Dimerization Cofactor Of HNF-1 Research Articles

Kinetic Stability May Determine the Interaction Dynamics of the Bifunctional Protein DCoH1, the Dimerization Cofactor of the Transcription Factor HNF-1α,

The two disparate functions of DCoH1 (dimerization cofactor of HNF-1)/PCD (pterin-4a-carbinolamine dehydratase) are associated with a change in oligomeric state. DCoH dimers enhance the activity of the diabetes-associated transcription factor HNF-1α (hepatocyte nuclear factor-1α), while the PCD activity of DCoH1 homotetramers aids in aromatic amino acid metabolism. These complexes compete for the same interface of the DCoH dimer. Formation of the DCoH1/HNF-1α complex requires cofolding. The homotetramer of the DCoH1 paralogue, DCoH2, interacts with HNF-1α through simple mixing. To further investigate regulation of DCoH/HNF-1α complex formation, we measured the stability of the DCoH1 homotetramer through unfolding studies by intrinsic tryptophan fluorescence. DCoH2 unfolding is reversible. Surprisingly, the DCoH1 homotetramer is resistant to guanidine unfolding but refolds at a much lower guanidine concentration. We show that a point mutation at the DCoH1 tetramer interface, Thr 51 Ser, overcomes the dissociation barrier of the homotetramer and increases the interaction with HNF-1α. The 1.8 Ǻ resolution crystal structure of DCoH1 T51S shows the presence of an ordered water molecule at the tetramer interface, as in DCoH2, which may destabilize the homotetramer. The equilibrium unfolding data were fit to a two-state model with no apparent intermediate. Folding intermediates were detectable by size exclusion chromatography. For wild-type DCoH1 the intermediates changed with time, suggesting a kinetic origin for the unfolding barrier of the homotetramer. We propose an unfolding pathway in which the tetramer unfolds slowly, but the dimer folds reversibly. Implications for regulation of DCoH1/HNF-1α complex formation are discussed.

Structure of the Conserved Transcriptional Repressor Enhancer of Rudimentary Homolog,

erh (enhancer of rudimentary homolog) is a ubiquitously expressed transcriptional coregulator that is highly conserved among eukaryotes, from humans to plants to protozoa. Functions attributed to erh include enhancement of pyrimidine biosynthesis, a role in cell cycle regulation, and repression of the tissue-specific transcription factor HNF-1 (hepatocyte nuclear factor-1) through binding the coactivator DCoH (dimerization cofactor of HNF1). No homologous sequences, other than erh orthologs, have been identified, and little is known about the interactions of erh. To further elucidate its function, we determined the crystal structure of erh to 2.0 A resolution. The erh structure is a novel alpha + beta fold consisting of a four-stranded antiparallel beta sheet with three amphipathic alpha helices situated on one face of the beta sheet. Structure-based searches of the Protein Data Bank, like sequence-based searches, failed to identify paralogs. We present structural and biochemical evidence that erh functions as a dimer. The dimer interface consists of a beta sandwich composed of the beta sheet from each monomer. Many of the surface residues of erh are conserved, including patches of hydrophobic and charged residues, suggesting protein-protein interaction interfaces. Two putative CKII phosphorylation sites are highly ordered in the structure and are predicted to disrupt dimerization and protein-protein interactions.

Biochemical and structural basis for partially redundant enzymatic and transcriptional functions of DCoH and DCoH2.

An inherited form of diabetes, maturity-onset diabetes of the young type 3 (MODY3), results from mutations in the transcriptional activator, hepatocyte nuclear factor-1alpha (HNF1alpha). Transcription by HNF1alpha is stimulated by the bifunctional coactivator DCoH (dimerization cofactor of HNF1). Strikingly, an HNF1alpha deletion in mice causes more severe phenotypes than a DCoH deletion. It has been hypothesized that a DCoH homolog, DCoH2, partially complements the DCoH deletion. To test this idea, we determined the biochemical properties and the 1.6-A-resolution crystal structure of DCoH2. Like DCoH, DCoH2 forms a tetramer, displays pterin-4alpha-carbinolamine dehydratase activity, and binds HNF1alpha in vivo and in vitro. DCoH and DCoH2 adopt identical folds with structural differences confined largely to the protein surfaces and the tetramer interface. In contrast to the hyperstable DCoH tetramer, DCoH2 readily disproportionates and forms a 2:2 complex with HNF1 in vitro. Phylogenetic analysis reveals six major subfamilies of DCoH proteins, including unique DCoH and DCoH2 branches in metazoans. These results suggest distinct roles for DCoH and DCoH2. Differences in conserved surface residues could mediate binding to different effectors. We propose that HNF1alpha binding kinetics may distinguish regulation by DCoH2, under thermodynamic control, from regulation by DCoH, under kinetic control.

