Structure-function analysis of the THAP zinc finger of THAP1, a large C2CH DNA-binding module linked to Rb/E2F pathways.
THAP1, the founding member of a previously uncharacterized large family of cellular proteins (THAP proteins), is a sequence-specific DNA-binding factor that has recently been shown to regulate cell proliferation through modulation of pRb/E2F cell cycle target genes. THAP1 shares its DNA-binding THAP zinc finger domain with Drosophila P element transposase, zebrafish E2F6, and several nematode proteins interacting genetically with the retinoblastoma protein pRb. In this study, we report the three-dimensional structure and structure-function relationships of the THAP zinc finger of human THAP1. Deletion mutagenesis and multidimensional NMR spectroscopy revealed that the THAP domain of THAP1 is an atypical zinc finger of approximately 80 residues, distinguished by the presence between the C2CH zinc coordinating residues of a short antiparallel beta-sheet interspersed by a long loop-helix-loop insertion. Alanine scanning mutagenesis of this loop-helix-loop motif resulted in the identification of a number of critical residues for DNA recognition. NMR chemical shift perturbation analysis was used to further characterize the residues involved in DNA binding. The combination of the mutagenesis and NMR data allowed the mapping of the DNA binding interface of the THAP zinc finger to a highly positively charged area harboring multiple lysine and arginine residues. Together, these data represent the first structure-function analysis of a functional THAP domain, with demonstrated sequence-specific DNA binding activity. They also provide a structural framework for understanding DNA recognition by this atypical zinc finger, which defines a novel family of cellular factors linked to cell proliferation and pRb/E2F cell cycle pathways in humans, fish, and nematodes.
Bessière
Damien
D
University of Toulouse, Institute of Pharmacology and Structural Biology, France.
Lacroix
Chrystelle
C
Campagne
Sébastien
S
Ecochard
Vincent
V
Guillet
Valérie
V
Mourey
Lionel
L
Lopez
Frédéric
F
Czaplicki
Jerzy
J
Demange
Pascal
P
Milon
Alain
A
Girard
Jean-Philippe
JP
Gervais
Virginie
V
D000595:Amino Acid Sequence; D051017:Apoptosis Regulatory Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D001483:Base Sequence; D015342:DNA Probes; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D050684:E2F Transcription Factors / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D016160:Retinoblastoma Protein / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D016335:Zinc Fingers
D051017:Apoptosis Regulatory Proteins; D015342:DNA Probes; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D009687:Nuclear Proteins; D016160:Retinoblastoma Protein; C474080:THAP1 protein, human