Distance determination in proteins using designed metal ion binding sites and site-directed spin labeling: application to the lactose permease of Escherichia coli.

As shown in the accompanying paper, the magnetic dipolar interaction between site-directed metal-nitroxide pairs can be exploited to measure distances in T4 lysozyme, a protein of known structure. To evaluate this potentially powerful method for general use, particularly with membrane proteins that are difficult to crystallize, both a paramagnetic metal ion binding site and a nitroxide side chain were introduced at selected positions in the lactose permease of Escherichia coli, a paradigm for polytopic membrane proteins. Thus, three individual cysteine residues were introduced into putative helix IV of a lactose permease mutant devoid of native cysteine residues containing a high-affinity divalent metal ion binding site in the form of six contiguous histidine residues in the periplasmic loop between helices III and IV. In addition, the construct contained a biotin acceptor domain in the middle cytoplasmic loop to facilitate purification. After purification and spin labeling, electron paramagnetic resonance spectra were obtained with the purified proteins in the absence and presence of Cu(II). The results demonstrate that positions 103, 111, and 121 are 8, 14, and > 23 A from the metal binding site. These data are consistent with an alpha-helical conformation of transmembrane domain IV of the permease. Application of the technique to determine helix packing in lactose permease is discussed.

MyoD forms micelles which can dissociate to form heterodimers with E47: implications of micellization on function.

MyoD is a member of a family of DNA-binding transcription factors that contain a helix-loop-helix (HLH) region involved in protein-protein interactions. In addition to self-association and DNA binding, MyoD associates with a number of other HLH-containing proteins, thereby modulating the strength and specificity of its DNA binding. Here, we examine the interactions of full-length MyoD with itself and with an HLH-containing peptide portion of an E2A gene product, E47-96. Analytical ultracentrifugation reveals that MyoD forms micelles that contain more than 100 monomers and are asymmetric and stable up to 36 degrees C. The critical micelle concentration increases slightly with temperature, but micelle size is unaffected. The micelles are in reversible equilibrium with monomer. Addition of E47-96 results in the stoichiometric formation of stable MyoD-E47-96 heterodimers and the depletion of micelles. Micelle formation effectively holds the concentration of free MyoD constant and equal to the critical micelle concentration. In the presence of micelles, the extent of all interactions involving free MyoD is independent of the total MyoD concentration and independent of one another. For DNA binding, the apparent relative specificity for different sites can be affected. In general, heterodimer-associated activities will depend on the self-association behavior of the partner protein.

Mxi2, a mitogen-activated protein kinase that recognizes and phosphorylates Max protein.

We describe Mxi2, a human protein that interacts with Max protein, the heterodimeric partner of the Myc oncoprotein. Mxi2 encodes a 297-residue protein whose sequence indicates that it is related to extracellular signal-regulated kinases (ERK protein kinases). Mxi2 in yeast interacts with Max and with the C terminus of c-Myc. Mxi2 phosphorylates Max both in vitro and in vivo. The Mxi2 putative substrate recognition region has sequence similarity to the helix-loop-helix region in Max and c-Myc, suggesting that substrate recognition might be mediated via this motif. Phosphorylation by Mxi2 may affect the ability of Max to oligomerize with itself and its partners, bind DNA, or regulate gene expression.

Association of erythroid transcription factors: complexes involving the LIM protein RBTN2 and the zinc-finger protein GATA1.

The RBTN2 LIM-domain protein, originally identified as an oncogenic protein in human T-cell leukemia, is essential for erythropoiesis. A possible role for RBTN2 in transcription during erythropoiesis has been investigated. Direct interaction of the RBTN2 protein was observed in vivo and in vitro with the GATA1 or -2 zinc-finger transcription factors, as well as with the basic helix-loop-helix protein TAL1. By using mammalian two-hybrid analysis, complexes involving RBTN2, TAL1, and GATA1, together with E47, the basic helix-loop-helix heterodimerization partner of TAL1, could be demonstrated. Thus, a molecular link exists between three proteins crucial for erythropoiesis, and the data suggest that variations in amounts of complexes involving RBTN2, TAL1, and GATA1 could be important for erythroid differentiation.

Transdifferentiation of myoblasts by the adipogenic transcription factors PPAR gamma and C/EBP alpha.

