The structure of the ultraspiracle ligand-binding domain reveals a nuclear receptor locked in an inactive conformation

Ultraspiracle (USP) is the invertebrate homologue of the mammalian retinoid X receptor (RXR). RXR plays a uniquely important role in differentiation, development, and homeostasis through its ability to serve as a heterodimeric partner to many other nuclear receptors. RXR is able to influence the activity of its partner receptors through the action of the ligand 9-cis retinoic acid. In contrast to RXR, USP has no known high-affinity ligand and is thought to be a silent component in the heterodimeric complex with partner receptors such as the ecdysone receptor. Here we report the 2.4-Å crystal structure of the USP ligand-binding domain. The structure shows that a conserved sequence motif found in dipteran and lepidopteran USPs, but not in mammalian RXRs, serves to lock USP in an inactive conformation. It also shows that USP has a large hydrophobic cavity, implying that there is almost certainly a natural ligand for USP. This cavity is larger than that seen previously for most other nuclear receptors. Intriguingly, this cavity has partial occupancy by a bound lipid, which is likely to resemble the natural ligand for USP.

Arrayed transposase-binding sequences on the ends of transposon Tn5090/Tn402

The transposon Tn5090/Tn402 encodes a 559 amino acid transposase, TniA, with a DDE motif. Gel mobility shifting and cleavage protection analysis with DNase I and hydroxyl radical probes revealed that TniA binds to multiple repeat sequences on either terminus of Tn5090/Tn402. Four of these TniA-binding 19mers occurred on the left-hand (t) end and two on the right-hand (i) end. Hydroxyl radical cleavage protection demonstrated the presence of 3–6 bp contact sequences on one face of the DNA helix. The binding pattern and organisation of repeats suggested parallels between Tn5090/Tn402 and Mu, which controls its transpositional activity in the assembly step of a higher order transpososome complex. The complex terminal structure and genes of transposase and nucleotide-binding proteins in tandem are hallmarks of the handful of Mu-like elements that are known to date.

Structure of human DNMT2, an enigmatic DNA methyltransferase homolog that displays denaturant-resistant binding to DNA

DNMT2 is a human protein that displays strong sequence similarities to DNA (cytosine-5)-methyltransferases (m5C MTases) of both prokaryotes and eukaryotes. DNMT2 contains all 10 sequence motifs that are conserved among m5C MTases, including the consensus S-adenosyl-l-methionine-binding motifs and the active site ProCys dipeptide. DNMT2 has close homologs in plants, insects and Schizosaccharomyces pombe, but no related sequence can be found in the genomes of Saccharomyces cerevisiae or Caenorhabditis elegans. The crystal structure of a deletion mutant of DNMT2 complexed with S-adenosyl-l-homocysteine (AdoHcy) has been determined at 1.8 Å resolution. The structure of the large domain that contains the sequence motifs involved in catalysis is remarkably similar to that of M.HhaI, a confirmed bacterial m5C MTase, and the smaller target recognition domains of DNMT2 and M.HhaI are also closely related in overall structure. The small domain of DNMT2 contains three short helices that are not present in M.HhaI. DNMT2 binds AdoHcy in the same conformation as confirmed m5C MTases and, while DNMT2 shares all sequence and structural features with m5C MTases, it has failed to demonstrate detectable transmethylase activity. We show here that homologs of DNMT2, which are present in some organisms that are not known to methylate their genomes, contain a specific target-recognizing sequence motif including an invariant CysPheThr tripeptide. DNMT2 binds DNA to form a denaturant-resistant complex in vitro. While the biological function of DNMT2 is not yet known, the strong binding to DNA suggests that DNMT2 may mark specific sequences in the genome by binding to DNA through the specific target-recognizing motif.

