Spectroscopic characterization of the heme binding (GAF) domain of two sensor kinases from Methanosarcina acetivorans

2019 ◽  
Vol 23 (07n08) ◽  
pp. 930-942
Author(s):  
Kerstin Fiege ◽  
Christian Twittenhoff ◽  
Kathrin Kwiatkowski ◽  
Nicole Frankenberg-Dinkel
Keyword(s):  
Spectroscopic Characterization ◽  
Binding Pocket ◽  
Cysteine Residue ◽  
Site Directed Mutagenesis ◽  
Heme Binding ◽  
Methanosarcina Acetivorans ◽  
Vis Spectroscopy ◽  
Sensor Kinases ◽  
Covalently Linked ◽  
Heme Cofactor

The sensor kinases MsmS and RdmS from the methanogenic archaeon Methanosarcina acetivorans are multidomain proteins containing a covalently linked heme cofactor. This cofactor is connected via a single cysteine residue in a GAF domain. Although both proteins were shown to display a redox-dependent control of the downstream kinase module, this property appears to be independent of the heme cofactor. We therefore envision an additional sensor role for the heme cofactor. In order to learn more about the heme binding pocket and its constitution, UV-vis spectroscopy in combination with site-directed mutagenesis was performed on the isolated heme-binding sGAF2 domain and the full-length protein. The data indicate a 6-coordinated heme with a proximal histidine ligand and a smaller ligand, likely a water molecule on the distal site. The latter is also thought to be the sensory site and is shown to easily undergo ligand exchange.

scholarly journals A binding hotspot inTrypanosoma cruzihistidyl-tRNA synthetase revealed by fragment-based crystallographic cocktail screens

2015 ◽  
Vol 71 (8) ◽  
pp. 1684-1698 ◽  
Author(s):  
Cho Yeow Koh ◽  
Latha Kallur Siddaramaiah ◽  
Ranae M. Ranade ◽  
Jasmine Nguyen ◽  
Tengyue Jian ◽  
...  
Keyword(s):  
Protozoan Parasite ◽  
Binding Pocket ◽  
Binding Mode ◽  
Current Treatment ◽  
Trna Synthetase ◽  
Cysteine Residue ◽  
Fragment Library ◽  
Covalently Linked ◽  
Poor Efficacy ◽  
Specific Inhibitors

American trypanosomiasis, commonly known as Chagas disease, is a neglected tropical disease caused by the protozoan parasiteTrypanosoma cruzi. The chronic form of the infection often causes debilitating morbidity and mortality. However, the current treatment for the disease is typically inadequate owing to drug toxicity and poor efficacy, necessitating a continual effort to discover and develop new antiparasitic therapeutic agents. The structure ofT. cruzihistidyl-tRNA synthetase (HisRS), a validated drug target, has previously been reported. Based on this structure and those of human cytosolic HisRS, opportunities for the development of specific inhibitors were identified. Here, efforts are reported to identify small molecules that bind toT. cruziHisRS through fragment-based crystallographic screening in order to arrive at chemical starting points for the development of specific inhibitors.T. cruziHisRS was soaked into 68 different cocktails from the Medical Structural Genomics of Pathogenic Protozoa (MSGPP) fragment library and diffraction data were collected to identify bound fragments after soaking. A total of 15 fragments were identified, all bound to the same site on the protein, revealing a fragment-binding hotspot adjacent to the ATP-binding pocket. On the basis of the initial hits, the design of reactive fragments targeting the hotspot which would be simultaneously covalently linked to a cysteine residue present only in trypanosomatid HisRS was initiated. Inhibition ofT. cruziHisRS was observed with the resultant reactive fragments and the anticipated binding mode was confirmed crystallographically. These results form a platform for the development of future generations of selective inhibitors for trypanosomatid HisRS.

