Abstract
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.
Highlights
We showed that the THAP domain of THAP1, the prototype of the THAP family [8], possesses zinc-dependent sequence-specific DNA binding activity and recognizes a consensus DNA target sequence of 11 nucleotides (THABS, for the THAP1 binding sequence) [7], considerably larger than the 3– 4 nucleotides motif typically recognized by classical C2H2 zinc fingers [2, 7]
We report here the three-dimensional structure and structure-function analysis of the sequence-specific DNA-binding THAP zinc finger of human THAP1, the prototype of a novel family of cellular factors involved in pRb/E2F cell cycle pathways
We recently demonstrated that THAP1 is a physiological regulator of cell proliferation
Summary
Plasmid Constructions—The THAP domain of human THAP1 (Met1–Phe or Met1–Lys; GenBankTM NP_060575) was amplified by PCR and cloned in-frame with a C-terminal His tag into a modified pET-26 plasmid (Novagen). For gel-shift assays, the recombinant THAP domain of human THAP1 was produced as previously described [7]. Binding analyses were performed with multiple injections of THAP domain (Met1–Lys90) at different protein concentrations over the immobilized surfaces at 25 °C. A DNA fragment with an unrelated sequence was reconstituted by hybridizing the oligonucleotides, 5Ј-GATTTGCATTTTAA-3Ј and 5Ј-TTAAAATGCAAATC-3Ј, and added to the THAP domain following the same procedure as described above. Electrophoretic Mobility Shift Assays (EMSA)—EMSA were performed with purified recombinant THAP domains produced in E. coli or with full-length THAP1 wild type or THAP1 mutants synthesized in vitro in RRL, using the following THABS probes, 25-bp (5Ј-AGCAAGTAAGGGCAAACTACTTCAT-3Ј) and 36-bp (5Ј-TATCAACTGTGGGCAAACTACGGGCAACAGGTAATG-3Ј), as previously described [7]. The final ensemble of 200 solutions was analyzed and clustered based on a pair-wise r.m.s.d. matrix calculated over the backbone atoms
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