Studies of the variability of the hepatocyte nuclear factor-1beta (HNF-1beta / TCF2) and the dimerization cofactor of HNF-1 (DcoH / PCBD) genes in relation to type 2 diabetes mellitus and beta-cell function.

Mutations in the homeodomain-containing transcription factor hepatocyte nuclear factor-1beta (HNF-1beta) are known to cause a rare subtype of maturity-onset diabetes of the young (MODY5), which is associated with early-onset progressive non-diabetic renal dysfunction. To investigate whether mutations in HNF-1 are implicated in the pathogenesis of MODY or late-onset diabetes with and without nephropathy in Danish Caucasians we examined the HNF-1beta (TCF2) and the dimerization cofactor of HNF-1 (DCoH, PCBD) genes for mutations in 11 MODY probands, 28 type 2 diabetic patients with nephropathy, and 46 type 2 diabetic patients with an impaired beta-cell function by combined single-strand conformation polymorphism (SSCP) and heteroduplex analysis. Analysis of the promoter and nine exons including intron-exon boundaries of the HNF-1beta gene revealed one novel silent polymorphism and three previously reported intronic variants. The silent polymorphism (I91I) was found in one patient with late-onset type 2 diabetes. One of the intronic variant (IVS6+26T-->C) was examined further. Among 584 type 2 diabetic patients the allelic frequency was 13.1% (11.2-15.0%) compared to 11.6% (8.6-14.5%) in 229 glucose tolerant control subjects (NS). No difference in insulin secretion during an OGTT was seen between carriers of the different IVS6+26T-->C genotypes among the 229 middle-aged control subjects, nor among 302 glucose tolerant 60-year-old Danish Caucasians. Mutation analysis of the four exons comprising the DCoH gene revealed a previously described A-->G polymorphism located in the 3' untranslated region, which was not investigated further. In conclusion, mutations in HNF-1beta and DCoH are not a major cause of MODY or late onset type 2 diabetes in Danish Caucasian subjects.

The dimerization domain of HNF-1α: structure and plasticity of an intertwined four-helix bundle with application to diabetes mellitus

Maturity-onset diabetes mellitus of the young (MODY) is a human genetic syndrome most commonly due to mutations in hepatocyte nuclear factor-1α (HNF-1α). Here, we describe the crystal structure of the HNF-1α dimerization domain at 1.7 Å resolution and assess its structural plasticity. The crystal’s low solvent content (23 %, v/v) leads to tight packing of peptides in the lattice. Two independent dimers, similar in structure, are formed in the unit cell by a 2-fold crystallographic symmetry axis. The dimers define a novel intertwined four-helix bundle (4HB). Each protomer contains two α-helices separated by a sharp non-canonical turn. Dimer-related α-helices form anti-parallel coiled-coils, including an N-terminal “mini-zipper” complementary in structure, symmetry and surface characteristics to transcriptional coactivator dimerization cofactor of HNF-1 (DCoH). A confluence of ten leucine side-chains (five per protomer) forms a hydrophobic core. Isotope-assisted NMR studies demonstrate that a similar intertwined dimer exists in solution. Comparison of structures obtained in multiple independent crystal forms indicates that the mini-zipper is a stable structural element, whereas the C-terminal α-helix can adopt a broad range of orientations. Segmental alignment of the mini-zipper (mean pairwise root-mean-square difference (rmsd) in Cα coordinates of 0.29 Å) is associated with a 2.1 Å mean Cα rmsd displacement of the C-terminal coiled-coil. The greatest C-terminal structural variation (4.1 Å Cα rmsd displacement) is observed in the DCoH-bound peptide. Diabetes-associated mutations perturb distinct structural features of the HNF-1α domain. One mutation (L12H) destabilizes the domain but preserves structural specificity. Adjoining H12 side-chains in a native-like dimer are predicted to alter the functional surface of the mini-zipper involved in DCoH recognition. The other mutation (G20R), by contrast, leads to a dimeric molten globule, as indicated by its 1H-NMR features and fluorescent binding of 1-anilino-8-naphthalene sulfonate. We propose that a glycine-specific turn configuration enables specific interactions between the mini-zipper and the C-terminal coiled-coil.

