CD30/TNF receptor-associated factor interaction: NF-kappa B activation and binding specificity.

CD30 is a member of the tumor necrosis factor (TNF) receptor superfamily. CD30 is expressed on normal activated lymphocytes, on several virally transformed T- or B-cell lines and on neoplastic cells of Hodgkin's lymphoma. The interaction of CD30 with its ligand induces pleiotropic effects on cells resulting in proliferation, differentiation, or death. The CD30 cytoplasmic tail interacts with TNF receptor-associated factors (TRAFs), which have been shown to transduce signals mediated by TNF-R2 and CD40. We demonstrate here that TRAF2 also plays an important role in CD30-induced NF-kappa B activation. We also show that TRAF2-mediated activation of NF-kappa B plays a role in the activation of HIV transcription induced by CD30 cross-linking. Detailed site-directed mutagenesis of the CD30 cytoplasmic tail reveals that there are two independent binding sites for TRAF, each interacting with a different domain of TRAF. Furthermore, we localized the TRAF-C binding site in CD30 to a 5-7 amino acid stretch.

Direct atomic force microscope imaging of EcoRI endonuclease site specifically bound to plasmid DNA molecules.

Direct imaging with the atomic force microscope has been used to identify specific nucleotide sequences in plasmid DNA molecules. This was accomplished using EcoRI (Gln-111), a mutant of the restriction enzyme that has a 1000-fold greater binding affinity than the wild-type enzyme but with cleavage rate constants reduced by a factor of 10(4). ScaI-linearized plasmids with single (pBS+) and double (pGEM-luc and pSV-beta-galactosidase) EcoRI recognition sites were imaged, and the bound enzyme was localized to a 50- to 100-nt resolution. The high affinity for the EcoRI binding site exhibited by this mutant endonuclease, coupled with an observed low level of nonspecific binding, should prove valuable for physically mapping large DNA clones by direct atomic force microscope imaging.

Unraveling principles of lead discovery: from unfrustrated energy landscapes to novel molecular anchors.

The search for novel leads is a critical step in the drug discovery process. Computational approaches to identify new lead molecules have focused on discovering complete ligands by evaluating the binding affinity of a large number of candidates, a task of considerable complexity. A new computational method is introduced in this work based on the premise that the primary molecular recognition event in the protein binding site may be accomplished by small core fragments that serve as molecular anchors, providing a structurally stable platform that can be subsequently tailored into complete ligands. To fulfill its role, we show that an effective molecular anchor must meet both the thermodynamic requirement of relative energetic stability of a single binding mode and its consistent kinetic accessibility, which may be measured by the structural consensus of multiple docking simulations. From a large number of candidates, this technique is able to identify known core fragments responsible for primary recognition by the FK506 binding protein (FKBP-12), along with a diverse repertoire of novel molecular cores. By contrast, absolute energetic criteria for selecting molecular anchors are found to be promiscuous. A relationship between a minimum frustration principle of binding energy landscapes and receptor-specific molecular anchors in their role as "recognition nuclei" is established, thereby unraveling a mechanism of lead discovery and providing a practical route to receptor-biased computational combinatorial chemistry.

Common molecular determinants of local anesthetic, antiarrhythmic, and anticonvulsant block of voltage-gated Na+ channels.

Voltage-gated Na+ channels are the molecular targets of local anesthetics, class I antiarrhythmic drugs, and some anticonvulsants. These chemically diverse drugs inhibit Na+ channels with complex voltage- and frequency-dependent properties that reflect preferential drug binding to open and inactivated channel states. The site-directed mutations F1764A and Y1771A in transmembrane segment IVS6 of type IIA Na+ channel alpha subunits dramatically reduce the affinity of inactivated channels for the local anesthetic etidocaine. In this study, we show that these mutations also greatly reduce the sensitivity of Na+ channels to state-dependent block by the class Ib antiarrhythmic drug lidocaine and the anticonvulsant phenytoin and, to a lesser extent, reduce the sensitivity to block by the class Ia and Ic antiarrhythmic drugs quinidine and flecainide. For lidocaine and phenytoin, which bind preferentially to inactivated Na+ channels, the mutation F1764A reduced the affinity for binding to the inactivated state 24.5-fold and 8.3-fold, respectively, while Y1771A had smaller effects. For quinidine and flecainide, which bind preferentially to the open Na+ channels, the mutations F1764A and Y1771A reduced the affinity for binding to the open state 2- to 3-fold. Thus, F1764 and Y1771 are common molecular determinants of state-dependent binding of diverse drugs including lidocaine, phenytoin, flecainide, and quinidine, suggesting that these drugs interact with a common receptor site. However, the different magnitude of the effects of these mutations on binding of the individual drugs indicates that they interact in an overlapping, but nonidentical, manner with a common receptor site. These results further define the contributions of F1764 and Y1771 to a complex drug receptor site in the pore of Na+ channels.

