Solution structure of the nonmethyl-CpG-binding CXXC domain of the leukaemia-associated MLL histone methyltransferase

Methylation of CpG dinucleotides is the major epigenetic modification of mammalian genomes, critical for regulating chromatin structure and gene activity. The mixed-lineage leukaemia (MLL) CXXC domain selectively binds nonmethyl-CpG DNA, and is required for transformation by MLL fusion proteins that commonly arise from recurrent chromosomal translocations in infant and secondary treatment-related acute leukaemias. To elucidate the molecular basis of nonmethyl-CpG DNA recognition, we determined the structure of the human MLL CXXC domain by multidimensional NMR spectroscopy. The CXXC domain has a novel fold in which two zinc ions are each coordinated tetrahedrally by four conserved cysteine ligands provided by two CGXCXXC motifs and two distal cysteine residues. We have identified the CXXC domain DNA binding interface by means of chemical shift perturbation analysis, cross-saturation transfer and site-directed mutagenesis. In particular, we have shown that residues in an extended surface loop are in close contact with the DNA. These data provide a template for the design of specifically targeted therapeutics for poor prognosis MLL-associated leukaemias.     

ZZ domain of dystrophin and utrophin: topology and mapping of a beta-dystroglycan interaction site

Dystrophin forms part of a vital link between actin cytoskeleton and extracellular matrix via the transmembrane adhesion receptor dystroglycan. Dystrophin and its autosomal homologue utrophin interact with beta-dystroglycan via their highly conserved C-terminal cysteine-rich regions, comprising the WW domain (protein-protein interaction domain containing two conserved tryptophan residues), EF hand and ZZ domains. The EF hand region stabilizes the WW domain providing the main interaction site between dystrophin or utrophin and dystroglycan. The ZZ domain, containing a predicted zinc finger motif, stabilizes the WW and EF hand domains and strengthens the overall interaction between dystrophin or utrophin and beta-dystroglycan. Using bacterially expressed ZZ domain, we demonstrate a conformational effect of zinc binding to the ZZ domain, and identify two zinc-binding regions within the ZZ domain by SPOTs overlay assays. Epitope mapping of the dystrophin ZZ domain was carried out with new monoclonal antibodies by ELISA, overlay assay and immunohistochemistry. One monoclonal antibody defined a discrete region of the ZZ domain that interacts with beta-dystroglycan. The epitope was localized to the conformationally sensitive second zinc-binding site in the ZZ domain. Our results suggest that residues 3326-3332 of dystrophin form a crucial part of the contact region between dystrophin and beta-dystroglycan and provide new insight into ZZ domain organization and function.     

The WW domain of dystrophin requires EF-hands region to interact with beta-dystroglycan.

Skeletal muscle dystrophin is a 427 kDa protein thought to act as a link between the actin cytoskeleton and the extracellular matrix. Perturbations of the dystrophin-associated complex, for example, between dystrophin and the transmembrane glycoprotein beta-dystroglycan, may lead to muscular dystrophy. Previously, the cysteine-rich region and first half of the carboxy-terminal domain of dystrophin were shown to interact with beta-dystroglycan through a stretch of fifteen amino acids at the carboxy-terminus of beta-dystroglycan. This region of dystrophin implicated in binding beta-dystroglycan contains four modular protein domains: a WW domain, two putative Ca2+-binding EF-hand motifs, and a putative zinc finger ZZ domain. The WW domain is a globular domain of 38-40 amino acids with two highly conserved tryptophan residues spaced 20-22 amino acids apart. A subset of WW domains was shown to bind ligands that contain a Pro-Pro-x-Tyr core motif (where x is any amino acid). Here we elucidate the role of the WW domain of dystrophin and surrounding sequence in binding beta-dystroglycan. We show that the WW domain of dystrophin along with the EF-hand motifs binds to the carboxy-terminus of beta-dystroglycan. Through site-specific mutagenesis and in vitro binding assays, we demonstrate that binding of dystrophin to the carboxy-terminus of beta-dystroglycan occurs via a beta-dystroglycan Pro-Pro-x-Tyr core motif. Targeted mutagenesis of conserved WW domain residues reveals that the dystrophin/beta-dystroglycan interaction occurs primarily through the WW domain of dystrophin. Precise mapping of this interaction could aid in therapeutic design.     

