Structural basis of ubiquitin recognition by the winged-helix domain of Cockayne syndrome group B protein.

Cockayne syndrome group B (CSB, also known as ERCC6) protein is involved in many DNA repair processes and essential for transcription-coupled repair (TCR). The central region of CSB has the helicase motif, whereas the C-terminal region contains important regulatory elements for repair of UV- and oxidative stress-induced damages and double-strand breaks (DSBs). A previous study suggested that a small part (∼30 residues) within this region was responsible for binding to ubiquitin (Ub). Here, we show that the Ub-binding of CSB requires a larger part of CSB, which was previously identified as a winged-helix domain (WHD) and is involved in the recruitment of CSB to DSBs. We also present the crystal structure of CSB WHD in complex with Ub. CSB WHD folds as a single globular domain, defining a class of Ub-binding domains (UBDs) different from 23 UBD classes identified so far. The second α-helix and C-terminal extremity of CSB WHD interact with Ub. Together with structure-guided mutational analysis, we identified the residues critical for the binding to Ub. CSB mutants defective in the Ub binding reduced repair of UV-induced damage. This study supports the notion that DSB repair and TCR may be associated with the Ub-binding of CSB.

Structural and functional studies of Stf76 from the Sulfolobus islandicus plasmid-virus pSSVx: a novel peculiar member of the winged helix-turn-helix transcription factor family.

The hybrid plasmid-virus pSSVx from Sulfolobus islandicus presents an open reading frame encoding a 76 amino acid protein, namely Stf76, that does not show significant sequence homology with any protein with known 3D structure. The recombinant protein recognizes specifically two DNA-binding sites located in its own promoter, thus suggesting an auto-regulated role of its expression. Circular dichroism, spectrofluorimetric, light scattering and isothermal titration calorimetry experiments indicated a 2:1 molar ratio (protein:DNA) upon binding to the DNA target containing a single site. Furthermore, the solution structure of Stf76, determined by nuclear magnetic resonance (NMR) using chemical shift Rosetta software, has shown that the protein assumes a winged helix-turn-helix fold. NMR chemical shift perturbation analysis has been performed for the identification of the residues responsible for DNA interaction. In addition, a model of the Stf76-DNA complex has been built using as template a structurally related homolog.

The C-terminal domain of the transcriptional regulator BldD from Streptomyces coelicolor A3(2) constitutes a novel fold of winged-helix domains.

BldD regulates transcription of key developmental genes in Streptomyces coelicolor. While the N-terminal domain is responsible for both dimerization and DNA binding, the structural and functional roles of the C-terminal domain (CTD) remain largely unexplored. Here, the solution structure of the BldD-CTD shows a novel winged-helix domain fold not compatible with DNA binding, due to the negatively charged surface and presence of an additional helix. Meanwhile, a small elongated groove with conserved hydrophobic patches surrounded by charged residues suggests that the BldD-CTD could be involved in protein-protein interactions that provide transcriptional regulation.

The wing of a winged helix-turn-helix transcription factor organizes the active site of BirA, a bifunctional repressor/ligase.

The BirA biotin protein ligase of Escherichia coli belongs to the winged helix-turn-helix (wHTH) family of transcriptional regulators. The N-terminal BirA domain is required for both transcriptional regulation of biotin synthesis and biotin protein ligase activity. We addressed the structural and functional role of the wing of the wHTH motif in both BirA functions. A panel of N-terminal deletion mutant proteins including a discrete deletion of the wing motif were unable to bind DNA. However, all the N-terminal deletion mutants weakly complemented growth of a ΔbirA strain at low biotin concentrations, indicating compromised ligase activity. A wing domain chimera was constructed by replacing the BirA wing with the nearly isosteric wing of the E. coli OmpR transcription factor. Although this chimera BirA was defective in operator binding, it was much more efficient in complementation of a ΔbirA strain than was the wing-less protein. The enzymatic activities of the wing deletion and chimera proteins in the in vitro synthesis of biotinoyl-5'-AMP differed greatly. The wing deletion BirA accumulated an off pathway compound, ADP, whereas the chimera protein did not. Finally, we report that a single residue alteration in the wing bypasses the deleterious effects caused by mutations in the biotin-binding loop of the ligase active site. We believe that the role of the wing in the BirA enzymatic reaction is to orient the active site and thereby protect biotinoyl-5'-AMP from attack by solvent. This is the first evidence that the wing domain of a wHTH protein can play an important role in enzymatic activity.

From keys to bulldozers: expanding roles for winged helix domains in nucleic-acid-binding proteins.

