Two new NMR structures of WW domains, the mouse formin binding protein and a putative 84.5 kDa protein from Saccharomyces cerevisiae, show that this domain, only 35 amino acids in length, defines the smallest monomeric triple-stranded antiparallel beta-sheet protein domain that is stable in the absence of disulfide bonds, tightly bound ions or ligands. The structural roles of conserved residues have been studied using site-directed mutagenesis of both wild type domains. Crucial interactions responsible for the stability of the WW structure have been identified. Based on a network of highly conserved long range interactions across the beta-sheet structure that supports the WW fold and on a systematic analysis of conserved residues in the WW family, we have designed a folded prototype WW sequence.

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 structurefunction 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 ϳ80 residues, distinguished by the presence between the C2CH zinc coordinating residues of a short antiparallel ␤-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.Zinc finger proteins represent the most abundant class of DNA-binding proteins in the human genome. Zinc fingers have been defined as small, functional, independently folded domains that require coordination of a zinc atom to stabilize their structure (1). The zinc finger superfamily includes the C2H2-type zinc finger, a compact ϳ30-amino acid DNA-binding module repeated in multiple copies in the protein structure (2, 3), the C4-type zinc finger found in the GATA family of transcription factors (4), and the zinc-coordinating DNA-binding domain of nuclear hormone receptors (5). We recently described an atypical zinc finger motif, characterized by a large C2CH module (Cys-X 2-4 -Cys-X 35-53 -Cys-X 2 -His) with a spacing of up to 53 amino acids between the zinc-coordinating C2 and CH residues (6). This motif, designated THAP domain or THAP zinc finger, defines a previously uncharacterized large family of cellular factors with more than 100 distinct members in the animal kingdom (6, 7). 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 typ...