisseria meningitidis research communications Source Type: research
Crystal structure of the GDP-bound GTPase domain of Rab5a from Leishmania donovani
Eukaryotic Rab5s are highly conserved small GTPase-family proteins that are involved in the regulation of early endocytosis. Leishmania donovani Rab5a regulates the sorting of early endosomes that are involved in the uptake of essential nutrients through fluid-phase endocytosis. Here, the 1.80 Å resolution crystal structure of the N-terminal GTPase domain of L. donovani Rab5a in complex with GDP is presented. The crystal structure determination was enabled by the design of specific single-site mutations and two deletions that were made to stabilize the protein for previous NMR studies. The structure of LdRab5a shows t...
Source: Acta Crystallographica Section F - October 29, 2020 Category: Biochemistry Authors: Zohib, M. Maheshwari, D. Pal, R.K. Freitag-Pohl, S. Biswal, B.K. Pohl, E. Arora, A. Tags: Leishmania donovani fluid-phase endocytosis early endosomes Rab5a crystal structure research communications Source Type: research
Chitoporin from Serratia marcescens: recombinant expression, purification and crystallization
This study reports the identification of a chitoporin (ChiP), termed SmChiP, from the outer membrane of S. marcescens. Sequence alignment with genetically characterized ChiPs suggests that SmChiP is more closely related to the monomeric EcChiP from Escherichia coli than to the trimeric VhChiP from Vibrio campbellii. A single crystal of SmChiP grown under the condition 22%(w/v) PEG 8000, 0.1 M calcium acetate, 0.1 M MES pH 6.0 diffracted X-ray synchrotron radiation to 1.85 Å resolution. SmChiP co-crystallized with chitohexaose under the condition 19%(w/v) PEG 1500, 2 M ammonium phosphate monobasic, 0.1 ...
Source: Acta Crystallographica Section F - October 29, 2020 Category: Biochemistry Authors: Amornloetwattana, R. Robinson, R.C. Soysa, H.S.M. van den Berg, B. Suginta, W. Tags: chitin chitoporin outer membrane proteins Serratia marcescens sugar transport chitoligosaccharides sugar-specific porins research communications Source Type: research
Structural and biochemical characterization of the class II fructose-1,6-bisphosphatase from Francisella tularensis
The crystal structure of the class II fructose-1,6-bisphosphatase (FBPaseII) from the important pathogen Francisella tularensis is presented at 2.4 Å resolution. Its structural and functional relationships to the closely related phosphatases from Mycobacterium tuberculosis (MtFBPaseII) and Escherichia coli (EcFBPaseII) and to the dual phosphatase from Synechocystis strain 6803 are discussed. FBPaseII from F. tularensis (FtFBPaseII) was crystallized in a monoclinic crystal form (space group P21, unit-cell parameters a = 76.30, b = 100.17, c = 92.02 Å , β = 90.003 ° ) with four chains in the asymmetri...
Source: Acta Crystallographica Section F - October 23, 2020 Category: Biochemistry Authors: Selezneva, A.I. Gutka, H.J. Wolf, N.M. Qurratulain, F. Movahedzadeh, F. Abad-Zapatero, C. Tags: class II FBPases fructose-1,6-bisphophatase Mycobacterium tuberculosis Francisella tularensis antibiotic targets research communications Source Type: research
X-ray structure analysis of a unique d-amino-acid oxidase from the thermophilic fungus Rasamsonia emersonii strain YA
In this study, ReDAAO was crystallized by the hanging-drop vapor-diffusion method and its crystal structure was determined at a resolution of 2.00 Å . The crystal structure of the enzyme revealed that unlike other DAAOs, ReDAAO forms a homotetramer and contains an intramolecular disulfide bond (Cys230 – Cys285), suggesting that this disulfide bond is involved in the higher thermal stability of ReDAAO. Moreover, the structure of the active site and its vicinity in ReDAAO indicates that Arg97, Lys99, Lys114 and Ser231 are candidates for recognizing the side chain of d-Glu. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - October 23, 2020 Category: Biochemistry Authors: Shimekake, Y. Hirato, Y. Funabashi, R. Okazaki, S. Goto, M. Furuichi, T. Suzuki, H. Kera, Y. Takahashi, S. Tags: d-amino-acid oxidases flavoenzymes d-amino acids substrate specificity thermal stability thermophilic fungi Rasamsonia emersonii research communications Source Type: research
Alternative conformation of the C-domain of the P140 protein from Mycoplasma genitalium
The human pathogen Mycoplasma genitalium is responsible for urethritis in men, and for cervicitis and pelvic inflammatory disease in women. The adherence of M. genitalium to host target epithelial cells is mediated through an adhesion complex called Nap, which is essential for infectivity. Nap is a transmembrane dimer of heterodimers of the immunodominant proteins P110 and P140. The M. genitalium genome contains multiple copies of portions that share homology with the extracellular regions of P140 and P110 encoded by the genes mg191 and mg192, respectively. Homologous recombination between the genes and the copies al...
