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.

Identification of ETS domain proteins in murine T lymphocytes that interact with the Moloney murine leukemia virus enhancer.

The enhancer of Moloney murine leukemia virus (Mo-MuLV) contains an array of transcriptional control elements that direct viral gene expression in diverse cell types. The murine transcription factor Ets-1 was shown to bind to the LVb and LVc elements of the enhancer by DNase I protection and methylation interference assays. Enhancers containing disrupted Ets-1 binding sites were tested in transient expression assays in the murine T-cell line EL4.E1; alterations in the LVb element affected constitutive enhancer activity, while mutation of either the LVb or LVc element disrupted phorbol ester-induced enhancer activity. Members of the ets gene family of proteins display similar DNA-binding properties; therefore, we speculated that ets proteins other than Ets-1 also might bind these elements. Crude nuclear extracts of EL4.E1 cells were assayed to identify the protein(s) that potentially functions at the LVb element. The predominant binding activity was not Ets-1 but rather two independent DNA-protein complexes that comigrated in mobility shift assays. UV cross-linking and denaturing gel electrophoresis sized the two DNA-binding species, which we denoted p55 and p100. Immunoprecipitation combined with UV cross-linking identified p55 as the alpha subunit of GA-binding protein. The DNA-binding properties of p100 and several ets proteins were compared. Similarities suggested that p100 is also an ETS domain protein, possibly Elf-1. This strategy could be used to identify other ETS domain proteins in crude nuclear extracts. These findings suggest multiple ETS domain proteins could regulate gene expression of Mo-MuLV.

Delineation of transcriptional control signals within the Moloney murine sarcoma virus long terminal repeat.

We identified three distinct elements within the Moloney murine sarcoma virus long terminal repeat that control transcription. The phenotypes of unidirectional deletion mutants of the long terminal repeat were assayed in microinjected frog oocytes and in transfected mouse fibroblasts. Steady-state levels of RNA bearing the same 5' terminus as the authentic Moloney murine sarcoma viral transcripts were measured by primer extension in assays that included a pseudo-wild-type internal reference. Mutant phenotypes define the boundaries of three functional elements. A region between 21 and 31 base pairs upstream from the mRNA cap site contains AT-rich sequences that function to establish the transcription start site. A second control element, termed the distal signal, lies between 31 and 84 base pairs upstream of the mRNA cap site. A CAT box consensus sequence is located at the 5' boundary of the distal signal. Additional components of the distal signal include a hexanucleotide sequence that is repeated four times. The distal signal augments transcription efficiency in oocytes but contributes only weakly to long terminal repeat-mediated expression in mouse fibroblasts. A third transcriptional control element lies between 156 and 364 base pairs upstream of the mRNA cap site. This element includes the 75-base-pair repeats previously identified as the Moloney murine sarcoma virus enhancer. In contrast to the distal signal, the Moloney murine sarcoma virus enhancer is crucial for significant expression in mouse fibroblasts but does not contribute to transcriptional expression in frog oocytes.

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.

Alternate utilization of two regulatory domains within the Moloney murine sarcoma virus long terminal repeat.

The Moloney murine sarcoma virus long terminal repeat (LTR) harbors two distinct positive activators of transcription, namely, a distal signal and an enhancer. In this report we demonstrate that infection by herpes simplex virus (HSV) can markedly affect the utilization of these two Moloney murine sarcoma virus transcription signals. We investigated the HSV-mediated trans-acting effects with two goals in mind: first, to gain insight into LTR function, and second, to probe the mechanisms used by HSV to establish its own transcription cascade. In mock-infected cells, LTR-mediated expression was heavily dependent on the Moloney murine sarcoma virus enhancer but was effectively distal signal independent. HSV infection mobilized the use of the LTR distal signal and concomitantly alleviated enhancer dependence. Indeed, enhancer function may actually be inhibited by HSV trans-acting factors. These results suggest that the two positive control signals of the Moloney murine sarcoma virus LTR facilitate transcriptional activation by two different pathways. We further observed that the identity of the structural gene driven by the LRT, as well as the state of integration of a transfected template, can exert a substantial effect on the response of a template to HSV infection. According to these findings, we propose a tentative model to account for the initial temporal shift of the HSV transcriptional cascade.

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.

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.

Conformation of pyridazine nucleosides: the molecular structure of 4-hydroxy-1-(beta-D-ribofuranosyl)pyridazin-6-one (3-deaza-6-azauridine).

The glycosyl torsiona angle in 4-hydroxy-1-(beta-D-ribofuranosyl)-pyridazin-6-one (or 3-deaza-6-azauridine) is in the "high-anti" region. This is similar to the torsional angles observed for 6-azapyrimidine and 8-azapurine nucleosides, but in marked contrast to those found in uridine, 3-deazauridine and other pyrimidine nucleosides.

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.

Orally absorbable cephalosporin antibiotics. 2. Structure-activity studies of bicyclic glycine derivatives of 7-aminodeacetoxycephalosporanic acid.

Three positional analogues (4-, 5-, and 7-) of benzothienylglycine and (N-acetylindolinyl)-5-glycine were prepared and coupled to 7-aminodeacetoxycephalosporanic acid (7-ADCA) to give the cephalosporins 17a-c. In addition two isomeric (2,3-b and 3,2-b) thienothiopheneglycines were synthesized and coupled to 7-ADCA to yield cephalosporins 30d and 30e. In vitro testing of these new cephalosporins indicates good activity against Gram-positive bacteria. Against Streptococcus pneumoniae infections compound 25 displayed better mouse protection (both orally and subcutaneously) than cephalexin.

Orally absorbable cephalosporin antibiotics. 1. Structure-activity relationships of benzothienyl- and naphthylglycine derivatives of 7-aminodeacetoxycephalosporanic acid.

A structure-activity relationship study of a number of orally absorbed cephalosporins together with their syntheses is described. These new cephalosporins are benzothienyl- and naphthylglycine derivatives of 7-aminodeacetoxycephalosporanic acid. Several different synthetic methods for the glycine side chains, their protection, and the final acylations are reported. Several of these analogues were more active than cephalexin both in vitro and in vivo against commonly encountered Gram-positive bacteria. (R)-7-(3-Benzothienylglycylamido)-3-methyl-3-cephem-4-carboxylic acid (1R) has emerged as a potent antibacterial agent and is currently undergoing preclinical evaluation.

Correlations between CNDO/2 charge distribution and 13C NMR chemical shift in 7-acylamino side chains of cephalosporins.

Molecular orbital calculations by the CNDO/2D method yield charge distributions which correlate well with the observed 13C NMR chemical shift for the amide carbon of acylamino side chains of cephalosporins. Acyl groups that withdraw electrons from the amide C-N bond and concomitantly make the amide nitrogen more negatively charged increase the chemical shift. The trends are related to the degree of amide resonance. No direct correlation was found between the chemical shift of the amide carbon and the antibacterial activity of the cephalosporins.

Alkylation interference identifies essential DNA contacts for sequence-specific binding of the eukaryotic transcription factor C/EBP.

Transcriptional regulators containing a "leucine zipper" and a flanking basic domain belong to a recently identified class of DNA-binding proteins. We have mapped the essential DNA contacts of one member of this group, the eukaryotic transcription factor C/EBP. Methylation and ethylation interference experiments detected major groove contacts over a full turn of the DNA helix on both an asymmetric and a symmetric C/EBP binding site. The contacts essential for C/EBP binding have two-fold symmetry yet differ significantly from the contacts of other dimeric DNA-binding proteins, including those bearing helix-turn-helix motifs and the type I restriction endonuclease EcoRI.

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.

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.