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PubMed2002

PurT-encoded glycinamide ribonucleotide transformylase. Accommodation of adenosine nucleotide analogs within the active site.

PurT-encoded glycinamide ribonucleotide transformylase, or PurT transformylase, functions in purine biosynthesis by catalyzing the formylation of glycinamide ribonucleotide through a catalytic mechanism requiring Mg(2+)ATP and formate. From previous x-ray diffraction analyses, it has been demonstrated that PurT transformylase from Escherichia coli belongs to the ATP-grasp superfamily of enzymes, which are characterized by three structural motifs referred to as the A-, B-, and C-domains. In all of the ATP-grasp enzymes studied to date, the adenosine nucleotide ligands are invariably wedged between the B- and C-domains, and in some cases, such as biotin carboxylase and carbamoyl phosphate synthetase, the B-domains move significantly upon nucleotide binding. Here we present a systematic and high-resolution structural investigation of PurT transformylase complexed with various adenosine nucleotides or nucleotide analogs including Mg(2+)ATP, Mg(2+)-5'-adenylylimidodiphosphate, Mg(2+)-beta,gamma-methyleneadenosine 5'-triphosphate, Mg(2+)ATPgammaS, or Mg(2+)ADP. Taken together, these studies indicate that the conformation of the so-called "T-loop," delineated by Lys-155 to Gln-165, is highly sensitive to the chemical identity of the nucleotide situated in the binding pocket. This sensitivity to nucleotide identity is in sharp contrast to that observed for the "P-loop"-containing enzymes, in which the conformation of the binding motif is virtually unchanged in the presence or absence of nucleotides.

Adenosine Diphosphate↗
PubMed1994

A genetic analysis of processes regulating cytochrome P4501A1 expression.

Cytochrome P4501A1 and its associated aryl hydrocarbon hydroxylase activity are highly inducible in the mouse hepatoma cell line, Hepa-1, by substrates of the enzyme and related compounds, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Mutants of this cell line, deficient in P4501A1 inducibility, were isolated. Some of the mutants show a dominant phenotype. Such mutants may have resulted from a genetic alteration leading to the inappropriate activation of a repressor gene that normally functions to restrict high level inducibility to the liver and certain other organs or to certain developmental stages. One dominant mutant was shown to express a protein that prevents binding of the liganded aryl hydrocarbon (Ah) receptor (which mediates induction of P4501A1) to its recognition sequence in DNA (the xenobiotic responsive element, or XRE). The majority of mutants are recessive, and were assigned to four different complementation groups (which probably correspond to four different genes). Gene A corresponds to the structural gene (Cyp1a-1) for P4501A1. Mutations in genes B, C and D all affect functioning of the Ah receptor. A cDNA for gene C was cloned. The encoded protein (ARNT) is required for ligand-dependent translocation of the Ah receptor to the nucleus and its binding to the XRE. ARNT and the Ah receptor form a heterodimeric complex which binds the XRE in a fashion such that both subunits bind the XRE directly. Both ARNT and the Ah receptor contain basic helix-loop-helix motifs. Such motifs have been identified in several transcription factors that bind DNA as heterodimers or homodimers. The roles of the proteins corresponding to the B and D genes are presently under investigation.

Animals↗
PubMed1996

Collier, a novel regulator of Drosophila head development, is expressed in a single mitotic domain.