Skeletal muscle and adipose tissue development often has a reciprocal relationship in vivo, particularly in myodystrophic states. We have investigated whether determined myoblasts with no inherent adipogenic potential can be induced to transdifferentiate into mature adipocytes by the ectopic expression of two adipogenic transcription factors, PPAR gamma and C/EBP alpha. When cultured under optimal conditions for muscle differentiation, murine G8 myoblasts expressing PPAR gamma and C/EBP alpha show markedly reduced levels of the myogenic basic helix-loop-helix proteins MyoD, myogenin, MRF4, and myf5 and are completely unable to differentiate into myotubes. Under conditions permissive for adipogenesis including a PPAR activator, these cells differentiate into mature adipocytes that express molecular markers characteristic of this lineage. Our results demonstrate that a developmental switch between these two related but highly specialized cell types can be controlled by the expression of key adipogenic transcription factors. These factors have an ability to inhibit myogenesis that is temporally and functionally separate from their ability to stimulate adipogenesis.

D1 cap region involved in the receptor recognition and neural cell survival activity of human ciliary neurotrophic factor.

Human ciliary neurotrophic factor (hCNTF), which promotes the cell survival and differentiation of motor and other neurons, is a protein belonging structurally to the alpha-helical cytokine family. hCNTF was subjected to three-dimensional structure modeling and site-directed mutagenesis to analyze its structure-function relationship. The replacement of Lys-155 with any other amino acid residue resulted in abolishment of neural cell survival activity, and some of the Glu-153 mutant proteins had 5- to 10-fold higher biological activity. The D1 cap region (around the boundary between the CD loop and helix D) of hCNTF, including both Glu-153 and Lys-155, was shown to play a key role in the biological activity of hCNTF as one of the putative receptor-recognition sites. In this article, the D1 cap region of the 4-helix-bundle proteins is proposed to be important in receptor recognition and biological activity common to alpha-helical cytokine proteins reactive with gp130, a component protein of the receptors.

The CuA center of cytochrome-c oxidase: electronic structure and spectra of models compared to the properties of CuA domains.

The electronic structure and spectrum of several models of the binuclear metal site in soluble CuA domains of cytochrome-c oxidase have been calculated by the use of an extended version of the complete neglect of differential overlap/spectroscopic method. The experimental spectra have two strong transitions of nearly equal intensity around 500 nm and a near-IR transition close to 800 nm. The model that best reproduces these features consists of a dimer of two blue (type 1) copper centers, in which each Cu atom replaces the missing imidazole on the other Cu atom. Thus, both Cu atoms have one cysteine sulfur atom and one imidazole nitrogen atom as ligands, and there are no bridging ligands but a direct Cu-Cu bond. According to the calculations, the two strong bands in the visible region originate from exciton coupling of the dipoles of the two copper monomers, and the near-IR band is a charge-transfer transition between the two Cu atoms. The known amino acid sequence has been used to construct a molecular model of the CuA site by the use of a template and energy minimization. In this model, the two ligand cysteine residues are in one turn of an alpha-helix, whereas one ligand histidine is in a loop following this helix and the other one is in a beta-strand.

Absence of yolk sac hematopoiesis from mice with a targeted disruption of the scl gene.

The scl gene encodes a basic-helix-loop-helix transcription factor which was identified through its involvement in chromosomal translocations in T-cell leukemia. To elucidate its physiological role, scl was targeted in embryonic stem cells. Mice heterozygous for the scl null mutation were intercrossed and their offspring were genotyped. Homozygous mutant (scl-/-) pups were not detected in newborn litters, and analysis at earlier time points demonstrated that scl-/- embryos were dying around embryonic day 9.5. The scl-/- embryos were pale, edematous, and markedly growth retarded after embryonic day 8.75. Histological studies showed complete absence of recognizable hematopoiesis in the yolk sac of these embryos. Early organogenesis appeared to be otherwise normal. Culture of yolk sac cells of wild-type, heterozygous, and homozygous littermates confirmed the absence of hematopoietic cells in scl-/- yolk sacs. Reverse transcription PCR was used to examine the transcripts of several genes implicated in early hematopoiesis. Transcripts of GATA-1 and PU.1 transcription factors were absent from RNA from scl-/- yolk sacs and embryos. These results implicate scl as a crucial regulator of early hematopoiesis.

Alternate protein frameworks for molecular recognition.