Analysis of the Xenopus laevis CCAAT-enhancer binding protein α gene promoter demonstrates species-specific differences in the mechanisms for both auto-activation and regulation by Sp1

Transcription factors belonging to the CCAAT-enhancer binding protein (C/EBP) family have been implicated in the regulation of gene expression during differentiation, development and disease. Autoregulation is relatively common in the modulation of C/EBP gene expression and the murine and human C/EBPα genes have been shown to be auto-activated by different mechanisms. In the light of this finding, it is essential that autoregulation of C/EBPα genes from a wider range of different species be investigated in order to gauge the degree of commonality, or otherwise, that may exist. We report here studies that investigate the regulation of the Xenopus laevis C/EBPα gene (xC/EBPα). The –1131/+41 promoter region was capable of directing high levels of expression in both the human hepatoma Hep3B and the Xenopus kidney epithelial A6 cell lines, and was auto-activated by expression vectors specifying for xC/EBPα or xC/EBPβ. Deletion analysis showed that the –321/+41 sequence was sufficient for both the constitutive promoter activity and auto-activation and electrophoretic mobility shift assays identified the interaction of C/EBPs and Sp1 to this region. Although deletion of either the C/EBP or the Sp1 site drastically reduced the xC/EBPα promoter activity, multimers of only the C/EBP site could confer autoregulation to a heterologous SV40 promoter. These results indicate that, in contrast to the human promoter and in common with the murine gene, the xC/EBPα promoter was subject to direct autoregulation. In addition, we demonstrate a novel species-specific action of Sp1 in the regulation of C/EBPα expression, with the factor able to repress the murine promoter but activate the Xenopus gene.

Identification of human DNA helicase V with the far upstream element-binding protein

The properties of human DNA helicase V (HDH V) were studied in greater detail following an improved purification procedure. From 450 g of cultured cells, <0.1 mg of pure protein was isolated. HDH V unwinds DNA unidirectionally by moving in the 3′ to 5′ direction along the bound strand in an ATP- and Mg2+-dependent fashion. The enzyme is not processive and can also unwind partial RNA–RNA duplexes such as HDH IV and HDH VIII. The Mr determined by SDS–PAGE (66 kDa) corresponds to that measured under native conditions, suggesting that HDH V exists as a monomer in the nucleus. Microsequencing of the purified HDH V shows that this enzyme is identical to the far upstream element-binding protein (FBP), a protein that stimulates the activity of the c-myc gene by binding specifically to the ‘FUSE’ DNA region localized upstream of its promoter. The sequence of HDH V/FBP contains RGG motifs like HDH IV/nucleolin, HDH VIII/G3BP as well as other human RNA and DNA helicases identified by other laboratories.

Determination of the binding constants of the centromere protein Cbf1 to all 16 centromere DNAs of Saccharomyces cerevisiae

Cbf1p is a Saccharomyces cerevisiae chromatin protein belonging to the basic region helix–loop–helix leucine zipper (bHLHzip) family of DNA binding proteins. Cbf1p binds to a conserved element in the 5′-flanking region of methionine biosynthetic genes and to centromere DNA element I (CDEI) of S.cerevisiae centromeric DNA. We have determined the apparent equilibrium dissociation constants of Cbf1p binding to all 16 CDEI DNAs in gel retardation assays. Binding constants of full-length Cbf1p vary between 1.7 and 3.8 nM. However, the dissociation constants of a Cbf1p deletion variant that has been shown to be fully sufficient for Cbf1p function in vivo vary in a range between 3.2 and 12 nM. In addition, native polyacrylamide gel electrophoresis revealed distinct changes in the 3D structure of the Cbf1p/CEN complexes. We also show that the previously reported DNA binding stimulation activity of the centromere protein p64 functions on both the Cbf1 full-length protein and a deletion variant containing only the bHLHzip domain of Cbf1p. Our results suggest that centromeric DNA outside the consensus CDEI sequence and interaction of Cbf1p with adjacent centromere proteins contribute to the complex formation between Cbf1p and CEN DNA.

