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Biomedical subjects

B J Graves

Publications and source records attributed to B J Graves.

At least 19 recordsLinked to original sources
PubMed2000

Carbocyclic influenza neuraminidase inhibitors possessing a C3-cyclic amine side chain: synthesis and inhibitory activity.

As part of our continuing work in the area of influenza neuraminidase inhibitors, a series of C3-aza inhibitors possessing a cyclic amine side chain was synthesized and evaluated for influenza neuraminidase inhibitory activity. Analogues possessing a six-, seven- and eight-membered ring, 4c-e, respectively, at the C3 position exhibited excellent influenza B neuraminidase inhibition.

Amines↗
PubMed2000

Phosphorylation represses Ets-1 DNA binding by reinforcing autoinhibition.

Phosphorylation of transcription factors is a key link between cell signaling and the control of gene expression. Here we report that phosphorylation regulates DNA binding of the Ets-1 transcription factor by reinforcing an autoinhibitory mechanism. Quantitative DNA-binding assays show that calcium-dependent phosphorylation inhibits Ets-1 DNA binding 50-fold. The four serines that mediate this inhibitory effect are distant from the DNA-binding domain but near structural elements required for autoinhibition. Mutational analyses demonstrate that an intact inhibitory module is required for phosphorylation-dependent regulation. Partial proteolysis studies indicate that phosphorylation stabilizes an inhibitory conformation. These findings provide a structural mechanism for phosphorylation-dependent inhibition of Ets-1 DNA binding and demonstrate a new function for inhibitory modules as structural mediators of negative signaling events.

Amino Acid Sequence↗
PubMed2000

Auto-inhibition of Ets-1 is counteracted by DNA binding cooperativity with core-binding factor alpha2.

Auto-inhibition is a common transcriptional control mechanism that is well characterized in the regulatory transcription factor Ets-1. Autoinhibition of Ets-1 DNA binding works through an inhibitory module that exists in two conformations. DNA binding requires a change in the inhibitory module from the packed to disrupted conformation. This structural switch provides a mechanism to tightly regulate Ets-1 DNA binding. We report that the Ets-1 partner protein core-binding factor alpha2 (CBFalpha2; also known as AML1 or PEBP2) stimulates Ets-1 DNA binding and counteracts auto-inhibition. Support for this conclusion came from three observations. First, the level of cooperative DNA binding (10-fold) was similar to the level of repression by auto-inhibition (10- to 20-fold). Next, a region necessary for cooperative DNA binding mapped to the inhibitory module. Third, an Ets-1 mutant with a constitutively disrupted inhibitory module did not bind DNA cooperatively with CBFalpha2. Furthermore, two additional lines of evidence indicated that CBFalpha2 affects the structural switch by direct interactions with Ets-1. First, the retention of cooperative DNA binding on nicked duplexes eliminated a potential role of through-DNA effects. Second, cooperative DNA binding was observed on composite sites with altered spacing or reversed orientation. We suggest that only protein interactions can accommodate this observed flexibility. These findings provide a mechanism by which CBF relieves the auto-inhibition of Ets-1 and illustrates one strategy for the synergistic activity of regulatory transcription factors.

Animals↗
PubMed2000

Auto-inhibition and partner proteins, core-binding factor beta (CBFbeta) and Ets-1, modulate DNA binding by CBFalpha2 (AML1).

Core-binding factor alpha2 (CBFalpha2; otherwise known as AML1 or PEBP2alphaB) is a DNA-binding subunit in the family of core-binding factors (CBFs), heterodimeric transcription factors that play pivotal roles in multiple developmental processes in mammals, including hematopoiesis and bone development. The Runt domain in CBFalpha2 (amino acids 51 to 178) mediates DNA binding and heterodimerization with the non-DNA-binding CBFbeta subunit. Both the CBFbeta subunit and the DNA-binding protein Ets-1 stimulate DNA binding by the CBFalpha2 protein. Here we quantify and compare the extent of cooperativity between CBFalpha2, CBFbeta, and Ets-1. We also identify auto-inhibitory sequences within CBFalpha2 and sequences that modulate its interactions with CBFbeta and Ets-1. We show that sequences in the CBFalpha2 Runt domain and sequences C terminal to amino acid 214 inhibit DNA binding. Sequences C terminal to amino acid 214 also inhibit heterodimerization with the non-DNA-binding CBFbeta subunit, particularly heterodimerization off DNA. CBFbeta rescinds the intramolecular inhibition of CBFalpha2, stimulating DNA binding approximately 40-fold. In comparison, Ets-1 stimulates CBFalpha2 DNA binding 7- to 10-fold. Although the Runt domain alone is sufficient for heterodimerization with CBFbeta, sequences N terminal to amino acid 41 and between amino acids 190 and 214 are required for cooperative DNA binding with Ets-1. Cooperative DNA binding with Ets-1 is less pronounced with the CBFalpha2-CBFbeta heterodimer than with CBFalpha2 alone. These analyses demonstrate that CBFalpha2 is subject to both negative regulation by intramolecular interactions, and positive regulation by two alternative partnerships.