Source: Acta Crystallographica Section F - October 9, 2020 Category: Biochemistry Authors: Vizarraga, D. P é rez-Luque, R. Mart í n, J. Fita, I. Aparicio, D. Tags: Mycoplasma genitalium adhesins Nap gliding motility infection research communications Source Type: research
Multi-crystal data collection using synchrotron radiation as exemplified with low-symmetry crystals of Dps
Multi-crystal data collection using synchrotron radiation was successfully applied to determine the three-dimensional structure of a triclinic crystal form of Dps from Escherichia coli at 2.0 Å resolution. The final data set was obtained by combining 261 partial diffraction data sets measured from crystals with an average size of approximately 5 µ m. The most important features of diffraction data measurement and processing for low-symmetry crystals are discussed. (Source: Acta Crystallographica Section F)
Source: Acta Crystallographica Section F - October 8, 2020 Category: Biochemistry Authors: Kovalenko, V. Popov, A. Santoni, G. Loiko, N. Tereshkina, K. Tereshkin, E. Krupyanskii, Y. Tags: protein crystallography muti-crystal data collection clustering analysis methods communications Source Type: research
Structure of the substrate-binding domain of Plasmodium falciparum heat-shock protein 70-x
The malaria parasite Plasmodium falciparum extensively modifies erythrocytes that it invades by exporting a large complement of proteins to the host cell. Among these exported components is a single heat-shock 70 kDa class protein, PfHsp70-x, that supports the virulence and growth rate of the parasite during febrile episodes. The ATP-binding domain of PfHsp70-x has previously been resolved and showed the presence of potentially druggable epitopes that differ from those on human Hsp70 chaperones. Here, the crystallographic structure of the substrate-binding domain (SBD) of PfHsp70-x is presented in complex with a hydrop...
Source: Acta Crystallographica Section F - September 27, 2020 Category: Biochemistry Authors: Schmidt, J. Vakonakis, I. Tags: malaria chaperones Plasmodium falciparum erythrocyte remodelling PfHsp70-x PfEMP-1 crystallography complexes research communications Source Type: research
Crystal structure of XCC3289 from Xanthomonas campestris: homology with the N-terminal substrate-binding domain of Lon peptidase
LonA peptidase is a major component of the protein quality-control mechanism in both prokaryotes and the organelles of eukaryotes. Proteins homologous to the N-terminal domain of LonA peptidase, but lacking its other domains, are conserved in several phyla of prokaryotes, including the Xanthomonadales order. However, the function of these homologous proteins (LonNTD-like proteins) is not known. Here, the crystal structure of the LonNTD-like protein from Xanthomonas campestris (XCC3289; UniProt Q8P5P7) is reported at 2.8 Å resolution. The structure was solved by molecular replacement and contains one polypeptide in the...
Source: Acta Crystallographica Section F - September 15, 2020 Category: Biochemistry Authors: Singh, R. Deshmukh, S. Kumar, A. Goyal, V.D. Makde, R.D. Tags: LonA cereblon protein binding XCC3289 Xanthomonas campestris research communications Source Type: research
Room-temperature neutron and X-ray data collection of 3CL Mpro from SARS-CoV-2
The replication of SARS-CoV-2 produces two large polyproteins, pp1a and pp1ab, that are inactive until cleavage by the viral chymotrypsin-like cysteine protease enzyme (3CL Mpro) into a series of smaller functional proteins. At the heart of 3CL Mpro is an unusual catalytic dyad formed by the side chains of His41 and Cys145 and a coordinated water molecule. The catalytic mechanism by which the enzyme operates is still unknown, as crucial information on the protonation states within the active site is unclear. To experimentally determine the protonation states of the catalytic site and of the other residues in the substrate-...
