First experimental evidence of a zinc binuclear cluster in AlcR protein, mutational and X-ray absorption studies.
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.
Ascone
I
I
Laboratoire d'Utilisation du Rayonnement Electromagnétique, Faculté d'Orsay, France. ascone@lure.u-psud.fr
Lenouvel
F
F
Sequeval
D
D
Dexpert
H
H
Felenbok
B
B
D000595:Amino Acid Sequence; D001233:Aspergillus nidulans / Q000737:chemistry*; D001665:Binding Sites; D003545:Cysteine / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000235:genetics / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013057:Spectrum Analysis; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014965:X-Rays; D015032:Zinc / Q000737:chemistry*
C074202:ALCR protein, Aspergillus nidulans; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D008024:Ligands; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D004247:DNA; D015032:Zinc; D003545:Cysteine