Animals↗
PubMed1999

Inhibition of Ets-1 DNA binding and ternary complex formation between Ets-1, NF-kappaB, and DNA by a designed DNA-binding ligand.

Sequence-specific pyrrole-imidazole polyamides can be designed to interfere with transcription factor binding and to regulate gene expression, both in vitro and in living cells. Polyamides bound adjacent to the recognition sites for TBP, Ets-1, and LEF-1 in the human immunodeficiency virus, type 1 (HIV-1), long terminal repeat inhibited transcription in cell-free assays and viral replication in human peripheral blood lymphocytes. The DNA binding activity of the transcription factor Ets-1 is specifically inhibited by a polyamide bound in the minor groove. Ets-1 is a member of the winged-helix-turn-helix family of transcription factors and binds DNA through a recognition helix bound in the major groove with additional phosphate contacts on either side of this major groove interaction. The inhibitory polyamide possibly interferes with phosphate contacts made by Ets-1, by occupying the adjacent minor groove. Full-length Ets-1 binds the HIV-1 enhancer through cooperative interactions with the p50 subunit of NF-kappaB, and the Ets-inhibitory polyamide also blocks formation of ternary Ets-1. NF-kappaB.DNA complexes on the HIV-1 enhancer. A polyamide bound adjacent to the recognition site for NF-kappaB also inhibits NF-kappaB binding and ternary complex formation. These results broaden the application range of minor groove-binding polyamides and demonstrate that these DNA ligands are powerful inhibitors of DNA-binding proteins that predominantly use major groove contacts and of cooperative protein-DNA ternary complexes.

Base Sequence↗
PubMed1998

Equilibrium analysis of high affinity interactions using BIACORE.

BIACORE biosensors are useful for measuring reaction kinetics and calculating affinity constants for macromolecular interactions. However, one drawback with the flow system used in these instruments is that the standard injection procedures limit the amount of time available to collect association-phase data. This is especially problematic during equilibrium analysis of high affinity interactions. Using protein-DNA interactions as a model system, we demonstrate a simple method for overcoming this limitation. By placing the analyte directly into the running buffer we were able to deliver a continuous supply of protein to the sensor surfaces for greater than 12 h at a time. Complete equilibrium binding profiles were generated by changing the concentration of analyte and allowing the surface reactions to reequilibrate. Analyte concentrations were also decreased to demonstrate that the binding reactions were fully reversible. This method of analysis is a simple and convenient way of directly measuring equilibrium dissociation constants for very high affinity interactions.

Base Sequence↗
PubMed1998

Structure of the Ets-1 pointed domain and mitogen-activated protein kinase phosphorylation site.

The Pointed (PNT) domain and an adjacent mitogen-activated protein (MAP) kinase phosphorylation site are defined by sequence conservation among a subset of ets transcription factors and are implicated in two regulatory strategies, protein interactions and posttranslational modifications, respectively. By using NMR, we have determined the structure of a 110-residue fragment of murine Ets-1 that includes the PNT domain and MAP kinase site. The Ets-1 PNT domain forms a monomeric five-helix bundle. The architecture is distinct from that of any known DNA- or protein-binding module, including the helix-loop-helix fold proposed for the PNT domain of the ets protein TEL. The MAP kinase site is in a highly flexible region of both the unphosphorylated and phosphorylated forms of the Ets-1 fragment. Phosphorylation alters neither the structure nor monomeric state of the PNT domain. These results suggest that the Ets-1 PNT domain functions in heterotypic protein interactions and support the possibility that target recognition is coupled to structuring of the MAP kinase site.

Amino Acid Sequence↗
PubMed1996

Solution structure of the ETS domain from murine Ets-1: a winged helix-turn-helix DNA binding motif.