BACKGROUND: Segmentation of the Drosophila embryo is based on a cascade of hierarchical gene interactions that is initiated by maternal morphogens; these interactions define spatially restricted domains of zygotic gene expression within the blastoderm. Although the hierarchy of the segmentation genes that subdivide the trunk is well established, the patterning of the head is less well understood. Seven head segments can be assigned on the basis of metameric patterns of segment-polarity gene expression and internal sensory organs. The domains of expression of head gap-like genes broadly overlap, with their posterior margins out of phase by one segment. Taken together with the lack of pair-rule gene expression in the head, these observations led to the suggestion that head gap genes act in a combinatorial manner, determining head segmental borders and segmental identity at the same time. RESULTS: We have identified a new Drosophila gene, collier (col), whose expression at the blastoderm stage is restricted to a single stripe of cells corresponding to part of the intercalary and mandibular segment primordia, possibly parasegment O. Reduction of col activity in early gastrula embryos by antisense RNA expression results in a specific lack of head structures derived from these segments. The expression of col coincides with a mitotic domain, which supports the proposal that cells in this domain undergo a concerted mitotic and differentiation program that is orchestrated at the transcriptional level. Col is an ortholog of mammalian early B-cell factor/Olfactory-1. These proteins define a new family of transcription factors that contain a helix-loop-helix dimerization motif and a new type of DNA-binding domain that is highly conserved during evolution. CONCLUSIONS: Here we describe Col, the first Drosophila member of a new family of transcription factors. Col may act as a "second-level regulator' of head patterning. The structural conservation of Col during evolution raises the questions of its conservation of function in head specification and its interactions with other factors conserved between insects and vertebrates.

Amino Acid Sequence↗
PubMed1994

NMR studies and conformational analysis of a DNA four-way junction formed in a linear synthetic oligonucleotide.

A linear DNA oligomer (M(r) 14,000, 46 nucleotides) was especially designed, chemically synthesized, and studied by means of 1H NMR spectroscopy. The design of the oligomer was guided by the idea that incorporation of three short palindromic sequences, each interspersed by 5'-CTTG-3' motifs at predetermined positions in the oligomer, would give rise to the formation of three stable minihairpin loops [Ippel, J. H., et al. (1992) J. Biomol. Struct. Dyn. 9, 1-16], which in turn were expected to encourage further folding of the strand into a stable four-way junction containing three "hairpin" arms and an open-ended duplex stem as the fourth arm. Linear DNA four-way junctions constructed according to this concept can be more compact and are therefore expected to be more suitable as model compounds for conformational studies compared to junctions that are built from two or more separate strands. A stable cruciform conformation was substantiated for the 46-mer in aqueous solution in the presence of Mg2+. Complete sequential 1H NMR assignments of the nonexchangeable protons (except H4', H5', and H5") were obtained with the aid of NOESY and HOHAHA experiments. The NMR data gave evidence for the expected existence of minihairpin-loop structures at the three 5'-CTTG-3' motifs in the sequence. The complementary stem domains adopt a regular B-DNA form. Watson-Crick type base pairing is preserved for all residues in the stem domains, including the residues at the center of the junction. A systematic investigation of the interresidual NOEs observed between the protons of the eight central residues revealed the complete stacking pattern of the residues at the branch point.

Base Sequence↗
PubMed1993

A cellular protein binds B1 and Alu small cytoplasmic RNAs in vitro.

B1 and Alu are sequence-homologous interspersed elements of unknown function that have expanded in the genomes of mice and humans, respectively. A minority of B1 and Alu sequences are expressed as small cytoplasmic RNAs. These RNAs have conserved a secondary structure motif also present in signal recognition particle (SRP) RNA despite substantial sequence divergence, whereas random B1 and Alu sequences have not. This RNA structure has also been conserved by the source sequences that gave rise to successive transpositions during B1 and Alu evolution. In the present work small cytoplasmic B1 and Alu RNAs synthesized in vitro were found to bind a cellular protein by mobility shift and UV cross-linking analyses. The mouse and human proteins demonstrate the same specificity to a panel of competitor RNAs. Results using mutated B1 RNA indicate that a single strand loop in the conserved Alu motif is essential for binding. Previous work by Strub et al. (Stub, K., Moss, J. B., and Walter, P. (1991) Mol. Cell. Biol. 11, 3949-3959) demonstrated that the Alu-specific protein SRP 9/14 does not footprint to this region of SRP RNA. This observation coupled with the failure of anti-SRP/9 antibodies to identify SRP 9/14 in the B1 RNA-protein complex as well as the apparent mass and other characteristics of the protein described here suggest that it is a novel B1-Alu RNA-binding protein. Conservation of primary and secondary structure by B1 and Alu small cytoplasmic RNAs as well as features of their specific expression and ability to interact with the conserved binding protein indicate that these RNAs are more homologous than previously appreciated.

