Metal-binding properties of the zinc fingers in metal responsive element-binding transcription factor-1 and design of single-stranded nucleic acid binding proteins using nucleocapsid-like CCHC zinc-binding domains.

摘要

The zinc-induced DNA binding activation of MTF1 was estimated to be half-maximal at a zinc concentration of 2–3 μM. We have measured the metal affinity of each of the six zinc fingers of murine MTF1 in isolation as well as in the context of a fragment of mMTF1 consisting of the six zinc fingers, mMTF1-ZF, and have found all dissociation constants for Co 2+ to be in the micromolar range. We have found the order of binding from tightest to weakest for the individual zinc fingers to be: finger 2 > 4 > 3 > 1 > 5 ≈ 6. In the context of mMTF1-ZF, all dissociation constants remain in the sub-micromolar range, and the order of binding from tightest to weakest is: finger 4 > 2 > 5 > 3 > 6 > 1. C₂H₂ zinc fingers have dissociation constants for Zn2+ that are 10 −3–10−4 times those for Co 2+. Therefore, given the affinities measured, the zinc fingers of mMTF1-ZF would all be fully bound at Zn2+ concentrations much less than the apparent 2–3 μM seen for half maximal DNA binding. These data suggest that the tetrahedral coordination of Zn2+ by the zinc fingers of mMTF1 is not responsible for this protein's ability to sense zinc levels.; We have used NC-like CCHC zinc-binding motifs as modules to design single-stranded nucleic acid binding proteins. The affinity for guanine-containing single-stranded oligonucleotides increases with the number of CCHC domains. We show the amino acid linker between domains determines the spacing of guanines recognized. We demonstrate that two CCHC domains, separated by a linker from a protein which recognizes the sequence GXXG, can discriminate between GXXG and guanines spaced one nucleotide closer or farther. By switching the linker region to that from a protein which recognizes GXG, we can change the specificity to GXG. These data provide a framework for using NC-like CCHC zinc fingers to design single-stranded nucleic acid binding proteins.