DNA-binding and strand-annealing activities of human Mre11: implications for its roles in DNA double-strand break repair pathways

DNA double-strand breaks (DSBs) in eukaryotic cells can be repaired by non-homologous end-joining or homologous recombination. The complex containing the Mre11, Rad50 and Nbs1 proteins has been implicated in both DSB repair pathways, even though they are mechanistically different. To get a better understanding of the properties of the human Mre11 (hMre11) protein, we investigated some of its biochemical activities. We found that hMre11 binds both double- and single-stranded (ss)DNA, with a preference for ssDNA. hMre11 does not require DNA ends for efficient binding. Interestingly, hMre11 mediates the annealing of complementary ssDNA molecules. In contrast to the annealing activity of the homologous recombination protein hRad52, the activity of hMre11 is abrogated by the ssDNA binding protein hRPA. We discuss the possible implications of the results for the role(s) of hMre11 in both DSB repair pathways.

A short segment within the cytoplasmic domain of the neural cell adhesion molecule (N-CAM) is essential for N-CAM-induced NF-κB activity in astrocytes

The neural cell adhesion molecule (N-CAM) is expressed on the surface of astrocytes, where its homophilic binding leads to the activation of the transcription factor NF-κB. Transfection of astrocytes with a construct encompassing the transmembrane region and the cytoplasmic domain of N-CAM (designated Tm-Cyto, amino acids 685–839 in the full-length molecule) inhibited this activation up to 40%, and inhibited N-CAM-induced translocation of NF-κB to the nucleus. N-CAM also activated NF-κB in astrocytes from N-CAM knockout mice, presumably through binding to a heterophile. This activation, however, was not blocked by Tm-Cyto expression, indicating that the inhibitory effect of the Tm-Cyto construct is specific for cell surface N-CAM. Deletions and point mutations of the cytoplasmic portion of the Tm-Cyto construct indicated that the region between amino acids 780 and 800 were essential for inhibitory activity. This region contains four threonines (788, 793, 794, and 797). Mutation to alanine of T788, T794, or T797, but not T793, abolished inhibitory activity, as did mutation of T788 or T797 to aspartic acid. A Tm-Cyto construct with T794 mutated to aspartic acid retained inhibitory activity but did not itself induce a constitutive NF-κB response. This result suggests that phosphorylation of T794 may be necessary but is not the triggering event. Overall, these findings define a short segment of the N-CAM cytoplasmic domain that is critical for N-CAM-induced activation of NF-κB and may be important in other N-CAM-mediated signaling.

Activity-dependent CREB phosphorylation: Convergence of a fast, sensitive calmodulin kinase pathway and a slow, less sensitive mitogen-activated protein kinase pathway

The cAMP-responsive element binding protein (CREB), a key regulator of gene expression, is activated by phosphorylation on Ser-133. Several different protein kinases possess the capability of driving this phosphorylation, making it a point of potential convergence for multiple intracellular signaling cascades. Previous work in neurons has indicated that physiologic synaptic stimulation recruits a fast calmodulin kinase IV (CaMKIV)-dependent pathway that dominates early signaling to CREB. Here we show in hippocampal neurons that the fast, CaMK-dependent pathway can be followed by a slower pathway that depends on Ras/mitogen-activated protein kinase (MAPK), along with CaMK. This pathway was blocked by dominant-negative Ras and was specifically recruited by depolarizations that produced strong intracellular Ca2+ transients. When both pathways were recruited, phosphorylated CREB (pCREB) formation was overwhelmingly dominated by the CaMK pathway between 0 and 10 min, and by the MAPK pathway at 60 min, whereas the two pathways acted in concert at 30 min. The Ca2+ signals that produced only rapid CaMK signaling to pCREB or both rapid CaMK and slow MAPK signaling deviated significantly for only ≈1 min, yet their differential impact on pCREB extended over a much longer period, between 20 and 60 min and beyond, which is of likely significance for gene expression. The CaMK-dependent MAPK pathway may inform the nucleus about stimulus amplitude. In contrast, the CaMKIV pathway may be well suited to conveying information on the precise timing of localized synaptic stimuli, befitting its greater speed and sensitivity, whereas the previously described calcineurin pathway may carry information about stimulus duration.

