Alternating zinc finger motifs in the male-associated protein ZFY: defining architectural rules by mutagenesis and design of an "aromatic swap" second-site revertant.
We describe spectroscopic and biochemical studies of native and mutant Zn finger peptides from ZFY, a putative transcription factor encoded by the sex-determining region of the human Y chromosome. The parent peptide, based on ZFY domain 6, exhibits metal-dependent helix formation within a rigid tertiary framework. Nonaromatic substitutions of the consensus aromatic group (Tyr 10----Ser or Lys) are surprisingly compatible with native architecture but result in loss of stability to pH or guanidine denaturation. Remarkably, these perturbations are reverted by a second-site mutation in which an alternative aromatic residue is introduced (Ser 12----Phe). Design of the second-site revertant ("aromatic swap") is based on the ZFY two-finger repeat, a conserved symmetry among the ZFY-related zinc finger proteins, and is in accord with recent 2D NMR structures of Zn finger peptides. These experiments suggest general rules for metal-dependent folding of the Zn finger motif.
Weiss
M A
MA
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Keutmann
H T
HT
D000595:Amino Acid Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D051741:Kruppel-Like Transcription Factors; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis; D011487:Protein Conformation; D015202:Protein Engineering; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics; D016335:Zinc Fingers / Q000235:genetics*
D004268:DNA-Binding Proteins; D051741:Kruppel-Like Transcription Factors; D010446:Peptide Fragments; D014157:Transcription Factors; C054430:ZFY protein, human