Specific DNA binding to a major histocompatibility complex enhancer sequence by a synthetic 57-residue double zinc finger peptide from a human enhancer binding protein.
Two 57-residue peptides containing one pair of "zinc fingers" from a human enhancer binding protein were prepared by solid-phase peptide synthesis. One peptide (MBP-DF) contained the native sequence, while the second peptide ([Abu11]MBP-DF) has an alpha-aminobutyric acid residue substituted for a nonconserved cysteine residue at position 11. The peptides were characterized by several chemical and physical methods, and their DNA binding properties were evaluated using gel retardation experiments. Spectroscopic studies demonstrated that addition of metal ions such as zinc and cobalt resulted in specific conformational changes in both peptides, indicating that cysteine-11 does not appear to be involved in metal chelation. One-dimensional 1H NMR studies indicate that a stable folded structure is formed upon addition of zinc, and the chemical shift pattern is consistent with that previously observed for one constituent single finger (Omichinski, J., Clore, G. M., Appella, E., Sakaguchi, K., and Gronenborn, A. M. (1990) Biochemistry 29, 9324-9334). Gel retardation experiments demonstrate that the peptides are capable of interacting with a 15-mer oligonucleotide comprising a portion of the major histocompatibility complex enhancer sequence and that the interaction is zinc-dependent. The dissociation constant for the [Abu11]MBP-DF peptide is 1.4 x 10(-7) M with maximal binding occurring at a zinc-to-peptide ratio of 2 to 1. The binding specificity observed with respect to related enhancer sequences exhibits the same relative order as noted previously for the whole protein. Studies with point mutants of the major histocompatibility complex enhancer binding sequence indicate that the last GC base pair in a four-guanine stretch plays a pivotal role in the interaction between the peptide and DNA.
Sakaguchi
K
K
Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Appella
E
E
Omichinski
J G
JG
Clore
G M
GM
Gronenborn
A M
AM
D000595:Amino Acid Sequence; D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008285:Major Histocompatibility Complex; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry; D011487:Protein Conformation; D014157:Transcription Factors / Q000378:metabolism*; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D015345:Oligonucleotide Probes; D010455:Peptides; D014157:Transcription Factors