Zinc metalloregulation of the zinc finger pair domain.
The yeast transcriptional activator Zap1 contains two uncommon structural motifs designated zinc finger pair domains. The hallmark of this domain is the packing of two zinc finger motifs in one globular unit. One finger pair domain in Zap1 contains the AD2 transactivation domain. Zn(II) binding to this domain (ZF1/2) is kinetically labile yielding a zinc-regulated transactivator. The second finger pair domain (ZF3/4) lies within the DNA-binding domain, and it stably binds Zn(II). The goal of this study was to map the determinant conferring lability in Zn(II) binding by using finger pair chimeras. Whereas ZF2 contains the transactivation function, zinc regulation is dependent on the presence of ZF1. ZF3 can functionally replace ZF1, and a ZF3/2 finger pair retains limited zinc regulation. Replacement of ZF3 by ZF1 creating a ZF1/4 chimera was found to stably bind Zn(II), suggesting that the presence of a stable motif (ZF4) can impart binding stability on a labile motif (ZF1). Zn(II) binding in finger pair domains is dependent on the presence of both motifs. Mutations in one finger motif markedly attenuate Zn(II) binding to the second motif. Kinetic lability in Zn(II) binding was mapped to the alpha-helix of ZF2. A ZF1/ZFbeta2alpha4 chimera resembles ZF3/4 in Zn(II) binding stability in incubation studies with the Zn(II) chelators. The present results demonstrate that zinc regulation of AD activity of ZF2 is dependent on determinants in ZF1 as well as the alpha-helix segment of ZF2.
Bird
Amanda J
AJ
University of Utah Health Sciences Center, Department of Medicine and Biochemistry, Salt Lake City, Utah 84132, USA.
Swierczek
Sabina
S
Qiao
Wei
W
Eide
David J
DJ
Winge
Dennis R
DR
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D004247:DNA / Q000737:chemistry; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism; D015533:Transcriptional Activation; D015032:Zinc / Q000737:chemistry*; D016335:Zinc Fingers
D011993:Recombinant Fusion Proteins; D004247:DNA; D015719:Single-Strand Specific DNA and RNA Endonucleases; D015032:Zinc