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Palmieri M.,Biological and Pharmaceutical science and Technology | Russo L.,Biological and Pharmaceutical science and Technology | Malgieri G.,Biological and Pharmaceutical science and Technology | Esposito S.,Biological and Pharmaceutical science and Technology | And 12 more authors.
Journal of Inorganic Biochemistry | Year: 2014

The zinc coordination sphere in prokaryotic zinc finger domain is extremely versatile and influences the stability and the folding property of the domain. Of a particular interest is the fourth zinc coordinating position, which is frequently occupied by two successive histidines, both able to coordinate the metal ion. To clarify their structural and functional role we report the NMR solution structure and the dynamics behavior of Ros87 H42A, which is a functional mutant of Ros87, the DNA binding domain of the Ros protein containing a prokaryotic Cys2His2 zinc finger domain. The structural analysis indicates that reducing the spacer among the two coordinating histidines from 4 (among His37 and His42) amino acids to 3 (among His37 and His41) increases the helicity of the first α-helix. At the same time, the second helix appears more mobile in the μs-ms timescale and the hydrophobic core is reduced. These data explain the high frequency of three-residue His spacers in the eukaryotic zinc finger domain and their absence in the prokaryotic counterpart. Furthermore, the structural comparison shows that the second coordination position is more sensitive to H42A mutation with respect to the first and the third position, providing the rationale of the high variability of the second and the fourth zinc coordinating position in Ros homologs, which adopt different metal coordination but preserve similar tertiary structures and DNA binding activities. Finally, H/D exchange measurements and NMR thermal unfolding analysis indicate that this mutant likely unfolds via a different mechanism with respect to the wild-type. © 2013 Published by Elsevier Inc.


PubMed | CNR Institute of Neuroscience, Biological and Pharmaceutical science and Technology and University of Naples Federico II
Type: | Journal: European journal of medicinal chemistry | Year: 2015

Eukaryotic Cys2His2 zinc finger domain is one of the most common and important structural motifs involved in protein-DNA interaction. The recognition motif is characterized by the tetrahedral coordination of a zinc ion by conserved cysteine and histidine residues. We have characterized the prokaryotic Cys2His2 zinc finger motif, included in the DNA binding region (Ros87) of Ros protein from Agrobacterium tumefaciens, demonstrating that, although possessing a similar zinc coordination sphere, this domain presents significant differences from its eukaryotic counterpart. Furthermore, basic residues flanking the zinc binding region on either side have been demonstrated, by Electrophoretic Mobility Shift Assay (EMSA) experiments, to be essential for Ros DNA binding. In spite of this wealth of knowledge, the structural details of the mechanism through which the prokaryotic zinc fingers recognize their target genes are still unclear. Here, to gain insights into the molecular DNA recognition process of prokaryotic zinc finger domains we applied a strategy in which we performed molecular docking studies using a combination of Nuclear Magnetic Resonance (NMR) and Molecular Dynamics (MD) simulations data. The results demonstrate that the MD ensemble provides a reasonable picture of Ros87 backbone dynamics in solution. The Ros87-DNA model indicates that the interaction involves the first two residue of the first -helix, and several residues located in the basic regions flanking the zinc finger domain. Interestingly, the prokaryotic zinc finger domain, mainly with the C-terminal tail that is wrapped around the DNA, binds a more extended recognition site than the eukaryotic counterpart. Our analysis demonstrates that the introduction of the protein flexibility in docking studies can improve, in terms of accuracy, the quality of the obtained models and could be particularly useful for protein showing high conformational heterogeneity as well as for computational drug design applications.


PubMed | CNR Institute of Neuroscience, Biological and Pharmaceutical science and Technology and University of Naples Federico II
Type: | Journal: Journal of inorganic biochemistry | Year: 2013

The zinc coordination sphere in prokaryotic zinc finger domain is extremely versatile and influences the stability and the folding property of the domain. Of a particular interest is the fourth zinc coordinating position, which is frequently occupied by two successive histidines, both able to coordinate the metal ion. To clarify their structural and functional role we report the NMR solution structure and the dynamics behavior of Ros87 H42A, which is a functional mutant of Ros87, the DNA binding domain of the Ros protein containing a prokaryotic Cys2His2 zinc finger domain. The structural analysis indicates that reducing the spacer among the two coordinating histidines from 4 (among His37 and His42) amino acids to 3 (among His37 and His41) increases the helicity of the first -helix. At the same time, the second helix appears more mobile in the s-ms timescale and the hydrophobic core is reduced. These data explain the high frequency of three-residue His spacers in the eukaryotic zinc finger domain and their absence in the prokaryotic counterpart. Furthermore, the structural comparison shows that the second coordination position is more sensitive to H42A mutation with respect to the first and the third position, providing the rationale of the high variability of the second and the fourth zinc coordinating position in Ros homologs, which adopt different metal coordination but preserve similar tertiary structures and DNA binding activities. Finally, H/D exchange measurements and NMR thermal unfolding analysis indicate that this mutant likely unfolds via a different mechanism with respect to the wild-type.


Petriccione M.,Italian Agricultural Research Council | Papa S.,Biological and Pharmaceutical Science and Technology | Ciniglia C.,Biological and Pharmaceutical Science and Technology
Environmental Science and Pollution Research | Year: 2014

Walnut husk washing waters (WHWW), a by-product of walnut production, are indiscriminately used for irrigation without preliminary risk assessment. Basing on previous in vitro results on the toxicity of this by-product, we have followed the morphophysiological development of Zea mays, Lactuca sativa cv. Gentilina and L. sativa cv. Canasta under diluted and undiluted WHWW irrigation. Significant development alterations have been observed in root and shoot elongations for all crops as well as in total biomass and chlorophyll content. The genotoxic potential of WHWW has been concurrently verified; acridine orange/ethidium bromide staining evidenced chromatin modifications and DNA degradation and also was confirmed by DNA laddering. The DNA instability was also assessed through RAPD, thus suggesting the danger of the by-product of walnut processing and focusing the attention on the necessity of an efficient treatment of WHWWs. The findings obtained by PCA of agronomic and physiological traits suggested that establishing guidelines for the administration of WHWW for irrigation is of great importance, and it is necessary to supervise their use in agricultural soils. © 2013 Springer-Verlag Berlin Heidelberg.

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