Solution structure of the cysteine-rich domain of the Escherichia coli chaperone protein DnaJ

The solution structure of the cysteine-rich (CR) domain of Escherichia coli DnaJ has been solved by NMR methods. The structure of a 79 residue CR domain construct shows a novel fold with an overall V-shaped extended beta-hairpin topology. The CR domain is characterized by four C-X-X-C-X-G-X-G sequence motifs that bind two zinc ions. Residues in these two zinc modules show strong similarities in the grouping of resonances in the (15)N-(1)H HSQC spectrum and display pseudo-symmetry of the motifs in the calculated structures. The conformation of the cysteine residues coordinated to the zinc ion resembles that of the rubredoxin-knuckle, but there are significant differences in hydrogen bonding patterns in the two motifs. Zinc (15)N-(1)H HSQC titrations indicate that the fold of the isolated DnaJ CR domain is zinc-dependent and that one zinc module folds before the other. The C-X-X-C-X-G-X-G sequence motif is highly conserved in CR domains from a wide variety of species. The three-dimensional structure of the E. coli CR domain indicates that this sequence conservation is likely to result in a conserved structural motif.     

Solution structure of the zinc finger HIT domain in protein FON

The zinc finger HIT domain is a sequence motif found in many proteins, including thyroid hormone receptor interacting protein 3 (TRIP-3), which is possibly involved in maturity-onset diabetes of the young (MODY). Novel zinc finger motifs are suggested to play important roles in gene regulation and chromatin remodeling. Here, we determined the high-resolution solution structure of the zinc finger HIT domain in ZNHIT2 (protein FON) from Homo sapiens, by an NMR method based on 567 upper distance limits derived from NOE intensities measured in three-dimensional NOESY spectra. The structure yielded a backbone RMSD to the mean coordinates of 0.19 A for the structured residues 12-48. The fold consists of two consecutive antiparallel beta-sheets and two short C-terminal helices packed against the second beta-sheet, and binds two zinc ions. Both zinc ions are coordinated tetrahedrally via a CCCC-CCHC motif to the ligand residues of the zf-HIT domain in an interleaved manner. The tertiary structure of the zinc finger HIT domain closely resembles the folds of the B-box, RING finger, and PHD domains with a cross-brace zinc coordination mode, but is distinct from them. The unique three-dimensional structure of the zinc finger HIT domain revealed a novel zinc-binding fold, as a new member of the treble clef domain family. On the basis of the structural data, we discuss the possible functional roles of the zinc finger HIT domain.     

Structure and ubiquitin interactions of the conserved zinc finger domain of Npl4

Ubiquitylated proteins are directed into a large number of different cellular pathways through interactions with effector proteins that contain conserved ubiquitin binding motifs. Here, we report the solution structure and ubiquitin binding properties of one such motif, the Npl4 zinc finger or RanBP2/Nup358 zinc finger (NZF) domain. Npl4 NZF forms a compact module composed of four antiparallel beta-strands linked by three ordered loops. A single zinc ion is coordinated by four conserved cysteines from the first and third loops, which form two rubredoxin knuckles. Npl4 NZF binds specifically, but weakly, to free ubiquitin using a conserved 13TF14 dipeptide to interact with the "Ile-44" surface of ubiquitin. Our studies reveal the structure of this versatile class of protein binding domains and provide a means for identifying the subset of NZF domains likely to bind ubiquitin.     

Mutation of the CH1 Domain in the Histone Acetyltransferase CREBBP Results in Autism-Relevant Behaviors in Mice

