Scissors-grip model for DNA recognition by a family of leucine zipper proteins.

C/EBP is a sequence-specific DNA binding protein that regulates gene expression in certain mammalian cells. The region of the C/EBP polypeptide required for specific recognition of DNA is related in amino acid sequence to other regulatory proteins, including the Fos and Jun transforming proteins. It has been proposed that these proteins bind DNA via a bipartite structural motif, consisting of a dimerization interface termed the "leucine zipper" and a DNA contact surface termed the "basic region." An evaluation of the properties of conserved amino acids within the basic region of 11 deduced protein sequences, coupled with the observation that they are located at an invariant distance from the leucine zipper, has led to the formulation of a "scissors-grip" model for DNA binding. The architectural features of this model are well suited for interaction with directly abutted, dyadsymmetric DNA sequences. Data supportive of the model were obtained with chemical probes of protein: DNA complexes.

Evidence of changes in protease sensitivity and subunit exchange rate on DNA binding by C/EBP.

The transcription factor C/EBP uses a bipartite structural motif to bind DNA. Two protein chains dimerize through a set of amphipathic alpha helices termed the leucine zipper. Highly basic polypeptide regions emerge from the zipper to form a linked set of DNA contact surfaces. In the recently proposed a "scissors grip" model, the paired set of basic regions begin DNA contact at a central point and track in opposite directions along the major groove, forming a molecular clamp around DNA. This model predicts that C/EBP must undertake significant changes in protein conformation as it binds and releases DNA. The basic region of ligand-free C/EBP is highly sensitive to protease digestion. Pronounced resistance to proteolysis occurred when C/EBP associated with its specific DNA substrate. Sequencing of discrete proteolytic fragments showed that prominent sites for proteolysis occur at two junction points predicted by the "scissors grip" model. One junction corresponds to the cleft where the basic regions emerge from the leucine zipper. The other corresponds to a localized nonhelical segment that has been hypothesized to contain an N-cap and facilitate the sharp angulation necessary for the basic region to track continuously in the major groove of DNA.

Interactions of coiled coils in transcription factors: where is the specificity?

Amphipathic alpha-helices create the dimerization interface in the bZIP and bHLH classes of DNA-binding proteins. These amphipathic helices have been shown to enter into a wide variety of specific dimerization interactions, and this large array of possible combinatorial interactions may provide for fine control of biological function. In bHLH-ZIP proteins, the addition of a leucine-zipper region immediately carboxyl-terminal to the helix-loop-helix region provides for an additional level of both dimerization specificity and control, again through the interaction of amphipathic alpha-helices. Interhelical electrostatic interactions have been implicated in regulating dimerization specificity.

Structure and oxygen equilibrium of the three coelomic cell hemoglobins of the echiuran worm Thalassema mellita (Conn).

1. The three coelomic cell hemoglobins from Thalassema mellita have been isolated to purity; the two major components have dimeric structure while the third minor component has monomeric structure. 2. Acid-urea Triton gel electrophoresis of the isolated hemoglobins identified three polypeptides among the three hemoglobins, one of the dimeric hemoglobins is a heterodimer (pI = 4.9) with one polypeptide sharing identity with the monomeric hemoglobin (pI = 6.3), while the other dimer is a homodimer (pI = 4.5) consisting of the third polypeptide. 3. SDS gel electrophoresis suggests that the two dimeric hemoglobins have interpolypeptide disulfide bonds. 4. Coelomic cell suspensions and lysed coelomic cells have PO2 at half saturation (P50) of 2.5-3.0 mmHg and cooperativity values (n) of 1.5-1.93. 5. All three isolated hemoglobins have higher oxygen affinities and lower cooperativity values (P50 = 1-2 mmHg, n = 1-1.3) than lysed coelomic cells suggesting some heterotrophic and homotrophic interactions.

Molecular model for DNA recognition by the family of basic-helix-loop-helix-zipper proteins.

The basic-helix-loop-helix-zipper (bHLH-Zip) motif is a conserved region of approximately 70 amino acids that mediates both sequence-specific DNA binding and protein dimerization. This motif is found in protein sequences from many eukaryotic organisms and is contained in the protein sequence of the oncogene myc and its partner max, and a shortened version of the motif (bHLH) is found in the muscle determination factor myoD and its partner E12. An evaluation of the conserved amino acids that define the motif coupled with the published mutagenic studies of this region has led to our formulation of a molecular model for the binding of this motif as a dimer to specific sequences of DNA. This model has the dimeric protein interacting with an abutted, dyad-symmetric DNA sequence. Helix 2 of each monomer is modeled as a coiled-coil extension of the C-terminal "leucine zipper." Helix 1 does not interact with helix 1 from its partner in the dimer but with the hydrophobic surface created when the helix 2 regions of the dimer interact with each other as a coiled-coil. Sequence-specific interactions are proposed between the basic region and the invariant cis elements that all bHLH-Zip proteins bind.