Triplex targeting of human PDGF-B (c-sis, proto-oncogene) promoter specifically inhibits factors binding and PDGF-B transcription

Human c-sis/PDGF-B proto-oncogene has been shown to be overexpressed in a large percentage of human tumor cells establishing a growth-promoting, autocrine growth circuit. Triplex forming oligonucleotides (TFOs) can recognize and bind sequences in duplex DNA, and have received considerable attention because of their potential for targeting specific genomic sites. The c-sis/PDGF-B promoter contains a unique homopurine/homopyrimidine sequence (SIS proximal element, SPE), which is crucial for binding nuclear factors that provoke transcription. In order to develop specific transcriptional inhibitors of the human c-sis/PDGF-B proto-oncogene, 20 potential TFOs targeting part or all of the SPE were screened by gel mobility analysis. DNase I footprinting shows that the TFOs we designed can form a sequence-specific triplex with the target. Protein binding assays demonstrate that triplex formation inhibits nuclear factors binding the c-sis/PDGF-B promoter. Both transient and stable transfection experiments demonstrate that the transcriptional activity of the promoter is considerably inhibited by the TFOs. We propose that TFOs represent a therapeutic potential to specifically diminish the expression of c-sis/PDGF-B proto-oncogene in various pathologic settings where constitutive expression of this gene has been observed.

Serine Phosphorylation of Focal Adhesion Kinase in Interphase and Mitosis: A Possible Role in Modulating Binding to p130Cas

Focal adhesion kinase (FAK) is an important regulator of integrin signaling in adherent cells and accordingly its activity is significantly modulated during mitosis when cells detach from the extracellular matrix. During mitosis, FAK becomes heavily phosphorylated on serine residues concomitant with its inactivation and dephosphorylation on tyrosine. Little is known about the regulation of FAK activity by serine phosphorylation. In this report, we characterize two novel sites of serine phosphorylation within the C-terminal domain of FAK. Phosphorylation-specific antibodies directed to these sites and against two previously characterized sites of serine phosphorylation were used to study the regulated phosphorylation of FAK in unsynchronized and mitotic cells. Among the four major phosphorylation sites, designated pS1-pS4, phosphorylation of pS1 (Ser722) is unchanged in unsynchronized and mitotic cells. In contrast, pS3 and pS4 (Ser843 and Ser910) exhibit increased phosphorylation during mitosis. In vitro peptide binding experiments provide evidence that phosphorylation of pS1 (Ser722) may play a role in modulating FAK binding to the SH3 domain of the adapter protein p130Cas.

The Catastrophe-promoting Activity of Ectopic Op18/Stathmin Is Required for Disruption of Mitotic Spindles But Not Interphase Microtubules

Oncoprotein18/stathmin (Op18) is a microtubule (MT) destabilizing protein that is inactivated during mitosis by phosphorylation at four Ser-residues. Op18 has at least two functions; the N-terminal region is required for catastrophe-promotion (i.e., transition from elongation to shortening), while the C-terminal region is required to inhibit MT-polymerization rate in vitro. We show here that a “pseudophosphorylation” derivative of Op18 (i.e., four Ser- to Glu-substitutions at phosphorylation sites) exhibits a selective loss of catastrophe-promoting activity. This is contrasted to authentic phosphorylation, which efficiently attenuates all activities except tubulin binding. In intact cells, overexpression of pseudophosphorylated Op18, which is not phosphorylated by endogenous kinases, is shown to destabilize interphase MTs but to leave spindle formation untouched. To test if the mitotic spindle is sensitive only to the catastrophe-promoting activity of Op18 and resistant to C-terminally associated activities, N- and C-terminal truncations with defined activity-profiles were employed. The cell-cycle phenotypes of nonphosphorylatable mutants (i.e., four Ser- to Ala-substitutions) of these truncation derivatives demonstrated that catastrophe promotion is required for interference with the mitotic spindle, while the C-terminally associated activities are sufficient to destabilize interphase MTs. These results demonstrate that specific Op18 derivatives with defined activity-profiles can be used as probes to distinguish interphase and mitotic MTs.