Animals↗
PubMed1994

Solution structure of the TR1C fragment of skeletal muscle troponin-C.

Residues 12-87 (TR1C fragment) of turkey skeletal muscle troponin-C comprises two helix-loop-helix calcium-binding motifs which are the regulatory calcium-binding sites in the N-terminal domain of the protein. We have used the combined distance geometry-simulated annealing protocol DGII (Havel, T. F. (1991) Prog. Biophys. Mol. Biol. 56, 43-78) to determine the structure of this 76-residue polypeptide in solution from 475 1H NMR-derived distance restraints. The nuclear Overhauser enhancement-derived distance constraints used in the DGII protocol were supplemented by introducing generic hydrogen bond distance restraints for slowly exchanging amide hydrogens in regular secondary structure elements, by restricting the available phi angle space to -180 degrees to 0 degrees for all residues except glycines, and by tailoring the distance boundaries used for quantitating the nuclear Overhauser enhancement intensities to correspond to characteristic distances found in helices. This improved the geometry of the four helices in the resulting structures. The relative positions of helices A and B which flank calcium-binding loop 1, helix D which follows calcium-binding loop 2, and the beta-sheet between the two calcium-binding loops were well defined and had an overall root-mean-square deviation for 20 converged structures of 1.4 +/- 0.2 A for backbone atoms. The structure and relative orientations of these regions are very similar to these of the corresponding regions of the protein in the crystal structure of intact turkey skeletal troponin C (Herzberg, O., and James, M. N. G. (1988) Nature 313, 653-659). The structure of helix C was well defined, but its relative position to the other helices was not defined. It occupied a range of positions in the set of 20 DGII structures, the average of which was quite similar to the orientation of helix C in the x-ray structure. The overall structure of the apo regulatory domain of troponin-C is therefore not affected by the loss of the N-helix, or the low pH conditions used for the x-ray structure, but may be more flexible in regions known to be involved in contacts with other skeletal muscle regulatory proteins.

Amino Acid Sequence↗
PubMed1998

Three-dimensional structure of adenosylcobinamide kinase/adenosylcobinamide phosphate guanylyltransferase from Salmonella typhimurium determined to 2.3 A resolution,.

The X-ray structure of adenosylcobinamide kinase/adenosylcobinamide phosphate guanylyltransferase (CobU) from Salmonella typhimurium has been determined to 2.3 A resolution. This enzyme of subunit molecular weight 19 770 plays a central role in the assembly of the nucleotide loop for adenosylcobalamin where it catalyzes both the phosphorylation of the 1-amino-2-propanol side chain of the corrin ring and the subsequent attachment of GMP to form the product adenosylcobinamide-GDP. The kinase activity is believed to be associated with a P-loop motif, whereas the transferase activity proceeds at a different site on the enzyme via a guanylyl intermediate. The enzyme was crystallized in the space group C2221 with unit cell dimensions of a = 96.4 A, b = 114.4 A, and c = 106.7 A, with three subunits per asymmetric unit. The structure reveals that the enzyme is a molecular trimer and appears somewhat like a propeller with overall molecular dimensions of approximately 64 A x 77 A x 131 A. Each subunit consists of a single domain that is dominated by a seven-stranded mixed beta-sheet flanked on either side by a total of five alpha-helices and one helical turn. Six of the seven beta-strands run parallel. The C-terminal strand lies at the edge of the sheet and runs antiparallel to the others. Interestingly, CobU displays a remarkable structural and topological similarity to the central domain of the RecA protein, although the reason for this observation is unclear. The structure contains a P-loop motif located at the base of a prominent cleft formed by the association of two subunits and is most likely the kinase active site. Each subunit of CobU contains a cis peptide bond between Glu80 and Cys81 where Glu80 faces the P-loop and might serve to coordinate the magnesium ion of the triphosphate substrate. Interestingly, His46, which is the putative site for guanylylation, lies approximately 21 A from the P-loop and is solvent-exposed. This suggests that the enzyme undergoes a conformational change when the substrates bind to bring these two active sites into closer proximity.