Ets-1 is the prototypic member of the ets family of transcription factors. This family is characterized by the conserved ETS domain that mediates specific DNA binding. Using NMR methods, we have determined the structure of a fragment of murine Ets-1 composed of the 85 residue ETS domain and a 25 amino acid extension that ends at its native C-terminus. The ETS domain folds into a helix-turn-helix motif on a four-stranded anti-parallel beta-sheet scaffold. This structure places Ets-1 in the winged helix-turn-helix (wHTH) family of DNA binding proteins and provides a model for interpreting the sequence conservation of the ETS domain and the specific interaction of Ets-1 with DNA. The C-terminal sequence of Ets-1, which is mutated in the v-Ets oncoprotein, forms an alpha-helix that packs anti-parallel to the N-terminal helix of the ETS domain. In this position, the C-terminal helix is poised to interact directly with an N-terminal inhibitory region in Ets-1 as well as the wHTH motif. This explains structurally the concerted role of residues flanking the ETS domain in the intramolecular inhibition of Ets-1 DNA binding.

Amino Acid Sequence↗
PubMed1996

Structural coupling of the inhibitory regions flanking the ETS domain of murine Ets-1.

Several members of the ets gene family of transcription factors show negative regulation of DNA binding by intramolecular interactions. A structural mechanism for this auto-inhibition is investigated using a 161-residue N-terminal deletion mutant of murine Ets-1, Ets-1 delta N280. This protein shows a similar reduced affinity for DNA as native Ets-1 because it contains the ETS domain in context of the flanking amino- and carboxy-terminal regions that together mediate repression of DNA binding. The secondary structure of Ets-1 delta N280 was determined using NMR chemical shift, NOE, J coupling, and amide hydrogen exchange information. In addition to the winged helix-turn-helix ETS domain, Ets-1 delta N280 contains two alpha-helices in the amino-terminal inhibitory region and one alpha-helix in the carboxy-terminal inhibitory region. Chemical shift comparisons were made between this protein and an activated form of Ets-1 lacking the amino-terminal inhibitory region. The spectral differences demonstrate that the amino- and carboxy-terminal inhibitory sequences are structurally coupled to one another, thus explaining the observation that both regions are required for the repression of DNA binding. Furthermore, these data show that the inhibitory sequences also interact directly with the first helix of the intervening ETS domain, thereby providing a pathway for the repression of DNA binding. These results lead to a model of an inhibitory module in Ets-1 composed of both the amino- and carboxy-terminal regions interfaced with the ETS domain. This establishes the structural framework for understanding the intramolecular inhibition of Ets-1 DNA binding.

Amino Acid Sequence↗
PubMed1996

Characterization of the cooperative function of inhibitory sequences in Ets-1.

DNA binding by the eukaryotic transcription factor Ets-1 is negatively regulated by an intramolecular mechanism. Quantitative binding assays compared the DNA-binding activities of native Ets-1, three deletion mutants, and three tryptic fragments. Ets-1 and activated Ets-1 polypeptides differed in DNA-binding affinity as much as 23-fold. Inhibition was mediated by two regions flanking the minimal DNA-binding domain. Both regions regulated affinity by enhancing dissociation of the protein-DNA complex. Three lines of evidence indicated that inhibition requires cooperative interaction between the two regions: first, the two inhibitory regions acted through a common mechanism; second, neither region functioned independently of the other; finally, mutation of the C-terminal inhibitory region altered the conformation of the N-terminal inhibitory region. In addition, partial proteolysis detected an identical altered conformation in the N-terminal inhibitory region of Ets-1 bound to DNA. This finding suggested that repression is transiently disrupted during DNA binding. These results provide evidence that the two inhibitory regions of Ets-1 are structurally, as well as functionally, coupled. In addition, conformational change is shown to be a critical component of the inhibition mechanism. A cooperative, allosteric model of autoinhibition is described. Autoinhibition of Ets-1 could be relieved by either protein partner(s) or posttranslational modifications.

Allosteric Regulation↗
PubMed1995

Modulation of transcription factor Ets-1 DNA binding: DNA-induced unfolding of an alpha helix.

Conformational changes, including local protein folding, play important roles in protein-DNA interactions. Here, studies of the transcription factor Ets-1 provided evidence that local protein unfolding also can accompany DNA binding. Circular dichroism and partial proteolysis showed that the secondary structure of the Ets-1 DNA-binding domain is unchanged in the presence of DNA. In contrast, DNA allosterically induced the unfolding of an alpha helix that lies within a flanking region involved in the negative regulation of DNA binding. These findings suggest a structural basis for the intramolecular inhibition of DNA binding and a mechanism for the cooperative partnerships that are common features of many eukaryotic transcription factors.

Base Sequence↗