Revisiting the interaction of heme with hemopexin

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Milena Sophie Detzel ◽  
Benjamin Franz Schmalohr ◽  
Francèl Steinbock ◽  
Marie-Thérèse Hopp ◽  
Anuradha Ramoji ◽  
...  
Keyword(s):  
Binding Sites ◽  
Protective Factor ◽  
Homology Model ◽  
Binding Pocket ◽  
Heme Binding ◽  
Vis Spectroscopy ◽  
Erythrocyte Lysis ◽  
Peptide Models ◽  
High Concentrations ◽  
Displacement Assays

Abstract In hemolytic disorders, erythrocyte lysis results in massive release of hemoglobin and, subsequently, toxic heme. Hemopexin is the major protective factor against heme toxicity in human blood and currently considered for therapeutic use. It has been widely accepted that hemopexin binds heme with extraordinarily high affinity of <1 pM in a 1:1 ratio. However, several lines of evidence point to a higher stoichiometry and lower affinity than determined 50 years ago. Here, we re-analyzed these data. SPR and UV/Vis spectroscopy were used to monitor the interaction of heme with the human protein. The heme-binding sites of hemopexin were characterized using hemopexin-derived peptide models and competitive displacement assays. We obtained a K D value of 0.32 ± 0.04 nM and the ratio for the interaction was determined to be 1:1 at low heme concentrations and at least 2:1 (heme:hemopexin) at high concentrations. We were able to identify two yet unknown potential heme-binding sites on hemopexin. Furthermore, molecular modelling with a newly created homology model of human hemopexin suggested a possible recruiting mechanism by which heme could consecutively bind several histidine residues on its way into the binding pocket. Our findings have direct implications for the potential administration of hemopexin in hemolytic disorders.

Characterization of Recombinant Human Coagulation Factor XFriuli

1996 ◽  
Vol 75 (02) ◽  
pp. 313-317 ◽  
Author(s):  
D J Kim ◽  
A Girolami ◽  
H L James
Keyword(s):  
Catalytic Domain ◽  
Coagulation Factor ◽  
Molecular Size ◽  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
Bleeding Tendency ◽  
Factor X ◽  
Amino Terminal ◽  
Normal Plasma ◽  
Plasma Factor

SummaryNaturally occurring plasma factor XFriuli (pFXFr) is marginally activated by both the extrinsic and intrinsic coagulation pathways and has impaired catalytic potential. These studies were initiated to obtain confirmation that this molecule is multi-functionally defective due to the substitution of Ser for Pro at position 343 in the catalytic domain. By the Nelson-Long site-directed mutagenesis procedure a construct of cDNA in pRc/CMV was derived for recombinant factor XFriuli (rFXFr) produced in human embryonic (293) kidney cells. The rFXFr was purified and shown to have a molecular size identical to that of normal plasma factor X (pFX) by gel electrophoretic, and amino-terminal sequencing revealed normal processing cleavages. Using recombinant normal plasma factor X (rFXN) as a reference, the post-translational y-carboxy-glutamic acid (Gla) and (β-hydroxy aspartic acid (β-OH-Asp) content of rFXFr was over 85% and close to 100%, respectively, of expected levels. The specific activities of rFXFr in activation and catalytic assays were the same as those of pFXFr. Molecular modeling suggested the involvement of a new H-bond between the side-chains of Ser-343 and Thr-318 as they occur in anti-parallel (3-pleated sheets near the substrate-binding pocket of pFXFr. These results support the conclusion that the observed mutation in pFXFr is responsible for its dysfunctional activation and catalytic potentials, and that it accounts for the moderate bleeding tendency in the homozygous individuals who possess this variant procoagulant.

scholarly journals Site-Directed Mutagenesis of the Heterotrimeric Killer Toxin Zymocin Identifies Residues Required for Early Steps in Toxin Action

2014 ◽  
Vol 80 (20) ◽  
pp. 6549-6559 ◽  
Author(s):  
Sabrina Wemhoff ◽  
Roland Klassen ◽  
Friedhelm Meinhardt
Keyword(s):  
Kluyveromyces Lactis ◽  
Killer Toxin ◽  
Cysteine Residue ◽  
Site Directed Mutagenesis ◽  
Target Cells ◽  
Directed Mutagenesis ◽  
Content Type ◽  
Protein Toxin ◽  
Assisted Delivery

