Cloning, purification and preliminary X-ray data analysis of the human ID2 homodimer.

The ID proteins are named for their role as inhibitors of DNA binding and differentiation. They contain a helix-loop-helix (HLH) domain but lack a basic DNA-binding domain. In complex with basic HLH (bHLH) transcription factors, gene expression is regulated by DNA-binding inactivation. Although the HLH domain is highly conserved and shares a similar topology, the IDs preferentially bind class I bHLH-group members such as E47 (TCF3) but not the class III bHLH member Myc. A structure of an ID protein could potentially shed light on its mechanism. Owing to their short half-lives in vivo and reported in vitro instability, this paper describes the strategies that went into expressing sufficient soluble and stable ID2 to finally obtain diffraction-quality crystals. A 2.1 Šresolution data set was collected from a crystal belonging to space group P3(1)21 with unit-cell parameters a=b=51.622, c=111.474 Å, α=ß=90, γ=120° that was obtained by hanging-drop vapour diffusion in a precipitant solution consisting of 0.1 M MES pH 6.5, 2.0 M potassium acetate. The solvent content was consistent with the presence of one or two molecules in the asymmetric unit.

A divalent ion is crucial in the structure and dominant-negative function of ID proteins, a class of helix-loop-helix transcription regulators.

Inhibitors of DNA binding and differentiation (ID) proteins, a dominant-negative group of helix-loop-helix (HLH) transcription regulators, are well-characterized key players in cellular fate determination during development in mammals as well as Drosophila. Although not oncogenes themselves, their upregulation by various oncogenic proteins (such as Ras, Myc) and their inhibitory effects on cell cycle proteins (such as pRb) hint at their possible roles in tumorigenesis. Furthermore, their potency as inhibitors of cellular differentiation, through their heterodimerization with subsequent inactivation of the ubiquitous E proteins, suggest possible novel roles in engineering induced pluripotent stem cells (iPSCs). We present the high-resolution 2.1Å crystal structure of ID2 (HLH domain), coupled with novel biochemical insights in the presence of a divalent ion, possibly calcium (Ca2+), in the loop of ID proteins, which appear to be crucial for the structure and activity of ID proteins. These new insights will pave the way for new rational drug designs, in addition to current synthetic peptide options, against this potent player in tumorigenesis as well as more efficient ways for stem cells reprogramming.

Crystal optimization and preliminary diffraction data analysis of the SCAN domain of Zfp206.

Zfp206 (also named Zscan10) is a transcription factor that plays an important role in maintaining the pluripotent state of embryonic stem cells. Zfp206 is a member of the SCAN-domain family of C(2)H(2) zinc-finger transcription factors. The SCAN domain is a highly conserved motif of 84 residues which mediates the self-association of and heterodimerization between SCAN-domain family transcription factors. The SCAN domain may therefore be the key to the selective oligomerization of and may combinatorially enhance the regulatory versatility of C(2)H(2) zinc fingers. This paper describes crystallization attempts with the SCAN domain of Zfp206 (Zfp206SCAN) and optimization strategies to obtain diffraction-quality crystals. The best diffracting crystal was grown in a solution consisting of 0.3 M ammonium sulfate, 0.1 M Tris-HCl pH 8.6, 25% PEG 3350, 0.1 M ethylenediaminetetraacetic acid disodium salt dehydrate (EDTA) using the hanging-drop vapour-diffusion technique. Optimized crystals diffracted to 1.85 Å resolution and belonged to space group I422, with unit-cell parameters a = 67.57, c = 87.54 Å. A Matthews analysis indicated the presence of one Zfp206SCAN molecule per asymmetric unit.

Conversion of Sox17 into a pluripotency reprogramming factor by reengineering its association with Oct4 on DNA.

Very few proteins are capable to induce pluripotent stem (iPS) cells and their biochemical uniqueness remains unexplained. For example, Sox2 cooperates with other transcription factors to generate iPS cells, but Sox17, despite binding to similar DNA sequences, cannot. Here, we show that Sox2 and Sox17 exhibit inverse heterodimerization preferences with Oct4 on the canonical versus a newly identified compressed sox/oct motif. We can swap the cooperativity profiles of Sox2 and Sox17 by exchanging single amino acids at the Oct4 interaction interface resulting in Sox2KE and Sox17EK proteins. The reengineered Sox17EK now promotes reprogramming of somatic cells to iPS, whereas Sox2KE has lost this potential. Consistently, when Sox2KE is overexpressed in embryonic stem cells it forces endoderm differentiation similar to wild-type Sox17. Together, we demonstrate that strategic point mutations that facilitate Sox/Oct4 dimer formation on variant DNA motifs lead to a dramatic swap of the bioactivities of Sox2 and Sox17.

Structure of Smad1 MH1/DNA complex reveals distinctive rearrangements of BMP and TGF-beta effectors.

Smad1 is a downstream effector of the BMP signaling pathway that binds regulatory DNA to execute gene expression programs leading to, for example, the maintenance of pluripotency in mice. On the contrary, the TGF-beta-activated Smad3 triggers strikingly different programs such as mesodermal differentiation in early development. Because Smad1 and Smad3 contain identical amino acids at the DNA contact interface it is unclear how they elicit distinctive bioactivities. Here, we report the crystal structure of the MH1 domain of Smad1 bound to a palindromic Smad binding element. Surprisingly, the DNA contact interface of Smad1 is drastically rearranged when compared to Smad3. The N-terminal helix 1 of Smad1 is dislodged from its intramolecular binding site and adopts a domain swapped arrangement with a symmetry-related molecule. As a consequence, helix 2 kinks away from the double helix disabling several key phosphate backbone interactions. Thermal melting analysis corroborates a decompacted conformation of Smad1 and DNA binding assays indicate a lower overall affinity of Smad1 to DNA but increased cooperativity when binding to palindromic DNA motifs. These findings suggest that Smad1 and Smad3 evolved differential qualities to assemble on composite DNA elements and to engage in co-factor interactions by remodeling their N-termini.

