The Drosophila juvenile hormone receptor candidates methoprene-tolerant (MET) and germ cell-expressed (GCE) utilize a conserved LIXXL motif to bind the FTZ-F1 nuclear receptor

Juvenile hormone (JH) has been implicated in many developmental processes in holometabolous insects, but its mechanism of signaling remains controversial. We previously found that in Drosophila Schneider 2 cells, the nuclear receptor FTZ-F1 is required for activation of the E75A gene by JH. Here, we utilized insect two-hybrid assays to show that FTZ-F1 interacts with two JH receptor candidates, the bHLH-PAS paralogs MET and GCE, in a JH-dependent manner. These interactions are severely reduced when helix 12 of the FTZ-F1 activation function 2 (AF2) is removed, implicating AF2 as an interacting site. Through homology modeling, we found that MET and GCE possess a C-terminal α-helix featuring a conserved motif LIXXL that represents a novel nuclear receptor (NR) box. Docking simulations supported by two-hybrid experiments revealed that FTZ-F1·MET and FTZ-F1·GCE heterodimer formation involves a typical NR box-AF2 interaction but does not require the canonical charge clamp residues of FTZ-F1 and relies primarily on hydrophobic contacts, including a unique interaction with helix 4. Moreover, we identified paralog-specific features, including a secondary interaction site found only in MET. Our findings suggest that a novel NR box enables MET and GCE to interact JH-dependently with the AF2 of FTZ-F1.     

A novel DNA-binding motif abuts the zinc finger domain of insect nuclear hormone receptor FTZ-F1 and mouse embryonal long terminal repeat-binding protein.

Fruit fly FTZ-F1, silkworm BmFTZ-F1, and mouse embryonal long terminal repeat-binding protein are members of the nuclear hormone receptor superfamily, which recognizes the same sequence, 5'-PyCAAGGPyCPu-3'. Among these proteins, a 30-amino-acid basic region abutting the C-terminal end of the zinc finger motif, designated the FTZ-F1 box, is conserved. Gel mobility shift competition by various mutant peptides of the DNA-binding region revealed that the FTZ-F1 box as well as the zinc finger motif is involved in the high-affinity binding of FTZ-F1 to its target site. Using a gel mobility shift matrix competition assay, we demonstrated that the FTZ-F1 box governs the recognition of the first three bases, while the zinc finger region recognizes the remaining part of the binding sequence. We also showed that the DNA-binding region of FTZ-F1 recognizes and binds to DNA as a monomer. Occurrence of the FTZ-F1 box sequence in other members of the nuclear hormone receptor superfamily raises the possibility that these receptors constitute a unique subfamily which binds to DNA as a monomer.     

Monomeric complex of human orphan estrogen related receptor-2 with DNA: a pseudo-dimer interface mediates extended half-site recognition

While most nuclear receptors bind DNA as homo or heterodimers, the human estrogen related receptors (hERRs) are members of a subfamily of orphan receptors that bind DNA as monomers. We have determined the solution structure of the DNA binding domain (DBD) of hERR2 bound to its cognate DNA. The structure and base interactions of the core DBD are similar to those of other nuclear receptors. However, high-affinity, sequence-specific DNA binding as a monomer necessitates formation of additional base contacts outside the core DBD. This is accomplished using a modified guanosine-binding "AT-hook" within the C-terminal extension (CTE) flanking the DBD, which makes base-specific minor groove interactions. The structure of the CTE is stabilized both by interactions with the DNA and by packing against a region of the core DBD normally reserved for dimerization. This pseudo-dimer interface provides a basis for the expansion of DNA recognition and suggests a mechanism through which dimerization may have evolved from an ancestral monomeric receptor.     