Identification of the zinc-dependent endothelial cell binding protein for high molecular weight kininogen and factor XII: identity with the receptor that binds to the globular "heads" of C1q (gC1q-R).

High molecular weight kininogen (HK) and factor XII are known to bind to human umbilical vein endothelial cells (HUVEC) in a zinc-dependent and saturable manner indicating that HUVEC express specific binding site(s) for those proteins. However, identification and immunochemical characterization of the putative receptor site(s) has not been previously accomplished. In this report, we have identified a cell surface glycoprotein that is a likely candidate for the HK binding site on HUVECs. When solubilized HUVEC membranes were subjected to an HK-affinity column in the presence or absence of 50 microM ZnCl2 and the bound membrane proteins eluted, a single major protein peak was obtained only in the presence of zinc. SDS/PAGE analysis and silver staining of the protein peak revealed this protein to be 33 kDa and partial sequence analysis matched the NH2 terminus of gC1q-R, a membrane glycoprotein that binds to the globular "heads" of C1q. Two other minor proteins of approximately 70 kDa and 45 kDa were also obtained. Upon analysis by Western blotting, the 33-kDa band was found to react with several monoclonal antibodies (mAbs) recognizing different epitopes on gC1q-R. Ligand and dot blot analyses revealed zinc-dependent binding of biotinylated HK as well as biotinylated factor XII to the isolated 33-kDa HUVEC molecule as well as recombinant gC1q-R. In addition, binding of 125I-HK to HUVEC cells was inhibited by selected monoclonal anti-gC1q-R antibodies. C1q, however, did not inhibit 125I-HK binding to HUVEC nor did those monoclonals known to inhibit C1q binding to gC1q-R. Taken together, the data suggest that HK (and factor XII) bind to HUVECs via a 33-kDa cell surface glycoprotein that appears to be identical to gC1q-R but interact with a site on gC1q-R distinct from that which binds C1q.

Interaction of the 82-kDa subunit of the vaccinia virus early transcription factor heterodimer with the promoter core sequence directs downstream DNA binding of the 70-kDa subunit.

The vaccinia virus early transcription factor (VETF), a heterodimeric protein composed of 82- and 70-kDa subunits, interacts with viral early promoters at both a sequence-specific core region upstream and a sequence-independent region downstream of the RNA start site. To determine the VETF subunit-promoter interactions, 32P-labeled DNA targets were chemically synthesized with uniquely positioned phosphorothioates to which azidophenacyl bromide moieties were coupled. After incubating the derivatized promoter with VETF and exposing the complex to 302-nm light, the protein was denatured and the individual subunits with or without covalently bound DNA were isolated with specific antiserum and analyzed by SDS/polyacrylamide gel electrophoresis. Using a set of 26 duplex probes, with uniquely positioned aryl azide moieties on the coding or template strands, we found that the 82-kDa subunit interacted primarily with the core region of the promoter, whereas the 70-kDa subunit interacted with the downstream region. Nucleotide substitutions in the core region that downregulate transcription affected the binding of both subunits: the 82-kDa subunit no longer exhibited specificity for upstream regions of the promoter but also bound to downstream regions, whereas the binding of the 70-kDa subunit was abolished even though the mutations were far upstream of its binding site. These results suggested mechanisms by which the interaction of the 82-kDa subunit with the core sequence directs binding of the 70-kDa subunit to DNA downstream.

The recombination signals for adeno-associated virus site-specific integration.