Crystal optimization and preliminary diffraction data analysis of the Smad1 MH1 domain bound to a palindromic SBE DNA element.

The bone morphogenetic protein (BMP) signalling pathway regulates diverse processes such as cell differentiation, anterior/posterior axis specification, cell growth and the formation of extra-embryonic tissues. The transcription factor Smad1 relays the BMP signal from the cytoplasm to the nucleus, where it binds short DNA-sequence motifs and regulates gene expression. However, how Smad1 selectively targets particular genomic regions is poorly understood. In order to understand the physical basis of the specific interaction of Smad1 with DNA and to contrast it with the highly homologous but functionally distinct Smad3 protein, the DNA-binding Mad-homology 1 (MH1) domain of Smad1 was cocrystallized with a 17-mer palindromic Smad-binding element (SBE). The extensive optimizations of the length, binding-site spacing and terminal sequences of the DNA element in combination with the other crystallization parameters necessary for obtaining diffraction-quality crystals are described here. A 2.7 angstrom resolution native data set was collected at the National Synchrotron Radiation Research Centre, Taiwan, from crystals grown in a solution containing 0.2 M ammonium tartrate dibasic, 20% PEG 3350, 3% 2-propanol and 10% glycerol. The data set was indexed and merged in space group P222, with unit-cell parameters a = 73.94, b = 77.49, c = 83.78 angstrom, alpha = beta = gamma = 90 degrees. The solvent content in the unit cell is consistent with the presence of two Smad1 MH1 molecules bound to the duplex DNA in the asymmetric unit.

The structure of Sox17 bound to DNA reveals a conserved bending topology but selective protein interaction platforms.

Sox17 regulates endodermal lineage commitment and is thought to function antagonistically to the pluripotency determinant Sox2. To investigate the biochemical basis for the distinct functions of Sox2 and Sox17, we solved the crystal structure of the high mobility group domain of Sox17 bound to a DNA element derived from the Lama1 enhancer using crystals diffracting to 2.7 A resolution. Sox17 targets the minor groove and bends the DNA by approximately 80 degrees . The DNA architecture closely resembles the one seen for Sox2/DNA structures, suggesting that the degree of bending is conserved between both proteins and nucleotide substitutions have only marginal effects on the bending topology. Accordingly, affinities of Sox2 and Sox17 for the Lama1 element were found to be identical. However, when the Oct1 contact interface of Sox2 is compared with the corresponding region of Sox17, a significantly altered charge distribution is observed, suggesting differential co-factor recruitment that may explain their biological distinctiveness.

Purification, crystallization and preliminary X-ray diffraction analysis of the HMG domain of Sox17 in complex with DNA.

Sox17 is a member of the SRY-related high-mobility group (HMG) of transcription factors that have been shown to direct endodermal differentiation in early mammalian development. The LAMA1 gene encoding the alpha-chain of laminin-1 has been reported to be directly bound and regulated by Sox17. This paper describes the details of initial crystallization attempts with the HMG domain of mouse Sox17 (mSox17-HMG) with a 16-mer DNA element derived from the LAMA1 enhancer and optimization strategies to obtain a better diffracting crystal. The best diffracting crystal was obtained in a condition containing 0.1 M Tris-HCl pH 7.4, 0.2 M MgCl(2), 30% PEG 3350 using the hanging-drop vapour-diffusion method. A highly redundant in-house data set was collected to 2.75 A resolution with 99% completeness. The presence of the mSox17-HMG-DNA complex within the crystals was confirmed and Matthews analysis indicated the presence of one complex per asymmetric unit.

Identification and characterization of a novel mutation in the carbonic anhydrase IV gene that causes retinitis pigmentosa.

PURPOSE: The autosomal dominant retinitis pigmentosa (adRP) gene on chromosome 17, region q22 (RP17), was recently identified as a glycosylphosphatidylinositol membrane-anchored zinc metalloenzyme (protein CAIV), highly expressed in the choriocapillaris of the eye and undetectable in the retina. Only two missense mutations have thus far been identified in the gene CA4. Functional analysis of these mutations demonstrated that retinal disease may result from perturbation of pH homeostasis in the outer retina, after disruption of CAIV and sodium bicarbonate cotransporter 1 (NBC1)-mediated bicarbonate transport. CA4 was screened in a panel of patients with RP, to expand the mutation spectrum of this novel adRP gene and understand its pathogenic mechanism. METHODS: A total of 96 patients with simplex RP and adRP of Chinese ethnicity were screened for mutations in the eight coding exons of the CA4 gene by bidirectional sequencing. Functional consequences of CA4 mutations on the NBC1-mediated bicarbonate transport were studied by measuring bicarbonate fluxes in HEK293 cells cotransfected with NBC1 and CA4 mutant cDNAs. RESULTS: Thirteen sequence alterations were identified, including a novel mutation within exon 3 of CA4 (R69H) in a patient with simplex RP. R69H was not found in 432 normal chromosomes. R69H CAIV impaired NBC1-mediated pH recovery after acid load. CONCLUSIONS: A novel mutation has been identified in CA4 that provides further evidence that impaired pH regulation may underlie photoreceptor degeneration in RP17. This study indicates that, as with European patients with RP, mutations in CA4 also account for