ABSTRACT
AlcR is the transcriptional activator of the ethanol utilization pathway in Aspergillus nidulans. The zinc DNA-binding domain contains ligands of zinc, six cysteines (Zn2Cys6) or five cysteines and one histidine (Zn2Cys5His). The utilisation of complementary approaches such as X-ray absorption spectroscopy, mutational analysis, zinc content evaluation, determination of specific binding connecting structural and biological data, have allowed to determine zinc environment and to analyse the involvement of amino acids. The determination by EXAFS of zinc ligands (four sulphur atoms), the Zn content in the protein (2:1), the evaluation of the distance between two zinc atoms (3.16 +/- 0.02 angstroms), together with the total loss of specific DNA-binding activity when one cysteine ligand is mutated, are in favour of a zinc cluster model in which six cysteine sulphurs ligate two zinc atoms. XANES spectra of wild type and H10A AlcR protein are virtually identical indicating that Histidine 10 does not have a direct contribution in zinc ligation but electrophoretic mobility shift assays show that His10 is involved in DNA-binding. In contrast, proline 25 does not seem to play any direct role in the DNA-binding activity but XANES spectra of Pro25A AlcR protein are slightly modified comparing to the wild type protein spectra. This suggests a role of the proline in the stabilisation of the Zn cluster structure. AlcR DNA-binding domain belongs to the zinc binuclear class family (Zn2Cys6) with unique characteristics resulting from its primary and secondary structures and its binding specificity toward direct and inverted repeat target.
Subject(s)
Aspergillus nidulans/chemistry , DNA-Binding Proteins/chemistry , Fungal Proteins/chemistry , Zinc/chemistry , Amino Acid Sequence , Binding Sites , Cysteine/metabolism , DNA/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Fungal Proteins/genetics , Fungal Proteins/metabolism , Ligands , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Oligodeoxyribonucleotides/genetics , Oligodeoxyribonucleotides/metabolism , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino Acid , Spectrum Analysis , Transcription Factors/chemistry , Transcription Factors/genetics , Transcription Factors/metabolism , X-RaysABSTRACT
The Cu(I)GSH complex has recently been shown to be a good candidate for delivering copper to the active site of Cu-free Cu,Zn superoxide dismutase both in vivo and in vitro. In this work X-ray absorption spectroscopy has been used to characterize the Cu(I)GSH complex and to follow in vitro the reconstitution of Cu,Zn superoxide dismutase from the copper-free protein and this complex. The results obtained indicate that the copper is directly transferred as Cu(I) from the GSH complex into the empty copper binding site. No evidence has been obtained for a ternary complex in which the metal is bound to both GSH and the protein.