The winged helix domain (WHD) is a widespread nucleic-acid-binding protein structural element found in all kingdoms of life. Although the overall structure of the WHD is conserved, its functional properties and interaction profiles are extremely versatile. WHD-containing proteins can exploit nearly the full spectrum of nucleic acid structural features for recognition and even covalent modification or noncovalent rearrangement of target molecules. WHD functions range from sequence-recognizing keys in transcription factors and bulldozer-like strand-separating wedges in helicases to mediators of protein-protein interactions (PPIs). Further investigations are needed to understand the contribution of WHD structural dynamics to nucleic-acid-modifying enzymatic functions.

Structure of a novel winged-helix like domain from human NFRKB protein.

The human nuclear factor related to kappa-B-binding protein (NFRKB) is a 1299-residue protein that is a component of the metazoan INO80 complex involved in chromatin remodeling, transcription regulation, DNA replication and DNA repair. Although full length NFRKB is predicted to be around 65% disordered, comparative sequence analysis identified several potentially structured sections in the N-terminal region of the protein. These regions were targeted for crystallographic studies, and the structure of one of these regions spanning residues 370-495 was determined using the JCSG high-throughput structure determination pipeline. The structure reveals a novel, mostly helical domain reminiscent of the winged-helix fold typically involved in DNA binding. However, further analysis shows that this domain does not bind DNA, suggesting it may belong to a small group of winged-helix domains involved in protein-protein interactions.

Structural and functional characterization of Rpn12 identifies residues required for Rpn10 proteasome incorporation.

The ubiquitin-proteasome system targets selected proteins for degradation by the 26S proteasome. Rpn12 is an essential component of the 19S regulatory particle and plays a role in recruiting the extrinsic ubiquitin receptor Rpn10. In the present paper we report the crystal structure of Rpn12, a proteasomal PCI-domain-containing protein. The structure helps to define a core structural motif for the PCI domain and identifies potential sites through which Rpn12 might form protein-protein interactions. We demonstrate that mutating residues at one of these sites impairs Rpn12 binding to Rpn10 in vitro and reduces Rpn10 incorporation into proteasomes in vivo.

Crystal structure of the N-terminal region of human Ash2L shows a winged-helix motif involved in DNA binding.

Ash2L is a core component of the MLL family histone methyltransferases and has an important role in regulating the methylation of histone H3 on lysine 4. Here, we report the crystal structure of the N-terminal domain of Ash2L and reveal a new function of Ash2L. The structure shows that Ash2L contains an atypical PHD finger that does not have histone tail-binding activity. Unexpectedly, the structure shows a previously unrecognized winged-helix motif that directly binds to DNA. The DNA-binding-deficient mutants of Ash2L reduced Ash2L localization to the HOX locus. Strikingly, a single mutation in Ash2L(WH) (K131A) breaks the chromatin domain boundary, suggesting that Ash2L also has a role in chromosome demarcation.

Solution structure of the human HSPC280 protein.

The human HSPC280 protein belongs to a new family of low molecular weight proteins, which is only present in eukaryotes, and is absent in fungi. The solution structure of HSPC280 was determined using multidimensional NMR spectroscopy. The overall structure consists of three α-helices and four antiparallel ß-strands and has a winged helix-like fold. However, HEPC280 is not a typical DNA-binding winged helix protein in that it lacks DNA-binding activity. Unlike most winged-helix proteins, HSPC280 has an unusually long 13-residue (P62-V74) wing 1 loop connecting the ß3 and ß4 strands of the protein. Molecules of HSPC280 have a positively charged surface on one side and a negatively charged surface on the other side of the protein structure. Comparisons with the C-terminal 80-residue domain of proteins in the Abra family reveal a conserved hydrophobic groove in the HSPC280 family, which may allow HSPC280 to interact with other proteins.

Structure of the LexA-DNA complex and implications for SOS box measurement.

The eubacterial SOS system is a paradigm of cellular DNA damage and repair, and its activation can contribute to antibiotic resistance. Under normal conditions, LexA represses the transcription of many DNA repair proteins by binding to SOS 'boxes' in their operators. Under genotoxic stress, accumulating complexes of RecA, ATP and single-stranded DNA (ssDNA) activate LexA for autocleavage. To address how LexA recognizes its binding sites, we determined three crystal structures of Escherichia coli LexA in complex with SOS boxes. Here we report the structure of these LexA-DNA complexes. The DNA-binding domains of the LexA dimer interact with the DNA in the classical fashion of a winged helix-turn-helix motif. However, the wings of these two DNA-binding domains bind to the same minor groove of the DNA. These wing-wing contacts may explain why the spacing between the two half-sites of E. coli SOS boxes is invariant.

The copper-responsive repressor CopR of Lactococcus lactis is a 'winged helix' protein.