The adeno-associated virus (AAV) genome integrates site specifically into a defined region of human chromosome 19 (termed AAVS1). Using a functional assay for AAV integration into AAVS1 DNA propagated as an episome, we obtained evidence that a 33-nucleotide AAVS1 DNA sequence contains the minimum signal required for targeted integration. The recombination signal comprises a DNA-binding motif for the AAV regulatory Rep protein [Rep binding site (RBS)] separated by an eight-nucleotide spacer from a sequence that can act as a substrate for Rep endonucleolytic activity [terminal resolution site (TRS)]. Mutations in either the AAVS1-encoded RBS or TRS elements abort targeted integration. Since both the RBS and TRS elements are present in the viral origin of replication and are required for AAV replication, targeted integration into chromosome 19 AAVS1 DNA may involve a replicative type of recombination that is discussed. An additional chromosome 19 element, which is responsible for DNA rearrangements in episomes propagating AAVS1 DNA, was identified and shown not to be required for AAV episomal integration, despite its location adjacent to the recombination signal.

Multivalent DNA-binding properties of the HMG-1 proteins.

HMG-I proteins are DNA-binding proteins thought to affect the formation and function of transcription complexes. Each protein contains three DNA-binding motifs, known as AT-hooks, that bind in the minor groove of AT tracts in DNA. Multiple AT-hooks within a polypeptide chain should contact multiple AT tracts, but the rules governing these interactions have not been defined. In this study, we demonstrate that high-affinity binding uses two or three appropriately spaced AT tracts as a single multivalent binding site. These principles have implications for binding to regulatory elements such as the interferon beta enhancer, TATA boxes, and serum response elements.

A single residue in the M2-M3 loop is a major determinant of coupling between binding and gating in neuronal nicotinic receptors.

Binding of agonists to nicotinic acetylcholine receptors generates a sequence of changes that activate a cation-selective conductance. By measuring electrophysiological responses in chimeric alpha7/alpha3 receptors expressed in Xenopus oocytes, we have showed the involvement of the M2-M3 loop in coupling agonist binding to the channel gate. An aspartate residue therein, Asp-266 in the alpha7 subunit, was identified by site-directed mutagenesis as crucial, since mutants at this position exhibited very poor functional responses to three different nicotinic agonists. We have extended this investigation to another neuronal nicotinic receptor (alpha3/beta4), and found that a homologous residue in the beta4 subunit, Asp-268, played a similar role in coupling. These findings are consistent with a hypothesis that the aspartate residue in the M2-M3 loop, which is conserved in all homomer-forming alpha-type subunits and all neuronal beta-type subunits that combine to form functional receptors, is a major determinant of information transmission from binding site to channel gate in all neuronal nicotinic receptors.

A functional peptide encoded in the Escherichia coli 23S rRNA.

A pentapeptide open reading frame equipped with a canonical ribosome-binding site is present in the Escherichia coli 23S rRNA. Overexpression of 23S rRNA fragments containing the mini-gene renders cells resistant to the ribosome-inhibiting antibiotic erythromycin. Mutations that change either the initiator or stop codons of the peptide mini-gene result in the loss of erythromycin resistance. Nonsense mutations in the mini-gene also abolish erythromycin resistance, which can be restored in the presence of the suppressor tRNA, thus proving that expression of the rRNA-encoded peptide is essential for the resistance phenotype. The ribosome appears to be the likely target of action of the rRNA-encoded pentapeptide, because in vitro translation of the peptide mini-gene decreases the inhibitory action of erythromycin on cell-free protein synthesis. Thus, the new mechanism of drug resistance reveals that in addition to the structural and functional role of rRNA in the ribosome, it may also have a peptide-coding function.

Complementation analysis of mutants of nitric oxide synthase reveals that the active site requires two hemes.