Directional non-cell autonomy and the transmission of polarity information by the frizzled gene of Drosophila.

The function of the frizzled (fz) locus is required for the development of a parallel array of bristles and hairs on the adult cuticle of Drosophila melanogaster. Marked fz mitotic clones from five alleles were generated and examined in the wing. Three alleles have a non-cell-autonomous hair polarity phenotype; wild-type cells distal to fz clones produce hairs that have an abnormal polarity. In contrast, fz clones of the other two fz alleles examined do not disrupt the polarity of neighbouring cells. These data suggest that fz has two mutably separate functions in establishing hair polarity on the wing. One function involves the transmittance and/or generation of a polarity signal along the proximal-distal axis of the wing. The second function involves the cellular interpretation of a polarity signal.

Thermodynamic characterization of the structural stability of the coiled-coil region of the bZIP transcription factor GCN4.

The thermal stability of a 56 amino acid fragment of GCN4 has been studied by high-sensitivity differential scanning calorimetry and circular dichroism spectroscopy. This fragment contains the leucine zipper and part of the basic region. The thermal unfolding of GCN4-56 is a reversible process and can be well represented by a reaction of the form N2<-->2U, indicating that the unfolding of the leucine zipper is a two-state process in which the helices are only stable when they are in the coiled-coil conformation. As expected, the transition temperature is concentration dependent. At pH 7.06 and a protein concentration of 5 x 10(-4) M the transition temperature is close to 70 degrees C while at 5 x 10(-6) M it is close to 50 degrees C. The enthalpy change for unfolding is 31.5 kcal mol-1 at 70 degrees C. Since the isolated helices are unstable, interactions at the interface between the two helices play a key role in the stabilization of the native dimer. These interactions primarily involve the burial of apolar surface from the solvent (hydrophobic effect) and electrostatic interactions. Structural thermodynamic calculations have permitted a dissection of the magnitude of the various contributions to the total Gibbs free energy of stabilization.

Dimerization specificity of the leucine zipper-containing bZIP motif on DNA binding: prediction and rational design.

We propose an interhelical salt bridge rule to explain the dimerization specificity between the two amphipathic alpha-helices in the leucine zipper structure. Using the bZIP class of DNA-binding proteins as a model system, we predicted and designed novel dimerization partners. We predicted that ATF4, a member of the ATF/CREB family of transcription factors, would preferentially form heterodimers with IGEBP1, a member of the C/EBP superfamily. These predictions were verified using a gel mobility-shift assay. To further test the value of this interhelical salt bridge rule, we modified the bZIP protein C/EBP attempting to design molecules that would form preferentially heterodimers with C/EBP or molecules that would not interact with C/EBP. These designed molecules behaved as predicted. Therefore, we conclude that this interhelical salt bridge rule is useful in understanding the dimerization specificity of bZIP proteins. In addition, we suggest that this rule could be used to design novel "dominant-negative" molecules to specifically inhibit the function of target leucine zipper proteins in vivo.

A Drosophila tissue polarity locus encodes a protein containing seven potential transmembrane domains.

The function of the frizzled (fz) locus in Drosophilia melanogaster is required to coordinate the cytoskeletons of epidermal cells to produce a parallel array of cuticular hairs and bristles (for example on the wild-type wing all hairs point towards the distal tip). In fz mutants it is not the structure of individual hairs and bristles that is altered, but their orientation with respect to their neighbours and the organism as a whole. Mitotic clone analysis indicates that fz has two functions in the developing wing. It is required for the proximal-distal transmission of an intercellular polarity signal, a process that is expected to be at least partly extracellular. It is also required for cells to respond to the polarity signal, which is expected to be a cytoplasmic function. The fz locus could encode either one bifunctional or two single-function proteins. We report here that, in pupae, fz produces a messenger RNA that encodes a protein with seven putative transmembrane domains. Thus, the Fz protein should contain both extracellular and cytoplasmic domains, which could function in the transmission and interpretation of polarity information, respectively. This is the first reported sequence for the protein product of a tissue polarity gene.

In situ detection of sequence-specific DNA binding activity specified by a recombinant bacteriophage.

We have used a recombinant bacteriophage that expresses the DNA-binding domain of C/EBP to optimize conditions for a screening technique that may facilitate the cloning of genes that encode sequence-specific DNA-binding proteins. The method relies on the expression of cDNA inserts in bacteriophage lambda gt11. Fusion protein adsorbed onto nitrocellulose filters is probed with radioactive, double-stranded DNA as a ligand. Two procedures greatly increase the level of binding between ligand and recombinant fusion protein. First, nitrocellulose filters are processed through a denaturation/renaturation regimen using 6 M guanidine hydrochloride. Second, synthetic DNA corresponding to the specific binding site is catenated extensively using DNA ligase. The combination of these procedures leads to remarkably strong detection signals. Specific DNA-binding signals can be detected on duplicate filters, and filters can be washed and reused by repeating the cycle of denaturation/renaturation.