High-resolution structure of the HNF-1alpha dimerization domain.

The N-terminal dimerization domain of the transcriptional activator hepatocyte nuclear factor-1alpha (HNF-1alpha) is essential for DNA binding and association of the transcriptional coactivator, DCoH (dimerization cofactor of HNF-1). To investigate the basis for dimerization of HNF-1 proteins, we determined the 1.2 A resolution X-ray crystal structure of the dimerization domain of HNF-1alpha (HNF-p1). Phasing was facilitated by devising a simple synthesis for Fmoc-selenomethionine and substituting leucine residues with selenomethionine. The HNF-1 dimerization domain forms a unique, four-helix bundle that is preserved with localized conformational shifts in the DCoH complex. In three different crystal forms, HNF-p1 displays subtle shifts in the conformation of the interhelix loop and the crossing angle between the amino- and carboxyl-terminal helices. In all three crystal forms, the HNF-p1 dimers pair through an exposed hydrophobic surface that also forms the binding site for DCoH. Conserved core residues in the dimerization domain of the homologous transcriptional regulator HNF-1beta rationalize the functional heterodimerization of the HNF-1alpha and HNF-1beta proteins. Mutations in HNF-1alpha are associated with maturity-onset diabetes of the young type 3 (MODY3), and the structure of HNF-p1 provides insights into the effects of three MODY3 mutations.

Structural basis of dimerization, coactivator recognition and MODY3 mutations in HNF-1alpha.

Maturity-onset diabetes of the young type 3 (MODY3) results from mutations in the transcriptional activator hepatocyte nuclear factor-1alpha (HNF-1alpha). Several MODY3 mutations target the HNF-1alpha dimerization domain (HNF-p1), which binds the coactivator, dimerization cofactor of HNF-1 (DCoH). To define the mechanism of coactivator recognition and the basis for the MODY3 phenotype, we determined the cocrystal structure of the DCoH-HNF-p1 complex and characterized biochemically the effects of MODY3 mutations in HNF-p1. The DCoH-HNF-p1 complex comprises a dimer of dimers in which HNF-p1 forms a unique four-helix bundle. Through rearrangements of interfacial side chains, a single, bifunctional interface in the DCoH dimer mediates both HNF-1alpha binding and formation of a competing, transcriptionally inactive DCoH homotetramer. Consistent with the structure, MODY3 mutations in HNF-p1 reduce activator function by two distinct mechanisms.

Mutations in the genes for hepatocyte nuclear factor (HNF)-1alpha, -4alpha, -1beta, and -3beta; the dimerization cofactor of HNF-1; and insulin promoter factor 1 are not common causes of early-onset type 2 diabetes in Pima Indians.