Autism spectrum disorders (ASDs) are a group of neurodevelopmental afflictions characterized by repetitive behaviors, deficits in social interaction, and impaired communication skills. For most ASD patients, the underlying causes are unknown. Genetic mutations have been identified in about 25 percent of ASD cases, including mutations in epigenetic regulators, suggesting that dysregulated chromatin or DNA function is a critical component of ASD. Mutations in the histone acetyltransferase CREB binding protein (CBP, CREBBP) cause Rubinstein-Taybi Syndrome (RTS), a developmental disorder that includes ASD-like symptoms. Recently, genomic studies involving large numbers of ASD patient families have theoretically modeled CBP and its paralog p300 (EP300) as critical hubs in ASD-associated protein and gene interaction networks, and have identified de novo missense mutations in highly conserved residues of the CBP acetyltransferase and CH1 domains. Here we provide animal model evidence that supports this notion that CBP and its CH1 domain are relevant to autism. We show that mice with a deletion mutation in the CBP CH1 (TAZ1) domain (CBP^ΔCH1/ΔCH1) have an RTS-like phenotype that includes ASD-relevant repetitive behaviors, hyperactivity, social interaction deficits, motor dysfunction, impaired recognition memory, and abnormal synaptic plasticity. Our results therefore indicate that loss of CBP CH1 domain function contributes to RTS, and possibly ASD, and that this domain plays an essential role in normal motor function, cognition and social behavior. Although the key physiological functions affected by ASD-associated mutation of epigenetic regulators have been enigmatic, our findings are consistent with theoretical models involving CBP and p300 in ASD, and with a causative role for recently described ASD-associated CBP mutations.     

Crystal structure of a novel zinc-binding ATP sulfurylase from Thermus thermophilus HB8

ATP sulfurylase (ATPS) is a ubiquitous enzyme that catalyzes the transfer of the adenylyl group from ATP to inorganic sulfate, producing adenosine 5'-phosphosulfate (APS) and pyrophosphate. The crystal structure of ATPS from Thermus thermophilus HB8 (TtATPS, 347 amino acid residues) in complex with APS was determined at 2.5 A resolution. TtATPS is composed of three domains [domain I (residues 1-134), domain II (residues 135-290), and domain III (residues 291-347)], like the Riftia pachyptila symbiont ATPS, but lacks a fourth domain present in ATPSs from the yeast Saccharomyces cerevisiae and from the fungus Penicillium chrysogenum. TtATPS forms a dimer in the crystal, and the manner of subunit association is different from that observed in dimeric R. pachyptila symbiont ATPS and in the hexameric S. cerevisiae and P. chrysogenum ATPSs. APS is located in the active site of TtATPS, which contains several motifs (QXRN, HXXH, and GRD) conserved in ATPSs. Unexpectedly, TtATPS binds one metal ion per subunit in domain III. XAFS measurement of the crystal and the Bijvoet difference Fourier map unambiguously characterized the metal ion as a zinc ion. The zinc ion is tetrahedrally coordinated by Cys294, Cys297, Cys306, and His310, and could not be removed from the protein by treatment with EDTA. The zinc ion binding site is far from the active site. Because all four residues coordinated to the zinc ion are conserved in the ATPSs from thermophilic bacteria such as Archaeoglobus fulgidus, Pyrococcus abyssi, and Sulfolobus solfataricus, zinc ion chelation may contribute to the thermal stability of these ATPSs.     

The High-Risk HPV16 E7 Oncoprotein Mediates Interaction between the Transcriptional Coactivator CBP and the Retinoblastoma Protein pRb

The oncoprotein E7 from human papillomavirus (HPV) strains that confer high cancer risk mediates cell transformation by deregulating host cellular processes and activating viral gene expression through recruitment of cellular proteins such as the retinoblastoma protein (pRb) and the cyclic-AMP response element binding binding protein (CBP) and its paralog p300. Here we show that the intrinsically disordered N-terminal region of E7 from high-risk HPV16 binds the TAZ2 domain of CBP with greater affinity than E7 from low-risk HPV6b. HPV E7 and the tumor suppressor p53 compete for binding to TAZ2. The TAZ2 binding site in E7 overlaps the LxCxE motif that is crucial for interaction with pRb. While TAZ2 and pRb compete for binding to a monomeric E7 polypeptide, the full-length E7 dimer mediates an interaction between TAZ2 and pRb by promoting formation of a ternary complex. Cell-based assays show that expression of full-length HPV16 E7 promotes increased pRb acetylation and that this response depends both on the presence of CBP/p300 and on the ability of E7 to form a dimer. These observations suggest a model for the oncogenic effect of high-risk HPV16 E7. The disordered region of one E7 molecule in the homodimer interacts with the pocket domain of pRb, while the same region of the other E7 molecule binds the TAZ2 domain of CBP/p300. Through its ability to dimerize, E7 recruits CBP/p300 and pRb into a ternary complex, bringing the histone acetyltransferase domain of CBP/p300 into proximity to pRb and promoting acetylation, leading to disruption of cell cycle control.