In an effort to determine whether proteins with structures other than the immunoglobulin fold can be used to mimic the ligand binding properties of antibodies, we generated a library from the four-helix bundle protein cytochrome b562 in which the two loops were randomized. Panning of this library against the bovine serum albumin (BSA) conjugate of N-methyl-p-nitrobenzylamine derivative 1 by phage display methods yielded cytochromes in which residues Trp-20, Arg-21, and Ser-22 in loop A and Arg-83 and Trp-84 in loop B were conserved. The individual mutants, which fold into native-like structure, bind selectively to the BSA-1 conjugate with micromolar dissociation constants (Kd), in comparison to a monoclonal antibody that binds selectively to 1 with a Kd of 290 nM. These and other antibody-like receptors may prove useful as therapeutic agents or as reagents for both intra- and extracellular studies.

Fusion of the TEL gene on 12p13 to the AML1 gene on 21q22 in acute lymphoblastic leukemia.

Chromosomal rearrangements involving band 12p13 are found in a wide variety of human leukemias but are particularly common in childhood acute lymphoblastic leukemia. The genes involved in these rearrangements, however, have not been identified. We now report the cloning of a t(12;21) translocation breakpoint involving 12p13 and 21q22 in two cases of childhood pre-B acute lymphoblastic leukemia, in which t(12;21) rearrangements were not initially apparent. The consequence of the translocation is fusion of the helix-loop-helix domain of TEL, an ETS-like putative transcription factor, to the DNA-binding and transactivation domains of the transcription factor AML1. These data show that TEL, previously shown to be fused to the platelet-derived growth factor receptor beta in chronic myelomonocytic leukemia, can be implicated in the pathogenesis of leukemia through its fusion to either a receptor tyrosine kinase or a transcription factor. The TEL-AML1 fusion also indicates that translocations affecting the AML1 gene can be associated with lymphoid, as well as myeloid, malignancy.

Identification of receptor-binding domains on human interleukin 5 and design of an interleukin 5-derived receptor antagonist.

A detailed structure-function analysis of human interleukin 5 (hIL5) has been performed. The hIL5 receptor is composed of two different polypeptide chains, the alpha and beta subunits. The alpha subunit alone is sufficient for ligand binding, but association with the beta subunit leads to a 2- to 3-fold increase in binding affinity. The beta chain is shared with the receptors for IL3 and granulocyte/macrophage-colony-stimulating factor--hence the descriptor beta C (C for common). All hIL5 mutants were analyzed in a solid-phase binding assay for hIL5R alpha interaction and in a proliferation assay using IL5-dependent cell lines for receptor-complex activation. Most residues affecting binding to the receptor alpha subunit were clustered in a loop connecting beta-strand 1 and helix B (mutants H38A, K39A, and H41A), in beta-strand 2 (E89A and R91A; weaker effect for E90A) and close to the C terminus (T109A, E110A, W111S, and I112A). Mutations at one position, E13 (Glu13), caused a reduced activation of the hIL5 receptor complex. In the case of E13Q, only 0.05% bioactivity was detected on a hIL5-responsive subclone of the mouse promyelocytic cell line FDC-P1. Moreover, on hIL5-responsive TF1 cells, the same mutant was completely inactive and proved to have antagonistic properties. Interactions of this mutant with both receptor subunits were nevertheless indistinguishable from those of nonmutated hIL5 by crosslinking and Scatchard plot analysis of transfected COS-1 cells.

Transplantation of a 17-amino acid alpha-helical DNA-binding domain into an antibody molecule confers sequence-dependent DNA recognition.

Recombinant antibodies capable of sequence-specific interactions with nucleic acids represent a class of DNA- and RNA-binding proteins with potential for broad application in basic research and medicine. We describe the rational design of a DNA-binding antibody, Fab-Ebox, by replacing a variable segment of the immunoglobulin heavy chain with a 17-amino acid domain derived from TFEB, a class B basic helix-loop-helix protein. DNA-binding activity was studied by electrophoretic mobility-shift assays in which Fab-Ebox was shown to form a specific complex with DNA containing the TFEB recognition motif (CACGTG). Similarities were found in the abilities of TFEB and Fab-Ebox to discriminate between oligodeoxyribonucleotides containing altered recognition sequences. Comparable interference of binding by methylation of cytosine residues indicated that Fab-Ebox and TFEB both contact DNA through interactions along the major groove of double-stranded DNA. The results of this study indicate that DNA-binding antibodies of high specificity can be developed by using the modular nature of both immunoglobulins and transcription factors.

