Zinc finger transcription factor Zn3-Sp1 reactions with Cd2+

Cadmium is a major environmental pollutant that causes kidney failure including the inability to resorb nutrients such as glucose. In a mouse kidney cell culture model, Cd(2+) inhibits Na(+)-dependent glucose uptake mediated by SGLT transporters. This defect has been traced to the down-regulation of SGLT mRNA synthesis mediated by the zinc-finger transcription factor, Zn(3)-Sp1. Incubation of Cd(2+) with Zn(2+)-Sp1 inhibited its capacity to bind to GC1, its binding site in the SGLT1 promoter. The extent of reaction was reduced as increasing concentrations of Zn(2+) are simultaneously present in the reaction mixture. The results are consistent with a Cd(2+)-Zn(2+) exchange reaction that inactivates the DNA binding function of the protein. The equilibrium constant for this reaction was calculated as 14 +/- 3 and 7 +/- 4 for the reactions measured by the binding to GC1 and an analogous SGLT2 promoter site. Sequential addition of Cd(2+) and Zn(2+) to Zn(3)-Sp1 failed to inhibit the reduction in DNA binding seen with Cd(2+) alone, indicating that substitution of Zn(2+) by Cd(2+) was followed by a second reaction that failed to respond to Zn(2+). Buffers for the DNA binding reaction (electrophoretic mobility shift assay) contain EDTA and Cd-EDTA is active in the same concentration range as Cd(2+). During the standard 15 min incubation, Cd(2+) down-regulates Zn(3)-Sp1 but is inactive against the adduct, Zn(3)-Sp1.GC1. Kinetic studies demonstrated that with 5 muM Cd(2+), Zn(3)-Sp1 was about 75% inactivated in 15 min, whereas, Zn(3)-Sp1.GC1 was slowly dissociated with 50% still remaining after 60 min. In contrast, Zn(3)-Sp1 bound to a cognate consensus site resisted any reaction over 60 min. An adduct of Zn(3)-Sp1.(polydI-dC) was just as reactive with Cd(2+) as Zn(3)-Sp1. Reexamination of the NMR structure of Zn- and Cd-finger peptides related to Sp1 fingers has revealed subtle changes in conformation of the metalbinding site and DNA-binding helix that occur when Cd(2+) is substituted by Zn(2+).