An initial ATP-independent step in the unwinding of a herpes simplex virus type I origin of replication by a complex of the viral origin-binding protein and single-strand DNA-binding protein

Using a spectrophotometric assay that measures the hyperchromicity that accompanies the unwinding of a DNA duplex, we have identified an ATP-independent step in the unwinding of a herpes simplex virus type 1 (HSV-1) origin of replication, Oris, by a complex of the HSV-1 origin binding protein (UL9 protein) and the HSV-1 single-strand DNA binding protein (ICP8). The sequence unwound is the 18-bp A + T-rich segment that links the two high-affinity UL9 protein binding sites, boxes I and II of Oris. P1 nuclease sensitivity of Oris and single-strand DNA-dependent ATPase measurements of the UL9 protein indicate that, at 37°C, the A + T-rich segment is sufficiently single stranded to permit the binding of ICP8. Binding of the UL9 protein to boxes I and II then results in the formation of the UL9 protein–ICP8 complex, that can, in the absence of ATP, promote unwinding of the A + T-rich segment. On addition of ATP, the helicase activity of the UL9 protein–ICP8 complex can unwind boxes I and II, permitting access of the replication machinery to the Oris sequences.

Activation of Trp3 by inositol 1,4,5-trisphosphate receptors through displacement of inhibitory calmodulin from a common binding domain

Mammalian homologues of Drosophila Trp form plasma membrane channels that mediate Ca2+ influx in response to activation of phospholipase C and internal Ca2+ store depletion. Previous studies showed that human Trp3 is activated by inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) and identified interacting domains, one on Trp and two on IP3R. We now find that Trp3 binds Ca2+-calmodulin (Ca2+/CaM) at a site that overlaps with the IP3R binding domain. Using patch-clamp recordings from inside-out patches, we further show that Trp3 has a high intrinsic activity that is suppressed by Ca2+/CaM under resting conditions, and that Trp3 is activated by the following: a Trp-binding peptide from IP3R that displaces CaM from Trp3, a myosin light chain kinase Ca2+/CaM binding peptide that prevents CaM from binding to Trp3, and calmidazolium, an inactivator of Ca2+/CaM. We conclude that inhibition of the inhibitory action of CaM is a key step of Trp3 channel activation by IP3Rs.

Allosteric crosstalk between peptide-binding, transport, and ATP hydrolysis of the ABC transporter TAP

The transporter associated with antigen processing (TAP) is essential for intracellular transport of protein fragments into the endoplasmic reticulum for loading of major histocompatibility complex (MHC) class I molecules. On the cell surface, these peptide–MHC complexes are monitored by cytotoxic T lymphocytes. To study the ATP hydrolysis of TAP, we developed an enrichment and reconstitution procedure, by which we fully restored TAP function in proteoliposomes. A TAP-specific ATPase activity was identified that could be stimulated by peptides and blocked by the herpes simplex virus protein ICP47. Strikingly, the peptide-binding motif of TAP directly correlates with the stimulation of the ATPase activity, demonstrating that the initial peptide-binding step is responsible for TAP selectivity. ATP hydrolysis follows Michaelis–Menten kinetics with a maximal velocity Vmax of 2 μmol/min per mg TAP, corresponding to a turnover number of approximately 5 ATP per second. This turnover rate is sufficient to account for the role of TAP in peptide loading of MHC molecules and the overall process of antigen presentation. Interestingly, sterically restricted peptides that bind but are not transported by TAP do not stimulate ATPase activity. These results point to coordinated dialogue between the peptide-binding site, the nucleotide-binding domain, and the translocation site via conformational changes within the TAP complex.