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Nemeth E.,University of Szeged | Kortvelyesi T.,University of Szeged | Kozisek M.,Gilead Sciences | Thulstrup P.W.,Copenhagen University | And 5 more authors.
JBIC Journal of Biological Inorganic Chemistry | Year: 2014

The nuclease domain of colicin E7 (NColE7) cleaves DNA nonspecifically. The active center is a Zn2+-containing HNH motif at the C-terminus. The N-terminal loop is essential for the catalytic activity providing opportunity for allosteric modulation of the enzyme. To identify the key residues responsible for the structural integrity of NColE7, a virtual alanine scan was performed on a semiempirical quantum chemical level within the 25 residue long N-terminal sequence (446-470). Based on the calculations the T454A/K458A/W464A-NColE7 triple mutant (TKW) was expressed and purified. According to the agarose gel electrophoresis experiments and linear dichroism spectra the catalytic activity of the TKW mutant decreased in comparison with wild-type NColE7. The distorted structure and weakened Zn2+ binding may account for this as revealed by circular dichroism spectra, mass spectrometry, fluorescence-based thermal analysis and isothermal microcalorimetric titrations. Remarkably, the substrate induced the folding of the mutant protein. © 2014 SBIC.


Enyedy E.A.,University of Szeged | Domotor O.,University of Szeged | Domotor O.,MTA SZTE Bioinorganic Chemistry Research Group | Bali K.,University of Szeged | And 3 more authors.
Journal of Biological Inorganic Chemistry | Year: 2015

Tris(8-quinolinolato)gallium(III) (KP46) and tris(maltolato)gallium(III) (GaM) are promising orally active antitumor metallodrugs currently undergoing clinical trials. Their interaction with human serum albumin (HSA) and transferrin (Tf) was studied in detail in aqueous solution by the combination of various methods such as spectrofluorometry, UV-vis spectrophotometry, 1H and saturation transfer difference NMR spectroscopy, and ultrafiltration-UV-vis spectrophotometry. Binding data were evaluated quantitatively. Tf was found to replace the original ligand much less efficiently in KP46 than in GaM, whereas a significant noncovalent binding of KP46 with HSA (log K′ = 4.04) retaining the coordination environment around gallium(III) was found. The interaction between HSA and KP46 was also confirmed by protein-complex modeling calculations. On the basis of the conditional stability constants, the distribution of gallium(III) in serum was computed and compared for these metallodrugs under physiological conditions, and revealed the prominent role of HSA in the case of KP46 and that of Tf for GaM. Graphical abstract: [Figure not available: see fulltext.] © 2014 SBIC.


Szunyogh D.,MTA SZTE Bioinorganic Chemistry Research Group | Gyurcsik B.,MTA SZTE Bioinorganic Chemistry Research Group | Gyurcsik B.,University of Szeged | Larsen F.H.,Copenhagen University | And 5 more authors.
Dalton Transactions | Year: 2015

Designed metal ion binding peptides offer a variety of applications in both basic science as model systems of more complex metalloproteins, and in biotechnology, e.g. in bioremediation of toxic metal ions, biomining or as artificial enzymes. In this work a peptide (HS: Ac-SCHGDQGSDCSI-NH2) has been specifically designed for binding of both ZnII and HgII, i.e. metal ions with different preferences in terms of coordination number, coordination geometry, and to some extent ligand composition. It is demonstrated that HS accommodates both metal ions, and the first coordination sphere, metal ion exchange between peptides, and speciation are characterized as a function of pH using UV-absorption-, synchrotron radiation CD-, 1H-NMR-, and PAC-spectroscopy as well as potentiometry. HgII binds to the peptide with very high affinity in a {HgS2} coordination geometry, bringing together the two cysteinates close to each end of the peptide in a loop structure. Despite the high affinity, HgII is kinetically labile, exchanging between peptides on the subsecond timescale, as indicated by line broadening in 1H-NMR. The ZnII-HS system displays more complex speciation, involving monomeric species with coordinating cysteinates, histidine, and a solvent water molecule, as well as HS-ZnII-HS complexes. In summary, the HS peptide displays conformational flexibility, contains many typical metal ion binding groups, and is able to accommodate metal ions with different structural and ligand preferences with high affinity. As such, the HS peptide may be a scaffold offering binding of a variety of metal ions, and potentially serve for metal ion sequestration in biotechnological applications. This journal is © The Royal Society of Chemistry.


Czene A.,MTA SZTE Bioinorganic Chemistry Research Group | Toth E.,University of Szeged | Gyurcsik B.,MTA SZTE Bioinorganic Chemistry Research Group | Gyurcsik B.,University of Szeged | And 6 more authors.
Acta Crystallographica Section F: Structural Biology and Crystallization Communications | Year: 2013

The metallonuclease colicin E7 is a member of the HNH family of endonucleases. It serves as a bacterial toxin in Escherichia coli, protecting the host cell from other related bacteria and bacteriophages by degradation of their chromosomal DNA under environmental stress. Its cell-killing activity is attributed to the nonspecific nuclease domain (NColE7), which possesses the catalytic ββα-type metal ion-binding HNH motif at its C-terminus. Mutations affecting the positively charged amino acids at the N-terminus of NColE7 (444-576) surprisingly showed no or significantly reduced endonuclease activity [Czene et al. (2013), J. Biol. Inorg. Chem. 18, 309-321]. The necessity of the N-terminal amino acids for the function of the C-terminal catalytic centre poses the possibility of allosteric activation within the enzyme. Precise knowledge of the intramolecular interactions of these residues that affect the catalytic activity could turn NColE7 into a novel platform for artificial nuclease design. In this study, the N-terminal deletion mutant ΔN4-NColE7-C*of the nuclease domain of colicin E7 selected by E. coli was overexpressed and crystallized at room temperature by the sitting-drop vapour-diffusion method. X-ray diffraction data were collected to 1.6 Å resolution and could be indexed and averaged in the trigonal space group P3121 or P3221, with unit-cell parameters a = b = 55.4, c = 73.1 Å. Structure determination by molecular replacement is in progress. © 2013.


Czene A.,MTA SZTE Bioinorganic Chemistry Research Group | Toth E.,University of Szeged | Nemeth E.,University of Szeged | Otten H.,Copenhagen University | And 7 more authors.
Metallomics | Year: 2014

The nuclease domain of colicin E7 metallonuclease (NColE7) contains its active centre at the C-terminus. The mutant ΔN4-NColE7-C∗-where the four N-terminal residues including the positively charged K446, R447 and K449 are replaced with eight residues from the GST tag-is catalytically inactive. The crystal structure of this mutant demonstrates that its overall fold is very similar to that of the native NColE7 structure. This implicates the stabilizing effect of the remaining N-terminal sequence on the structure of the C-terminal catalytic site and the essential role of the deleted residues in the mechanism of the catalyzed reaction. Complementary QM/MM calculations on the protein-DNA complexes support the less favourable cleavage by the mutant protein than by NColE7. Furthermore, a water molecule as a possible ligand for the Zn2+-ion is proposed to play a role in the catalytic process. These results suggest that the mechanism of the Zn2+-containing HNH nucleases needs to be further studied and discussed. © 2014 the Partner Organisations.

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