Mechanisms of Aurothiomalate-Cys sub(2)His sub(2) Zinc Finger Interactions

Zinc finger motifs are present in a wide variety of regulatory proteins and generally function as interaction modules between macromolecules. These functional interactions are controlled by mechanisms of zinc (Zn super(2+))-binding and release. Besides Zn super(2+) certain electrophilic metals can potentially react with zinc finger domains and lead to changes in the structure and function of those domains. In these studies, the Cys sub(2)His sub(2) zinc finger was chosen as a model for understanding how the gold (I) (Au super(1+)) drug, aurothiomalate (AuTM), interacts mechanistically with the Zn super(2+) coordination sphere. DNA binding assays were used to analyze functional interactions between AuTM and two model Cys sub(2)His sub(2) zinc finger transcription factors, TFIIIA and Sp1; inhibition in the micromolar range of AuTM was observed in both cases. Electrospray ionization mass spectrometry (ESI-MS) was utilized to examine molecular interactions between AuTM and a zinc finger peptide modeled after the third finger of Sp1 (Sp1-3). These experiments demonstrated Au super(1+) ions can bind the zinc finger structure and trigger the release of the Zn super(2+) ion. Quantifying the ESI-MS data allowed for a relative affinity value between Zn super(2+) and Au super(1+) ions to be calculated and shows Au super(1+) has a 4-fold higher affinity for Sp1-3 than Zn super(2+). Mechanistic differences between Zn super(2+) and Au super(1+) binding to the model Sp1-3 zinc finger were analyzed at isotopic resolution, and the metal-coordination spheres were probed with small molecules (H super(+), hydrogen peroxide, glutathione disulfide, and iodoacetamide). Natural isotope cluster analysis suggested the presence of a metal-thiol bond in the Cys sub(2)His sub(2) zinc finger structure. Metal exchange reactions between zinc fingers demonstrated Zn super(2+) ions exchanged more rapidly than Au super(1+) ions. Circular dichroism (CD) exhibited differences in the secondary structure of the Sp1-3 model peptide when binding Zn super(2+) or Au super(1+) ions.