The nuclear receptor coactivator PGC-1alpha exhibits modes of interaction with the estrogen receptor distinct from those of SRC-1

Estrogen receptor (ER) function is mediated by multi-domain co-regulator proteins. A fluorescently labelled fragment of the human PGC-1alpha co-regulator (residues 91-408) bearing the two motifs most strongly implicated in interactions with nuclear receptors (NR box2 and NR box3), was used to characterize in vitro binding of PGC-1alpha to ER. Anisotropy measurements revealed that the affinity of this PGC-1alpha fragment for human ERalpha and beta was fairly strong in the presence of estradiol (approximately 5 nM), and that unlike a similar fragment of SRC-1 (570-780), PGC-191-408 exhibited ligand-independent interactions with ER, particularly with ERbeta (Kd approximately 30 nM). Competition experiments of the complex between ERalpha and fluorescently labelled PGC-1 91-408 with unlabelled SRC-1 570-780 showed that PGC-1 91-408 was an efficient competitor of SRC-1 570-780, while the inverse was not true, underscoring their distinct modes of binding. The anisotropy data provide strong evidence for a ternary complex between ERalpha, SRC-1 570-780 and PGC-1 91-408. GST-pull-down experiments with deletion mutants of ERalpha revealed that the constitutive binding of PGC-1 91-408 requires the presence of the linker domain between the DNA binding and ligand binding domains (DBD and LBD). Homology modeling studies of the different regions of full length PGC-1alpha confirmed the lack of compact tertiary structure of the N-terminal region bearing the NR box motifs, and suggested a slightly different mode of interaction compared to the NR box motifs of SRC-1. They also provided reasonable structural models for the coiled-coil dimerization motif at residues 633-675, as well as the C-terminal putative RNA binding domain, raising important questions concerning the stoichiometry of its complex with the nuclear receptors.     

Molecular dynamics simulations of the glucocorticoid receptor DNA-binding domain in complex with DNA and free in solution.

Molecular dynamics simulations have been performed on the glucocorticoid receptor DNA binding domain (GR DBD) in aqueous solution as a dimer in complex with DNA and as a free monomer. In the simulated complex, we find a slightly increased bending of the DNA helix axis compared with the crystal structure in the spacer region of DNA between the two half-sites that are recognized by GR DBD. The bend is mainly caused by an increased number of interactions between DNA and the N-terminal extended region of the sequence specifically bound monomer. The recognition helices of GR DBD are pulled further into the DNA major groove leading to a weakening of the intrahelical hydrogen bonds in the middle of the helices. Many ordered water molecules with long residence times are found at the intermolecular interfaces of the complex. The hydrogen-bonding networks (including water bridges) on either side of the DNA major groove involve residues that are highly conserved within the family of nuclear receptors. Very similar hydrogen-bonding networks are found in the estrogen receptor (ER) DBD in complex with DNA, which suggests that this is a common feature for proper positioning of the recognition helix in ER DBD and GR DBD.     

Structural dynamics of the two-component response regulator RstA in recognition of promoter DNA element

The RstA/RstB system is a bacterial two-component regulatory system consisting of the membrane sensor, RstB and its cognate response regulator (RR) RstA. The RstA of Klebsiella pneumoniae (kpRstA) consists of an N-terminal receiver domain (RD, residues 1–119) and a C-terminal DNA-binding domain (DBD, residues 130–236). Phosphorylation of kpRstA induces dimerization, which allows two kpRstA DBDs to bind to a tandem repeat, called the RstA box, and regulate the expression of downstream genes. Here we report the solution and crystal structures of the free kpRstA RD, DBD and DBD/RstA box DNA complex. The structure of the kpRstA DBD/RstA box complex suggests that the two protomers interact with the RstA box in an asymmetric fashion. Equilibrium binding studies further reveal that the two protomers within the kpRstA dimer bind to the RstA box in a sequential manner. Taken together, our results suggest a binding model where dimerization of the kpRstA RDs provides the platform to allow the first kpRstA DBD protomer to anchor protein–DNA interaction, whereas the second protomer plays a key role in ensuring correct recognition of the RstA box.     