Adenosine Triphosphate↗
PubMed1999

A conserved motif present in a class of helix-loop-helix proteins activates transcription by direct recruitment of the SAGA complex.

The class I helix-loop-helix (HLH) proteins, which include E2A, HEB, and E2-2, have been shown to be required for lineage-specific gene expression during T and B lymphocyte development. Additionally, the E2A proteins function to regulate V(D)J recombination, possibly by allowing access of variable region segments to the recombination machinery. The mechanisms by which E2A regulates transcription and recombination, however, are largely unknown. Here, we identify a novel motif, LDFS, present in the vertebrate class I HLH proteins as well as in a yeast HLH protein that is essential for transactivation. We provide both genetic and biochemical evidence that the highly conserved LDFS motif stimulates transcription by direct recruitment of the SAGA histone acetyltransferase complex.

Amino Acid Sequence↗
PubMed2005

Regulation of the multidrug resistance transporter P-glycoprotein in multicellular prostate tumor spheroids by hyperthermia and reactive oxygen species.

Hyperthermia is an important component of many cancer treatment protocols. In our study the regulation of the multidrug resistance (MDR) transporter P-glycoprotein by hyperthermia was studied in multicellular prostate tumor spheroids. Hyperthermia treatment of small (50-100 microm) tumor spheroids significantly increased P-glycoprotein and mdr-1 mRNA expression with a maximum effect at 42 degrees C, whereas only moderate elevation of P-glycoprotein was found in large (350-450 microm) tumor spheroids. Hyperthermia caused an elevation of intracellular reactive oxygen species (ROS). Inhibition of ROS generation with NADPH-oxidase inhibitors diphenylen iodonium (DPI) and 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF) abolished P-glycoprotein expression but did not affect its transcript levels following heat treatment. This indicates that P-glycoprotein levels are controlled by regulating its translation rate or stability. Hyperthermia incubation resulted in a differential activation of p38 mitogen-activated protein kinase (MAPK), extracellular regulated kinase 1,2 (ERK1,2), and c-jun N-terminal kinase (JNK) immediately, 4 hr and 24 hr after treatment. Furthermore, upregulation of hypoxia-inducible factor 1alpha (HIF-1alpha) was observed. Elevation of HIF-1alpha and P-glycoprotein expression following hyperthermia treatment were abolished upon coadministration of the p38 inhibitor SB203580. In contrast the JNK inhibitor SP600125 and the ERK1,2 inhibitor UO126 resulted in increase of HIF-1alpha and P-glycoprotein in the control as well as the hyperthermia-treated samples, indicating negative regulation of intrinsic HIF-1alpha and P-glycoprotein expression by ERK1,2 and JNK signaling cascades. In summary our data demonstrate that hyperthermia-induced upregulation of P-glycoprotein and HIF-1alpha is mediated by activation of p38, whereas ERK1,2 and JNK are involved in repression of P-glycoprotein and HIF-1alpha under control conditions.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1995

Structure of the first C2 domain of synaptotagmin I: a novel Ca2+/phospholipid-binding fold.

C2 domains are regulatory sequence motifs that occur widely in nature. Synaptotagmin I, a synaptic vesicle protein involved in the Ca2+ regulation of exocytosis, contains two C2 domains, the first of which acts as a Ca2+ sensor. We now describe the three-dimensional structure of this C2 domain at 1.9 A resolution in both the Ca(2+)-bound and Ca(2+)-free forms. The C2 polypeptide forms an eight-stranded beta sandwich constructed around a conserved four-stranded motif designated as a C2 key. Ca2+ binds in a cup-shaped depression between two polypeptide loops located at the N- and C-termini of the C2-key motif.

