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 pathw ay in Aspergillus nidulans. The zinc DNA-binding domain contains ligan ds of zinc, six cysteines (Zn(2)Cys(6)) or five cysteines and one hist idine (Zn(2)Cys(5)His). The utilisation of complementary approaches su ch as X-ray absorption spectroscopy, mutational analysis, zinc content evaluation, determination of specific binding connecting structural a nd 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 Angstrom), together with the total loss of specific DNA-binding activ ity when one cysteine ligand is mutated, are in favour of a zinc clust er model in which six cysteine sulphurs ligate two zinc atoms. XANES s pectra of wild type and H10A AlcR protein are virtually identical indi cating that Histidine 10 does not have a direct contribution in zinc l igation but electrophoretic mobility shift assays show that His10 is i nvolved in DNA-binding. In contrast, proline 25 does not seem to play any direct role in the DNA-binding activity but XANES spectra of Pro25 A AlcR protein are slightly modified comparing to the wild type protei n 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 (Zn(2)Cys(6)) with unique characteristics resu lting from its primary and secondary structures and its binding specif icity toward direct and inverted repeat target. (C) 1997 Elsevier Scie nce B.V.