Glycosylation of the c-Myc transactivation domain.

O-linked N-acetylglucosamine (O-GlcNAc) is an abundant and dynamic posttranslational modification composed of a single monosaccharide, GlcNAc, glycosidically composed of a single monosaccharide, GlcNAc, glycosidically linked to the side-chain hydroxyl of serine or threonine residues. Although O-GlcNAc occurs on a myriad of nuclear and cytoplasmic proteins, only a few have thus far been identified. These O-GlcNAc-bearing proteins are also modified by phosphorylation and form reversible multimeric complexes. Here we present evidence for O-GlcNAc glycosylation of the oncoprotein c-Myc, a helix-loop-helix/leucine zipper phosphoprotein that heterodimerizes with Max and participates in the regulation of gene transcription in normal and neoplastic cells. O-GlcNAc modification of c-Myc is shown by three different methods: (i) demonstration of lectin binding to in vitro translated protein using a protein-protein interaction mobility-shift assay; (ii) glycosidase or glycosyltransferase treatment of in vitro translated protein analyzed by lectin affinity chromatography; and (iii) direct characterization of the sugar moieties on purified recombinant protein overexpressed in either insect cells or Chinese hamster ovary cells. Analyses of serial deletion mutants of c-Myc further suggest that the O-GlcNAc site(s) are located within or near the N-terminal transcription activation/malignant transformation domain, a region where mutations of c-Myc that are frequently found in Burkitt and AIDS-related lymphomas cluster.

Heat shock protein hsp90 regulates dioxin receptor function in vivo.

The dioxin (aryl hydrocarbon) receptor is a ligand-dependent basic helix-loop-helix (bHLH) factor that binds to xenobiotic response elements of target promoters upon heterodimerization with the bHLH partner factor Arnt. Here we have replaced the bHLH motif of the dioxin receptor with a heterologous DNA-binding domain to create fusion proteins that mediate ligand-dependent transcriptional enhancement in yeast (Saccharomyces cerevisiae). Previously, our experiments indicated that the ligand-free dioxin receptor is stably associated with the 90-kDa heat shock protein, hsp90. To investigate the role of hsp90 in dioxin signaling we have studied receptor function in a yeast strain where hsp90 expression can be down-regulated to about 5% relative to wild-type levels. At low levels of hsp90, ligand-dependent activation of the chimeric dioxin receptor construct was almost completely inhibited, whereas the activity of a similar chimeric construct containing the structurally related Arnt factor was not affected. Moreover, a chimeric dioxin receptor construct lacking the central ligand- and hsp90-binding region of the receptor showed constitutive transcriptional activity in yeast that was not impaired upon down-regulation of hsp90 expression levels. Thus, these data suggest that hsp90 is a critical determinant of conditional regulation of dioxin receptor function in vivo via the ligand-binding domain.

Expression at the Imprinted Dlk1-Gtl2 Locus Is Regulated by Proneural Genes in the Developing Telencephalon

Imprinting is an epigenetic mechanism that restrains the expression of about 100 genes to one allele depending on its parental origin. Several imprinted genes are implicated in neurodevelopmental brain disorders, such as autism, Angelman, and Prader-Willi syndromes. However, how expression of these imprinted genes is regulated during neural development is poorly understood. Here, using single and double KO animals for the transcription factors Neurogenin2 (Ngn2) and Achaete-scute homolog 1 (Ascl1), we found that the expression of a specific subset of imprinted genes is controlled by these proneural genes. Using in situ hybridization and quantitative PCR, we determined that five imprinted transcripts situated at the Dlk1-Gtl2 locus (Dlk1, Gtl2, Mirg, Rian, Rtl1) are upregulated in the dorsal telencephalon of Ngn2 KO mice. This suggests that Ngn2 influences the expression of the entire Dlk1-Gtl2 locus, independently of the parental origin of the transcripts. Interestingly 14 other imprinted genes situated at other imprinted loci were not affected by the loss of Ngn2. Finally, using Ngn2/Ascl1 double KO mice, we show that the upregulation of genes at the Dlk1-Gtl2 locus in Ngn2 KO animals requires a functional copy of Ascl1. Our data suggest a complex interplay between proneural genes in the developing forebrain that control the level of expression at the imprinted Dlk1-Gtl2 locus (but not of other imprinted genes). This raises the possibility that the transcripts of this selective locus participate in the biological effects of proneural genes in the developing telencephalon.