Maturity-onset diabetes of the young (MODY) is a genetically heterogeneous subtype of type 2 diabetes characterized by an early age at onset and autosomal dominant inheritance. MODY can result from heterozygous mutations in at least five genes. The purpose of this study was to determine whether alterations in known MODY genes and two MODY candidate genes contribute to the development of early-onset type 2 diabetes in Pima Indians. The coding regions of the known MODY genes hepatocyte nuclear factor (HNF)-1alpha, HNF-4alpha, HNF-1beta, and insulin promoter factor 1 and the coding regions of two MODY candidate genes, HNF-3beta and the dimerization cofactor of HNF-1, were sequenced in genomic DNA from Pima Indians. The primary "affected" study population consisted of 46 Pima Indians whose age at onset of type 2 diabetes was < or =20 years. DNA sequence variants identified in the affected group were then analyzed in a group of 80 "unaffected" Pima Indians who were at least 40 years old and had normal glucose tolerance. A total of 11 polymorphisms were detected in these genes. However, none of the polymorphisms differed in frequency among Pima Indians with an early age at onset of diabetes compared with older Pima Indians with normal glucose tolerance. Mutations in these known MODY or MODY candidate genes are not a common cause of early-onset diabetes in Pima Indians.

Human White Blood Cells and Hair Follicles Are Good Sources of mRNA for the Pterin Carbinolamine Dehydratase/Dimerization Cofactor of HNF1 for Mutation Detection

Pterin carbinolamine dehydratase/dimerization cofactor of HNF1 (PCD/DCoH) is a protein that has a dual function. It is a pterin 4α-carbinolamine dehydratase that is involved in the regeneration of the cofactor tetrahydrobiopterin during the phenylalanine hydroxylase- catalyzed hydroxylation of phenylalanine. In addition, it is the dimerization cofactor of HNF1 that is able to activate the transcriptional activity of HNF1. Deficiencies in the gene for this dual functional protein result in hyperphenylalaninemia. Here we report for the first time that the PCD/DCoH mRNA is present in human white blood cells and hair follicles. Taking advantage of this finding, a sensitive, rapid and convenient method for screening mutations occurring in the coding region of this gene has been described.

Expression of 4α-Carbinolamine Dehydratase in Human Epidermal Keratinocytes

4α-Carbinolamine dehydratase is a bifunctional protein involved in the regeneration of tetrahydrobiopterin during the hydroxylation of the aromatic amino acids. It is also a dimerization cofactor of HNF1 and therefore is believed to function as part of the hepatic gene transcription system. In view of the recent discoveries that the distribution and developmental pattern of the dehydratase do not correlate strictly with those of the aromatic amino acid hydroxylases and HNF1, the hypothesis that the dehydratase may have other unknown functions has been put forward. In the present paper, we demonstrate unambiguously that human epidermal keratinocytes express detectable levels of this protein as indicated by enzyme assay, immunoprecipitation, Western blot, and RT-PCR. Its complete coding sequence has been cloned and was found to be identical with the human liver counterpart. The possible function of the dehydratase in skin is discussed.

PCD/DCoH: more than a second molecular saddle.

Two recent crystal structures of the tetrameric pterin-4 alpha-carbinolamine dehydratase (PCD)/dimerization cofactor of HNF-1 (DCoH) protein provide fresh insights into how this multifunctional enzyme/transcriptional coactivator works.

Three-dimensional structure of the bifunctional protein PCD/DCoH, a cytoplasmic enzyme interacting with transcription factor HNF1.

The bifunctional protein pterin-4a-carbinolamine dehydratase (PCD)/dimerization cofactor of HNF1 (DCoH) is a cytoplasmic enzyme involved in the tetrahydrobiopterin regeneration and is found in complex with the transcription factor HNF1 in liver cell nuclei. An atypical hyperphenylalaninemia and the depigmentation disorder vitiligo are related to a deficiency of PCD/DCoH activity. The crystal structure of PCD/DCoH was solved by multiple isomorphous replacement and refined to a crystallographic R-factor of 20.5% at 2.7 A resolution. The single domain monomer comprises three alpha-helices packed against one side of a four-stranded, antiparallel beta-sheet. The functional enzyme is a homo-tetramer of 222 symmetry where each of the monomers contributes one helix to a central four helix bundle. In the tetramer two monomers form an eight-stranded, antiparallel beta-sheet with six helices packing against it from one side. The concave, hydrophobic surface of the eight-stranded beta-sheet with its two protruding loops at either end is reminiscent of the saddle-like shape seen in the TATA-box binding protein. PCD/DCoH binds as a dimer to the helical dimerization domain of dimeric HNF1 forming a hetero-tetramer possibly through a mixed four helix bundle.