Amino Acid Sequence↗
PubMed2000

Thymocyte selection is regulated by the helix-loop-helix inhibitor protein, Id3.

E2A, HEB, E2-2, and daughterless are basic helix-loop-helix (bHLH) proteins that play key roles in multiple developmental pathways. The DNA binding activity of E2A, HEB, and E2-2 is regulated by a distinct class of inhibitor HLH proteins, the Id gene products. Here, we show that Id3 is required for major histocompatability (MHC) class I- and class II-restricted thymocyte positive selection. Additionally, H-Y TCR-mediated negative selection is severely perturbed in Id3 null mutant mice. Finally, we show that E2A and Id3 interact genetically to regulate thymocyte development. These observations identify the HLH inhibitory protein Id3 as an essential component required for proper thymocyte maturation.

Animals↗
PubMed2001

In vitro RNA synthesis from exogenous dengue viral RNA templates requires long range interactions between 5'- and 3'-terminal regions that influence RNA structure.

Viral replicases of many positive-strand RNA viruses are membrane-bound complexes of cellular and viral proteins that include viral RNA-dependent RNA polymerase (RdRP). The in vitro RdRP assay system that utilizes cytoplasmic extracts from dengue viral-infected cells and exogenous RNA templates was developed to understand the mechanism of viral replication in vivo. Our results indicated that in vitro RNA synthesis at the 3'-untranslated region (UTR) required the presence of the 5'-terminal region (TR) and the two cyclization (CYC) motifs suggesting a functional interaction between the TRs. In this study, using a psoralen-UV cross-linking method and an in vitro RdRP assay, we analyzed structural determinants for physical and functional interactions. Exogenous RNA templates that were used in the assays contained deletion mutations in the 5'-TR and substitution mutations in the 3'-stem-loop structure including those that would disrupt the predicted pseudoknot structure. Our results indicate that there is physical interaction between the 5'-TR and 3'-UTR that requires only the CYC motifs. RNA synthesis at the 3'-UTR, however, requires long range interactions involving the 5'-UTR, CYC motifs, and the 3'-stem-loop region that includes the tertiary pseudoknot structure.

3' Untranslated Regions↗
PubMed1996

Positioning of positively charged residues in the V3 loop correlates with HIV type 1 syncytium-inducing phenotype.

Although the V3 loop of the envelope glycoprotein (gp120) plays a role in determining the phenotype, pathogenesis, and tropism of human immunodeficiency virus-1 (HIV-1), there has not been any consistent correlation between structure and phenotype. Theoretically determined structures of the V3 loop of gp120, from 20 different viral strains, 10 syncytium-inducing (SI) and 10 non-syncytium-inducing (NSI) phenotype, revealed that all V3 loops from SI phenotypic strains had at least two positively charged residues in close proximity, on the same face of the loop. All of the SI phenotypic V3 loop structures were capable of forming strong divalent electrostatic interactions with disulfated sugars. The ability to form this interaction may be a determinant of the phenotype, tropism, and pathogenicity of HIV-1 viral strains. This structural motif was absent in all V3 loops from viral strains with the NSI phenotype.

Amino Acid Sequence↗
PubMed1995

Synthesis of the basic-helix-loop-helix region of the immunoglobulin enhancer binding protein E47 and evaluation of its structural and DNA binding properties.

The basic-helix-loop-helix (bHLH) region of the immunoglobulin enhancer binding protein E47 (IEB E47) was prepared in high yield by a solid-phase peptide synthesis methodology. Size-exclusion chromatography, sedimentation equilibrium and cross-linking data showed that the synthetic bHLH protein, 1, was dimeric, and higher-order aggregates of trimer and tetramer were also observed. The circular dichroism spectrum of 1 showed a high helical content, which increased upon addition of DNA containing the kappa E2 sequence. Gel mobility shift experiments showed that protein 1 bound sequence specifically to the kappa E2 sequence with a binding constant of 10(-10) M2, and had an affinity for other E box sequences as well. Comparisons between the co-crystal structure of IEB E47 with DNA and structural studies in solution showed lower helical contents in solution as would have been predicted from the crystal structure.