CopR of Lactococcus lactis is a copper-responsive repressor involved in copper homoeostasis. It controls the expression of a total of 11 genes, the CopR regulon, in a copper-dependent manner. In the absence of copper, CopR binds to the promoters of the CopR regulon. Copper releases CopR from the promoters, allowing transcription of the downstream genes to proceed. CopR binds through its N-terminal domain to a 'cop box' of consensus TACANNTGTA, which is conserved in Firmicutes. We have solved the NMR solution structure of the N-terminal DNA-binding domain of CopR. The protein fold has a winged helix structure resembling that of the BlaI repressor which regulates antibiotic resistance in Bacillus licheniformis. CopR differs from other copper-responsive repressors, and the present structure represents a novel family of copper regulators, which we propose to call the CopY family.

Solution structure of the region 51-160 of human KIN17 reveals an atypical winged helix domain.

Human KIN17 is a 45-kDa eukaryotic DNA- and RNA-binding protein that plays an important role in nuclear metabolism and in particular in the general response to genotoxics. Its amino acids sequence contains a zinc finger motif (residues 28-50) within a 30-kDa N-terminal region conserved from yeast to human, and a 15-kDa C-terminal tandem of SH3-like subdomains (residues 268-393) only found in higher eukaryotes. Here we report the solution structure of the region 51-160 of human KIN17. We show that this fragment folds into a three-alpha-helix bundle packed against a three-stranded beta-sheet. It belongs to the winged helix (WH) family. Structural comparison with analogous WH domains reveals that KIN17 WH module presents an additional and highly conserved 3(10)-helix. Moreover, KIN17 WH helix H3 is not positively charged as in classical DNA-binding WH domains. Thus, human KIN17 region 51-160 might rather be involved in protein-protein interaction through its conserved surface centered on the 3(10)-helix.

Structural and biochemical characterization of a cyanobacterium circadian clock-modifier protein.

Circadian clocks are self-sustained biochemical oscillators. The oscillator of cyanobacteria comprises the products of three kai genes (kaiA, kaiB, and kaiC). The autophosphorylation cycle of KaiC oscillates robustly in the cell with a 24-h period and is essential for the basic timing of the cyanobacterial circadian clock. Recently, period extender (pex), mutants of which show a short period phenotype, was classified as a resetting-related gene. In fact, pex mRNA and the pex protein (Pex) increase during the dark period, and a pex mutant subjected to diurnal light-dark cycles shows a 3-h advance in rhythm phase. Here, we report the x-ray crystallographic analysis and biochemical characterization of Pex from cyanobacterium Synechococcus elongatus PCC 7942. The molecule has an (alpha+beta) structure with a winged-helix motif and is indicated to function as a dimer. The subunit arrangement in the dimer is unique and has not been seen in other winged-helix proteins. Electrophoresis mobility shift assay using a 25-base pair complementary oligonucleotide incorporating the kaiA upstream sequence demonstrates that Pex has an affinity for the double-stranded DNA. Furthermore, mutation analysis shows that Pex uses the wing region to recognize the DNA. The in vivo rhythm assay of Pex shows that the constitutive expression of the pex gene harboring the mutation that fails to bind to DNA lacks the period-prolongation activity in the pex-deficient Synechococcus, suggesting that Pex is a DNA-binding transcription factor.

The carboxyl-terminal nucleoplasmic region of MAN1 exhibits a DNA binding winged helix domain.

MAN1 is an integral protein of the inner nuclear membrane that interacts with nuclear lamins and emerin, thus playing a role in nuclear organization. It also binds to chromatin-associated proteins and transcriptional regulators, including the R-Smads, Smad1, Smad2, and Smad3. Mutations in the human gene encoding MAN1 cause sclerosing bone dysplasias, which sometimes have associated skin abnormalities. At the molecular level, these mutations lead to loss of the MAN1-R-Smads interaction, thus perturbing transforming growth factor beta superfamily signaling pathway. As a first step to understanding the physical basis of MAN1 interaction with R-Smads, we here report the structural characterization of the carboxyl-terminal nucleoplasmic region of MAN1, which is responsible for Smad binding. This region exhibits an amino-terminal globular domain adopting a winged helix fold, as found in several Smad-associated sequence-specific DNA binding factors. Consistently, it binds to DNA through the positively charged recognition helix H3 of its winged helix motif. However, it does not show the predicted carboxyl-terminal U2AF homology domain in solution, suggesting that the folding and stability of such a domain in MAN1 depend upon binding to an unidentified partner. Modeling the complex between DNA and the winged helix domain shows that the regions involved in DNA binding are essentially distinct from those reported to be involved in Smad binding. This suggests that MAN1 binds simultaneously to R-Smads and their targeted DNA sequences.