ABSTRACTZymocin is aKluyveromyces lactisprotein toxin composed of αβγ subunits encoded by the cytoplasmic virus-like element k1 and functions by αβ-assisted delivery of the anticodon nuclease (ACNase) γ into target cells. The toxin binds to cells' chitin and exhibits chitinase activityin vitrothat might be important during γ import.Saccharomyces cerevisiaestrains carrying k1-derived hybrid elements deficient in either αβ (k1ORF2) or γ (k1ORF4) were generated. Loss of either gene abrogates toxicity, and unexpectedly, Orf2 secretion depends on Orf4 cosecretion. Functional zymocin assembly can be restored by nuclear expression of k1ORF2 or k1ORF4, providing an opportunity to conduct site-directed mutagenesis of holozymocin. Complementation required active site residues of α's chitinase domain and the sole cysteine residue of β (Cys250). Since βγ are reportedly disulfide linked, the requirement for the conserved γ C231 was probed. Toxicity of intracellularly expressed γ C231A indicated no major defect in ACNase activity, while complementation of k1ΔORF4 by γ C231A was lost, consistent with a role of β C250 and γ C231 in zymocin assembly. To test the capability of αβ to carry alternative cargos, the heterologous ACNase fromPichia acaciae(P. acaciaeOrf2 [PaOrf2]) was expressed, along with its immunity gene, in k1ΔORF4. While efficient secretion of PaOrf2 was detected, suppression of the k1ΔORF4-derived k1Orf2 secretion defect was not observed. Thus, the dependency of k1Orf2 on k1Orf4 cosecretion needs to be overcome prior to studying αβ's capability to deliver other cargo proteins into target cells.

Modification of a novel x-type high-molecular-weight glutenin subunit gene from Aegilops markgrafii to improve dough strength of wheat flour

2018 ◽  
Vol 69 (9) ◽  
pp. 873
Author(s):  
Xin Ma ◽  
Xuye Du ◽  
Cunyao Bo ◽  
Hongwei Wang ◽  
Anfei Li ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Wheat Flour ◽  
Cysteine Residue ◽  
Site Directed Mutagenesis ◽  
Cysteine Residues ◽  
Dough Strength ◽  
Typical Structure ◽  
Dough Quality

High-molecular-weight glutenin subunits (HMW-GS) in bread wheat are major determinants of dough viscoelastic properties and the end-use quality of wheat flour. Cysteine residues, which form intermolecular disulphide bonds in HMW-GS, could improve the strength of gluten. To our knowledge, the number and position of cysteine residues in HMW-GS are conserved between wheat (Triticum aestivum) and Aegilops markgrafii. In the present study, we modified a gene (1Cx1.1) from Ae. markgrafii for an HMW-GS that possessed the typical structure and conserved number of cysteines. Site-directed mutagenesis was carried out in 1Cx1.1 to investigate how the position of cysteine residues in HMW-GS affects the mixing properties of dough. Six HMW-GS containing an extra cysteine residue were expressed in Escherichia coli, and the proteins were purified at sufficient scale for incorporation into flour to test dough quality. There were large differences in dough property among samples containing different modified subunits. Cysteine substituting in the N-terminal or repetitive-domain of HMW-GS could significantly improve dough quality. The results showed that the strategy was useful for providing genetic resources for gene engineering, and hence could be valuable for improving the processing quality of wheat.

scholarly journals Identification and Analysis of Genes Involved in Anaerobic Toluene Metabolism by Strain T1: Putative Role of a Glycine Free Radical

1998 ◽  
Vol 64 (5) ◽  
pp. 1650-1656 ◽  
Author(s):  
Peter W. Coschigano ◽  
Thomas S. Wehrman ◽  
L. Y. Young
Keyword(s):  
Free Radical ◽  
Amino Acid ◽  
Molecular Mass ◽  
Cysteine Residue ◽  
Open Reading Frames ◽  
Site Directed Mutagenesis ◽  
Pyruvate Formate Lyase ◽  
Calculated Molecular Mass ◽  
Acid Protein ◽  
Amino Acid Protein

ABSTRACT The denitrifying strain T1 is able to grow with toluene serving as its sole carbon source. Two mutants which have defects in this toluene utilization pathway have been characterized. A clone has been isolated, and subclones which contain tutD and tutE, two genes in the T1 toluene metabolic pathway, have been generated. ThetutD gene codes for an 864-amino-acid protein with a calculated molecular mass of 97,600 Da. The tutE gene codes for a 375-amino-acid protein with a calculated molecular mass of 41,300 Da. Two additional small open reading frames have been identified, but their role is not known. The TutE protein has homology to pyruvate formate-lyase activating enzymes. The TutD protein has homology to pyruvate formate-lyase enzymes, including a conserved cysteine residue at the active site and a conserved glycine residue that is activated to a free radical in this enzyme. Site-directed mutagenesis of these two conserved amino acids shows that they are also essential for the function of TutD.