Source: Acta Crystallographica Section F - September 14, 2020 Category: Biochemistry Authors: Kneller, D.W. Phillips, G. Kovalevsky, A. Coates, L. Tags: neutron diffraction X-ray diffraction SARS-CoV-2 3CL Mpro research communications Source Type: research
A new soaking procedure for X-ray crystallographic structural determination of protein – peptide complexes
Interactions between a protein and a peptide motif of its protein partner are prevalent in nature. Often, a protein also has multiple interaction partners. X-ray protein crystallography is commonly used to examine these interactions in terms of bond distances and angles as well as to describe hotspots within protein complexes. However, the crystallization process presents a significant bottleneck in structure determination since it often requires notably time-consuming screening procedures, which involve testing a broad range of crystallization conditions via a trial-and-error approach. This difficulty is also increased as...
Source: Acta Crystallographica Section F - September 14, 2020 Category: Biochemistry Authors: Ballone, A. Lau, R.A. Zweipfenning, F.P.A. Ottmann, C. Tags: co-crystallization crystal soaking protein – peptide complexes research communications Source Type: research
Multiple crystal forms of human MacroD2
In this study, new crystallization conditions for MacroD2 were found and three crystal structures of human MacroD2 in the apo state were solved in space groups P41212, P43212 and P43, and refined at 1.75, 1.90 and 1.70 Å resolution, respectively. Structural comparison of the apo crystal structures with the previously reported crystal structure of MacroD2 in complex with ADP-ribose revealed conformational changes in the side chains of Val101, Ile189 and Phe224 induced by the binding of ADP-ribose in the active site. These conformational variations may potentially facilitate design efforts of a MacroD2 inhibitor. (Sourc...
Source: Acta Crystallographica Section F - September 14, 2020 Category: Biochemistry Authors: Wazir, S. Maksimainen, M.M. Lehti ö , L. Tags: macrodomain ADP-ribosylation ADP-ribosyl-hydrolase crystal forms apo structure research communications Source Type: research
Crystallization and structure of ebselen bound to Cys141 of human inositol monophosphatase
Inositol monophosphatase (IMPase) is inhibited by lithium, which is the most efficacious treatment for bipolar disorder. Several therapies have been approved, or are going through clinical trials, aimed at the replacement of lithium in the treatment of bipolar disorder. One candidate small molecule is ebselen, a selenium-containing antioxidant, which has been demonstrated to produce lithium-like effects both in a murine model and in clinical trials. Here, the crystallization and the first structure of human IMPase covalently complexed with ebselen, a 1.47 Å resolution crystal structure (PDB entry 6zk0), are presented....
Source: Acta Crystallographica Section F - September 14, 2020 Category: Biochemistry Authors: Fenn, G.D. Waller-Evans, H. Atack, J.R. Bax, B.D. Tags: inositol monophosphatase IMPase ebselen bipolar disorder tetramer research communications Source Type: research
The coiled-coil domain of glycosomal membrane-associated Leishmania donovani PEX14: cloning, overexpression, purification and preliminary crystallographic analysis
The glycosomal membrane-associated Leishmania donovani protein PEX14, which plays a crucial role in protein import from the cytosol to the glycosomal matrix, consists of three domains: an N-terminal domain where the signalling molecule binds, a transmembrane domain and an 84-residue coiled-coil domain (CC) that is responsible for oligomerization. CCs are versatile domains that participate in a variety of functions including supramolecular assembly, cellular signalling and transport. Recombinant PEX14 CC was cloned, overexpressed, affinity-purified with in-column thrombin cleavage and further purified by size-exclusion chro...
Source: Acta Crystallographica Section F - September 14, 2020 Category: Biochemistry Authors: Shakya, A.K. Pratap, J.V. Tags: Leishmania donovani coiled-coil domain PEX14 peroxisomes glycosomes research communications Source Type: research