孤儿核受体hB1F／hLRH-1铰链区一个保守结构域对其转录功能的抑制作用

孤儿核受体hB1F(NR5A2 ,也称之为LRH 1、CPF或FTF)在胆汁酸合成代谢、乙肝病毒基因和部分肝特异性基因表达的调控中起着重要的作用。为理解hB1F激活转录的分子机制 ,对其铰链区潜在的功能结构域进行了分析。利用GAL4 DBD融合的hB1F缺失片段所进行的报告基因分析 ,发现了一个位于铰链区的强烈抑制hB1F反式激活能力的结构域。该结构域核心残基的定点突变导致了hB1F反式激活能力的显著上升 ,而且明显地增强hB1F对乙肝病毒增强子II 核心启动子的转录激活能力。生物信息学分析显示该结构域不存在明显的二级结构 ,但有意思的是 ,其氨基酸序列在核受体FTZ F1亚家族的成员中高度保守 ,且不见于其他蛋白质中。转染分析发现 ,该结构域的抑制活性存在于测试的五个不同细胞株中 ,但抑制的强度表现出明显差异。半定量RT PCR分析表明 ,与SF 1不同 ,该结构域抑制转录活性的强度与潜在的结合因子DP10 3的表达水平之间没有相关性。共转染实验还表明 ,参与hB1F转录活性调控的辅激活子SRC 1和辅抑制子SMRT与该抑制作用不直接相关。实验结果提示 ,孤儿核受体hB1F转录活性可能存在一种新的调控机制。     

Molecular Dynamics Simulation in Solvent of the Estrogen Receptor Protein DNA Binding Domain in Complex with a Non-Consensus Estrogen Response Element DNA Sequence

Abstract

We investigated protein/DNA interactions, using molecular dynamics simulations computed between a 10 Angstom water layer model of the estrogen receptor (ER) protein DNA binding domain (DBD) amino acids and DNA of a non-consensus estrogen response element (ERE) consisting of 29 nucleotide base pairs. This ERE nucleotide sequence occurs naturally upstream of the Xenopus laevis Vitelligenin AI gene. The ER DBD is encoded by three exons. Namely, exons 2 and 3 which encode the two zinc binding motifs and a sequence of exon 4 which encodes a predicted alpha helix. We generated a computer model of the ER DBD using atomic coordinates derived from the average of 30 nuclear magnetic resonance (NMR) spectroscopy coordinate sets. Amino acids on the carboxyl end of the ER DBD were disordered in both X-ray crystallography and NMR determinations and no coordinates were reported. This disordered region includes 10 amino acids of a predicted alpha helix encoded in exon 4 at the exon 3/4 splice junction. These amino acids are known to be important in DNA binding and are also believed to function as a nuclear translocation signal sequence for the ER protein. We generated a computer model of the predicted alpha helix consisting of the 10 amino acids encoded in exon 4 and attached this helix to the carboxyl end of the ER DBD at the exon 3/4 splice junction site. We docked the ER DBD model within the DNA major groove halfsites of the 29 base pair non-consensus ERE and flanking nucleotides. We constructed a solvated model with the ER DBD/ERE complex surrounded by a ten Angstrom water layer and conducted molecular dynamics simulations. Hydrogen bonding interactions were monitored. In addition, van der Waals and electrostatic interaction energies were calculated. Amino acids of the ER DBD DNA recognition helix formed both direct and water mediated hydrogen bonds at cognate codon-anticodon nucleotide base and backbone sites within the ERE DNA right major groove halfsite. Amino acids of the ER DBD exon 4 encoded predicted alpha helix formed direct and water mediated H-bonds with base and backbone sites of their cognate codon-anticodon nucleotides within the minor grooves flanking the ERE DNA major groove halfsites. These interactions together induced bending of the DNA into the protein.     

Determinants of target gene specificity for ROR alpha 1: monomeric DNA binding by an orphan nuclear receptor.

