NMR Structure of Transcription Factor Sp1 DNA Binding Domain^,

Abstract

To understand the DNA recognition mechanism of zinc finger motifs of transcription factor Sp1, we have determined the solution structure of DNA-binding domain of the Sp1 by solution NMR techniques. The DNA-binding domain of Sp1 consists of three Cys(2)His(2)-type zinc finger motifs. They have typical betabetaalpha zinc finger folds and relatively random orientations. From DNA-binding analysis performed by NMR and comparison between structures determined here and previously reported structures of other zinc fingers, it was assumed that DNA recognition modes of fingers 2 and 3 would be similar to those of fingers of Zif268, in which each finger recognizes four base pairs strictly by using residues at positions -1, 2, 3, and 6 of the recognition helix. On the contrary, finger 1 can use only two residues for DNA recognition, Lys550 and His553 at positions -1 and 3 of the helix, and has more relaxed sequence and site specificity than other Cys(2)His(2) zinc fingers. It is proposed that this relaxed property of finger 1 allows transcription factor Sp1 to bind various DNA sequences with high affinity.

Balancing between affinity and speed in target DNA search by zinc-finger proteins via modulation of dynamic conformational ensemble

“…S7. The human Sp1 ZF-DBD (Sp1 residue 616-709) comprising three ZFs was expressed in E. coli strain BL21(DE3) via a pET-49b-derivative plasmid and chromatographically purified using cation-exchange and size-exclusion chromatography as described (57 N-labeled Egr-1 ZF-DBDs with 28-bp DNA duplexes were prepared as described previously (28). For each sample of the complexes, the molar ratio of protein to DNA was 1:2 to ensure the DNA-bound states of the isotope-labeled proteins.…”

The mammalian zinc finger transcription factor Krüppel‐like factor 3 (KLF3/BKLF)

“…Sumoylation of KLF3 at K10 and K197 is required for full repressor activity. analysis of SP1 (22), it is known that C2H2 zinc fingers consist of two antiparallel b-sheets and an a-helix, with these structures being co-ordinated and stabilized by the zinc ion. The a-helix of each finger makes contact with the major groove of DNA, with specific residues binding to guanine-rich three base pair sites (21).…”

“…This finger is 23 amino acids long and residues K273, H276, and A279 of human KLF3 are predicted to make primary contact with DNA, having a preference for the sequence NGA/G. Three-dimensional NMR analysis of SP1 (22) and mutagenesis studies have indicated that finger 1 may have reduced DNA contact and contribute least to binding affinity and specificity (23).…”

Zinc binding ligands and cellular zinc trafficking: Apo-metallothionein, glutathione, TPEN, proteomic zinc, and Zn-Sp1

“…The EMSA technique measures the capacity of control and treated Sp1 to bind to a cognate DNA binding site, in this case one of the Sp1 recognition sequences (GC-1) in the sodium glucose co-transporter 1 gene of mouse kidney [25]. Zn-finger mini-domains depend upon structural Zn 2+ to establish their correct DNA-binding conformation [4,34]. Thus, the loss of DNA binding activity resulting from reaction with competing Zn 2+ binding ligands can be attributed to peptide unfolding in the absence of Zn 2+ .…”

“…Besides its role as a component of enzyme active sites it also provides critical structural stabilization to a multitude of proteins, including a multitude of zinc-finger transcription factors [3,4]. Despite its familiar role in protein structure and function, relatively little is known about Zn 2+ trafficking that brings Zn 2+ to such sites.…”