Journal of Molecular Biology
ZZ Domain of CBP: an Unusual Zinc Finger Fold in a Protein Interaction Module
Introduction
Transcriptional activation in eukaryotes involves interactions between DNA-bound activators, co-activators and components of the basal transcription complex. One of the most extensively studied transcriptional coactivators is CBP (CREB-binding protein)1 and its paralog p300,2 which function as acetyltransferases and mediate interactions between a number of transcription factors that bind specific DNA enhancer elements and the general RNA polymerase II complex that binds the promoter (reviewed by Goodman and Smolik3). CBP is a large (2441 residue) multidomain protein that contains three regions that bind zinc ions, utilizing the side-chains of conserved cysteine and histidine residues. These three regions were named CH1, CH2 and CH3, on the basis of their content of Cys and His residues.2 An alternative nomenclature identifies the CH1 domain with the TAZ1 domain and the CH2 region with a PHD zinc finger motif.4 The CH3 region (known also as the E1A-binding domain2) contains two separate zinc-binding motifs, termed ZZ and TAZ2.4 The structures of the homologous TAZ1 and TAZ2 domains in solution have been elucidated recently, both free and in complex with physiological partners.5, 6, 7, 8, 9 The structure of the ZZ motif, located between the histone acetyl transferase (HAT) domain and TAZ2, is unknown.
The ZZ domain is a small ∼45 amino acid residue motif that was identified initially in dystrophin.4 The ZZ domain is found in eukaryotic proteins of widely varying function and domain composition (Figure 1). Many of these proteins contain additional zinc-binding motifs, such as TAZ, PHD, RING, SWIM, and C2H2, and the majority serve as scaffolds in pathways involving acetyltransferase, protein kinase or ubiquitin-related activity. ZZ proteins can be grouped into four main functional classes: (1) chromatin modifying; (2) cytoskeletal scaffolding; (3) ubiquitin binding or conjugating; and (4) membrane receptor or ion channel modifying proteins. CBP/p300 and Ada2p/RSC810, 11 are chromatin remodeling proteins that function in transcriptional activation via acetylation of histone or protein targets. Unlike CBP/p300, Ada2p does not contain an acetyltransferase domain. It is, however, an integral component of the yeast SAGA and SLIK acetyltransferase complexes, the latter of which has recently been shown to exhibit histone deubiquitinase activity.12 Cytoskeletal proteins of the dystrophin family,13 including utrophin, α and β-dystrobrevin, and dystrophin-related protein 2 (DRP2), contain a highly conserved ZZ motif. These proteins maintain cellular integrity during muscle contraction by connecting the actin cytoskeleton to membrane glycoproteins. The scaffold proteins p62/ZIP/SQSTM114 recruit atypical protein kinases to membrane receptor signaling complexes and ion channels. These proteins contain a C-terminal UBA (ubiquitin-associated) domain and at least one (p62) has been shown to bind poly-ubiquitin. Mindbomb,15 HERC216 and PRT117 are E3 ubiquitin ligases. These and related proteins contain RING or HECT domains in addition to the ZZ motif. Other poorly characterized ZZ domain proteins include a potassium channel modulatory factor,18 vascular endothelial receptor related protein 2 (VER2),19 which is a putative protein kinase, and several predicted proteins with multiple ZZ domains.
We have prepared recombinant protein corresponding to the murine CBP ZZ-domain and determined its solution structure by NMR. The domain contains both β-sheet and helix, and binds two mole equivalents of zinc with Cys4 and Cys2His2 coordination in a cross-brace zinc finger architecture. The two histidine residues form an unusual HXH motif, in which the first histidine residue coordinates the zinc atom through the Nε2, and the second histidine residue coordinates through the Nδ1 atom. In addition, we show that the two components of the CH3 domain, the ZZ motif (this work) and the TAZ2 motif,5 are folded independently and that the site for the interaction of CBP binding partners p53 and E1A resides exclusively in the TAZ2 domain and not in ZZ.
Section snippets
Expression of the murine CBP ZZ domain
The domain boundaries of the CBP ZZ domain are not readily identifiable from amino acid sequence alignments alone. Several constructs were therefore investigated, containing 67 (1685–1751), 57 (1695–1751) and 52 (1700–1751) residues. The 67 and 57 residue constructs were expressed only at low levels in Escherichia coli, but the 52 residue construct, corresponding to the sequence shown in the first line of Figure 1, was expressed at high levels, with about 60% of the protein in the soluble
Structure of the ZZ domain
A search of the DALI database failed to find a match to a known structure.26 However, visual inspection of the ZZ domain structure shows that it belongs to the family of “cross-brace” zinc finger proteins, with interleaved zinc binding sites. This family includes the RING,27 PHD28, 29 and FYVE domain30 zinc fingers. In these domains, as in the ZZ motif, the two Zn atoms are coordinated in a tetrahedral geometry by cysteine and histidine ligands, and are located at opposite ends of a small
Preparation of the ZZ domain
Constructs of the ZZ domain were designed on the basis of sequence conservation and stability of the recombinant domain. The ZZ domain (residues 1700–1751 of mouse CBP) was cloned into pET21a and overexpressed in Escherichia coli BL21(DE3) [DNAY] in M9 minimal medium supplemented with 150 μM zinc sulfate. Binding of the two zinc atoms was confirmed by electrospray mass spectrometry. Both 15N and 15N/13C isotopically labeled proteins were produced upon low-temperature induction in minimal media,
Acknowledgements
We thank Linda Tennant, Cheyenne Reyes, and Ted Foss for technical assistance, and John Chung and Gerard Kroon for assistance with NMR experiments. This work was supported by grant CA96865 from the National Institutes of Health and by the Skaggs Institute for Chemical Biology. We thank the anonymous referees who pointed out the structural similarity to the RING and PHD motifs.
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Present addresses: G. B. Legge, Department of Biology and Biochemistry, 353 SR2 Houston, TX 77204-5001, USA; D. M. Hambly, Division of Biological and Biomedical Sciences, Department of Chemistry, Washington University Medical School, Box 1134, 1 Brookings Drive, St. Louis, MO 63130, USA.