The ROR alpha isoforms are orphan members of the steroid/thyroid/retinoid receptor superfamily. Previous DNA-binding studies indicated that ROR alpha isoforms bind to response elements consisting of a single copy of the core recognition sequence AGGTCA preceded by a 6-bp A/T-rich sequence and that the distinct amino-terminal domains of each isoform influence DNA-binding specificity. In this report, we have investigated in detail the protein determinants of target gene specificity for the ROR alpha 1 isoform and have now identified the minimal sequence both in its amino- and carboxy-terminal domains required for high-affinity DNA binding. High-resolution methylation and ethylation interference analyses and mixing of truncated proteins in a DNA-binding assay show that ROR alpha 1 presumably binds along one face of the DNA helix as a monomer. By analogy to previous studies of the orphan receptors NGFI-B and FTZ-F1, extensive mutational analysis of the ROR alpha 1 protein shows that a domain extending from the carboxy-terminal end of the second conserved zinc-binding motif is required for specific DNA recognition. However, point mutations and domain swap experiments between ROR alpha 1 and NGFI-B demonstrated that sequence-specific recognition dictated by the carboxy-terminal extension is determined by distinct subdomains in the two receptors. These results demonstrate that monomeric nuclear receptors utilize diverse mechanisms to achieve high-affinity and specific DNA binding and that ROR alpha 1 represents the prototype for a distinct subfamily of monomeric orphan nuclear receptors.     

A helix-turn-helix structure unit in human centromere protein B (CENP-B).

CENP-B has been suggested to organize arrays of centromere satellite DNA into a higher order structure which then directs centromere formation and kinetochore assembly in mammalian chromosomes. The N-terminal portion of CENP-B is a 15 kDa DNA binding domain (DBD) consisting of two repeating units, RP1 and RP2. The DBD specifically binds to the CENP-B box sequence (17 bp) in centromere DNA. We determined the solution structure of human CENP-B DBD RP1 by multi-dimensional 1H, 13C and 15N NMR methods. The CENP-B DBD RP1 structure consists of four helices and has a helix-turn-helix structure. The overall folding is similar to those of some other eukaryotic DBDs, although significant sequence homology with these proteins was not found. The DBD of yeast RAP1, a telomere binding protein, is most similar to CENP-B DBD RP1. We studied the interaction between CENP-B DBD RP1 and the CENP-B box by the use of NMR chemical shift perturbation. The results suggest that CENP-B DBD RP1 interacts with one of the essential regions of the CENP-B box DNA, mainly at the N-terminal basic region, the N-terminal portion of helix 2 and helix 3.     

Proposed mechanism for the stabilization of nuclear receptor DNA binding via protein dimerization.

Hepatocyte nuclear factor 4 (HNF-4) defines a new subgroup of nuclear receptors that exist in solution and bind DNA exclusively as homodimers. We recently showed that the putative ligand binding domain (LBD) of HNF-4 is responsible for dimerization in solution and prevents heterodimerization with other receptors. In this report, the role of the LBD in DNA binding by HNF-4 is further investigated by using electrophoretic mobility shift analysis. A comparison of constructs containing either the DNA binding domain (DBD) alone or the DBD plus the LBD of HNF-4 showed that dimerization via the DBD was sufficient to provide nearly the full DNA binding affinity of the full-length HNF-4. In contrast, dimerization via the DBD was not sufficient to produce a stable protein-DNA complex, whereas dimerization via the LBD increased the half-life of the complex by at least 100-fold. Circular permutation analysis showed that full-length HNF-4 bent DNA by approximately 80 degrees while the DBD bent DNA by only 24 degrees. Nonetheless, analysis of other constructs indicated that the increase in stability afforded by the LBD could be explained only partially by an increased ability to bend DNA. Coimmunoprecipitation studies, on the other hand, showed that dimerization via the LBD produced a protein-protein complex that was much more stable than the corresponding protein-DNA complex. These results led us to propose a model in which dimerization via the LBD stabilizes the receptor on DNA by converting an energetically favorable two-step dissociation event into an energetically unfavorable single-step event. Implications of this one-step model for other nuclear receptors are discussed.