scholarly journals Crystal structure, Hirshfeld surface analysis and spectroscopic characterization of the di-enol tautomeric form of the compound 3,3′-[(2-sulfanylidene-1,3-dithiole-4,5-diyl)bis(sulfanediyl)]bis(pentane-2,4-dione)

2020 ◽  
Vol 76 (9) ◽  
pp. 1427-1432
Author(s):  
Keysha T. Cordero Giménez ◽  
Victoria Y. Soto Díaz ◽  
Jean C. González Espiet ◽  
Alexis Lavín Flores ◽  
Jesbaniris Bas Concepción ◽  
...  
Keyword(s):  
Title Compound ◽  
Tautomeric Form ◽  
Spectroscopic Characterization ◽  
Inversion Center ◽  
Torsion Angles ◽  
Triclinic Space Group ◽  
Covalently Linked ◽  
Intramolecular Hydrogen ◽  
Short Contacts

The reaction between [TBA]2[Zn(dmit)2] and 3-chloro-2,4-pentanedione yielded single crystals of the title compound, (3E,3′E)-3,3′-[(2-sulfanylidene-1,3-dithiole-4,5-diyl)bis(sulfanediyl)]bis(4-hydroxypent-3-en-2-one), C13H14O4S5, after solvent evaporation. The title compound crystallizes in the triclinic space group P\overline{1} with two molecules related by an inversion center present in the unit cell. The central thione ring moiety contains a carbon–carbon double bond covalently linked to two sulfoxide substituents located outside of the plane of the ring. The S—C—C—S torsion angles are −176.18 (8) and −0.54 (18)°. Intramolecular hydrogen bonds occur within the two dione substituents (1.67–1.69 Å). Adjacent asymmetric units are linked by C—H...S (2.89–2.90 Å), S...S [3.569 (1) Å] and O...H [2.56–2.66 Å between non-stacked thione rings] short contacts.

scholarly journals Synthesis, spectroscopic characterization and thermogravimetric analysis of two series of substituted (metallo)tetraphenylporphyrins

2017 ◽  
Vol 8 ◽  
pp. 1191-1204 ◽  
Author(s):  
Rasha K Al-Shewiki ◽  
Carola Mende ◽  
Roy Buschbeck ◽  
Pablo F Siles ◽  
Oliver G Schmidt ◽  
...  
Keyword(s):  
Thermogravimetric Analysis ◽  
Molecular Beam ◽  
Elevated Temperatures ◽  
Oxalyl Chloride ◽  
Spectroscopic Characterization ◽  
Subsequent Treatment ◽  
Ionization Mass ◽  
Molecular Beam Deposition ◽  
Vis Spectroscopy ◽  
Beam Deposition

Subsequent treatment of H2TPP(CO2H)4 (tetra(p-carboxylic acid phenyl)porphyrin, 1) with an excess of oxalyl chloride and HNR2 afforded H2TPP(C(O)NR2)4 (R = Me, 2; iPr, 3) with yields exceeding 80%. The porphyrins 2 and 3 could be converted to the corresponding metalloporphyrins MTPP(C(O)NR2)4 (R = Me/iPr for M = Zn (2a, 3a); Cu (2b, 3b); Ni (2c, 3c); Co (2d, 3d)) by the addition of 3 equiv of anhydrous MCl2 (M = Zn, Cu, Ni, Co) to dimethylformamide solutions of 2 and 3 at elevated temperatures. Metalloporphyrins 2a–d and 3a–d were obtained in yields exceeding 60% and have been, as well as 2 and 3, characterized by elemental analysis, electrospray ionization mass spectrometry (ESIMS) and IR and UV–vis spectroscopy. Porphyrins 2, 2a–d and 3, 3a–d are not suitable for organic molecular beam deposition (OMBD), which is attributed to their comparatively low thermal stability as determined by thermogravimetric analysis (TG) of selected representatives.

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