Amino Acid Sequence↗
PubMed2001

Negative regulation of CD4 gene expression by a HES-1-c-Myb complex.

Expression of the CD4 gene is tightly controlled throughout thymopoiesis. The downregulation of CD4 gene expression in CD4(-) CD8(-) and CD4(-) CD8(+) T lymphocytes is controlled by a transcriptional silencer located in the first intron of the CD4 locus. Here, we determine that the c-Myb transcription factor binds to a functional site in the CD4 silencer. As c-Myb is also required for CD4 promoter function, these data indicate that depending on the context, c-Myb plays both positive and negative roles in the control of CD4 gene expression. Interestingly, a second CD4 silencer-binding factor, HES-1, binds to c-Myb in vivo and induces it to become a transcriptional repressor. We propose that the recruitment of HES-1 and c-Myb to the silencer leads to the formation of a multifactor complex that induces silencer function and repression of CD4 gene expression.

Basic Helix-Loop-Helix Proteins↗
PubMed1999

The kissing-loop motif is a preferred site of 5' leader recombination during replication of SL3-3 murine leukemia viruses in mice.

A panel of mouse T-cell lymphomas induced by SL3-3 murine leukemia virus (MLV) and three primer binding site mutants thereof (A. H. Lund, J. Schmidt, A. Luz, A. B. Sorensen, M. Duch, and F. S. Pedersen, J. Virol. 73:6117-6122, 1999) were analyzed for the occurrence of recombination between the exogenous input virus and endogenous MLV-like sequences within the 5' leader region. Evidence of recombination within the region studied was found in 14 of 52 tumors analyzed. Sequence analysis of a approximately 330-bp fragment of 44 chimeric proviruses, encompassing the U5, the primer binding site, and the upstream part of the 5' untranslated region, enabled us to map recombination sites, guided by distinct scattered nucleotide differences. In 30 of 44 analyzed sequences, recombination was mapped to a 33-nucleotide similarity window coinciding with the kissing-loop stem-loop motif implicated in dimerization of the diploid genome. Interestingly, the recombination pattern preference found in replication-competent viruses from T-cell tumors is very similar to the pattern previously reported for retroviral vectors in cell culture experiments. The data therefore sustain the hypothesis that the kissing loop, presumably via a role in RNA dimer formation, constitutes a hot spot for reverse transcriptase-mediated recombination in MLV.

5' Untranslated Regions↗
PubMed2001

Effect of pH and salt bridges on structural assembly: molecular structures of the monomer and intertwined dimer of the Eps8 SH3 domain.

The SH3 domain of Eps8 was previously found to form an intertwined, domain-swapped dimer. We report here a monomeric structure of the EPS8 SH3 domain obtained from crystals grown at low pH, as well as an improved domain-swapped dimer structure at 1.8 A resolution. In the domain-swapped dimer the asymmetric unit contains two "hybrid-monomers." In the low pH form there are two independently folded SH3 molecules per asymmetric unit. The formation of intermolecular salt bridges is thought to be the reason for the formation of the dimer. On the basis of the monomer SH3 structure, it is argued that Eps8 SH3 should, in principle, bind to peptides containing a PxxP motif. Recently it was reported that Eps8 SH3 binds to a peptide with a PxxDY motif. Because the "SH3 fold" is conserved, alternate binding sites may be possible for the PxxDY motif to bind. The strand exchange or domain swap occurs at the n-src loops because the n-src loops are flexible. The thermal b-factors also indicate the flexible nature of n-src loops and a possible handle for domain swap initiation. Despite the loop swapping, the typical SH3 fold in both forms is conserved structurally. The interface of the acidic form of SH3 is stabilized by a tetragonal network of water molecules above hydrophobic residues. The intertwined dimer interface is stabilized by hydrophobic and aromatic stacking interactions in the core and by hydrophilic interactions on the surface.

Adaptor Proteins, Signal Transducing↗