RESUMO
The HNH family of endonucleases is characterized by a ßßα metal-finger structural motif. Colicin E7 is a representative member of this family containing the strictly conserved HNH motif at its C-terminus. Structural and biochemical studies suggested that the HNH motif could contain all the residues necessary for metal ion binding and nuclease activity. In this work a 43 amino acid peptide extending from V534 to K576 of colicin E7 and encompassing the HNH motif was cloned and expressed in Escherichia coli as a ubiquitin fusion protein. The N-terminal fusion tag was cleaved off by a specific protease, and the HNH peptide was purified free of ubiquitin. Circular dichroism, fluorescence and mass spectrometry showed that the zinc-ion binding affinity of the purified HNH peptide was much weaker than that of the intact nuclease domain suggesting that the N-terminal part of the nuclease domain is essential for stabilizing the structure of the HNH motif. The coordination sphere of the metal ion was found to be not fully equipped by the ligand - leaving a free coordination site for the substrate. Neither DNA binding nor DNAse activity of the purified HNH peptide was detected. Comparison of the glutathion-S-transferase-fused N-terminal deletion mutants of the colicin E7 nuclease domain suggested that the presence of the DNA-binding site is still not sufficient for the catalytic activity.
Assuntos
Clonagem Molecular , Colicinas/genética , Colicinas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Zinco/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Colicinas/química , Colicinas/isolamento & purificação , DNA/metabolismo , Desoxirribonucleases/química , Desoxirribonucleases/metabolismo , Escherichia coli/química , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/genética , Peptídeos/isolamento & purificação , Peptídeos/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Ubiquitina/química , Ubiquitina/genética , Ubiquitina/isolamento & purificação , Ubiquitina/metabolismoRESUMO
Colicin E7 (ColE7) is a metallonuclease toxin of Escherichia coli belonging to the HNH superfamily of nucleases. It contains highly conserved amino acids in its HHX(14)NX(8)HX(3)H ßßα-type metal ion binding C-terminal active centre. However, the proximity of the arginine at the N-terminus of the nuclease domain of ColE7 (NColE7, 446-576) is necessary for the hydrolytic activity. This poses a possibility of allosteric activation control in this protein. To obtain more information on this phenomenon, two protein mutants were expressed, i.e. four and 25 N-terminal amino acids were removed from NColE7. The effect of the N-terminal truncation on the Zn(2+) ion and DNA binding as well as on the activity was investigated in this study by mass spectrometry, synchrotron-radiation circular dichroism and fluorescence spectroscopy and agarose gel mobility shift assays. The dynamics of protein backbone movement was simulated by molecular dynamics. Semiempirical quantum chemical calculations were performed to obtain better insight into the structure of the active centre. The longer protein interacted with both Zn(2+) ion and DNA more strongly than its shorter counterpart. The results were explained by the structural stabilization effect of the N-terminal amino acids on the catalytic centre. In agreement with this, the absence of the N-terminal sequences resulted in significantly increased movement of the backbone atoms compared with that in the native NColE7: in ΔN25-NColE7 the amino acid strings between residues 485-487, 511-515 and 570-571, and in ΔN4-NColE7 those between residues 467-468, 530-535 and 570-571.