For catalytic activity, nitric oxide synthases (NOSs) must be dimeric. Previous work revealed that the requirements for stable dimerization included binding of tetrahydrobiopterin (BH4), arginine, and heme. Here we asked what function is served by dimerization. We assessed the ability of individually inactive mutants of mouse inducible NOS (iNOS; NOS2), each deficient in binding a particular cofactor or cosubstrate, to complement each other by generating NO upon cotransfection into human epithelial cells. The ability of the mutants to homodimerize was gauged by gel filtration and/or PAGE under partially denaturing conditions, both followed by immunoblot. Their ability to heterodimerize was assessed by coimmunoprecipitation. Heterodimers that contained only one COOH-terminal hemimer and only one BH4-binding site could both form and function, even though the NADPH-, FAD-, and FMN-binding domains (in the COOH-terminal hemimer) and the BH4-binding sites (in the NH2-terminal hemimer) were contributed by opposite chains. Heterodimers that contained only one heme-binding site (Cys-194) could also form, either in cis or in trans to the nucleotide-binding domains. However, for NO production, both chains had to bind heme. Thus, NO production by iNOS requires dimerization because the active site requires two hemes.

Fos and Jun do not bend the AP-1 recognition site.

We have used a solution-based DNA cyclization assay and a gel-phasing method to show that contrary to previous reports [Kerppola, T. K. & Curran, T. (1991) Cell 66, 317-326], basic region leucine zipper proteins Fos and Jun do not significantly bend their AP-1 recognition site. We have constructed two sets of DNA constructs that contain the 7-bp 5'-TGACTCA-3' AP-1 binding site, from either the yeast or the human collagenase gene, which is well separated from and phased by 3-4 helical turns against an A tract-directed bend. The cyclization probabilities of DNAs with altered phasings are not significantly affected by Fos-Jun binding. Similarly, Fos-Jun and Jun-Jun bound to differently phased DNA constructs show insignificant variations in gel mobilities. Both these methods independently indicate that Fos and Jun bend their AP-1 target site by <5 degrees, an observation that has important implications in understanding their mechanism of transcriptional regulation.

A mammalian adaptor protein with conserved Src homology 2 and phosphotyrosine-binding domains is related to Shc and is specifically expressed in the brain.

The Shc adaptor protein, hereafter referred to as ShcA, possesses two distinct phosphotyrosine-recognition modules, a C-terminal Src homology 2 (SH2) domain and an N-terminal phosphotyrosine-binding (PTB) domain, and is itself phosphorylated on tyrosine in response to many extracellular signals. Phosphorylation of human ShcA at Tyr-317 within its central (CH1) region induces binding to the Grb2 SH2 domain and is thereby implicated in activation of the Ras pathway. Two shc-related genes (shcB and shcC) have been identified in the mouse. shcB is closely related to human SCK, while shcC has not yet been found in other organisms. The ShcC protein is predicted to have a C-terminal SH2 domain, a CH1 region with a putative Grb2-binding site, and an N-terminal PTB domain. The ShcC and ShcB SH2 domains bind phosphotyrosine-containing peptides and receptors with a specificity related to, but distinct from, that of the ShcA SH2 domain. The ShcC PTB domain specifically associates in vitro with the autophosphorylated receptors for nerve growth factor and epidermal growth factor. These results indicate that ShcC has functional SH2 and PTB; domains. In contrast to shcA, which is widely expressed, shcC RNA and proteins are predominantly expressed in the adult brain. These results suggest that ShcC may mediate signaling from tyrosine kinases in the nervous system, such as receptors for neurotrophins.

Multiplex selection technique (MuST): an approach to clone transcription factor binding sites.

We have used a multiplex selection approach to construct a library of DNA-protein interaction sites recognized by many of the DNA-binding proteins present in a cell type. An estimated minimum of two-thirds of the binding sites present in a library prepared from activated Jurkat T cells represent authentic transcription factor binding sites. We used the library for isolation of "optimal" binding site probes that facilitated cloning of a factor and to identify binding activities induced within 2 hr of activation of Jurkat cells. Since a large fraction of the oligonucleotides obtained appear to represent "optimal" binding sites for sequence-specific DNA-binding proteins, it is feasible to construct a catalog of consensus binding sites for DNA-binding proteins in a given cell type. Qualitative and quantitative comparisons of the catalogs of binding site sequences from various cell types could provide valuable insights into the process of differentiation acting at the level of transcriptional control.

Transcriptional activation of the mouse obese (ob) gene by CCAAT/enhancer binding protein alpha.

Like other adipocyte genes that are transcriptionally activated by CCAAT/enhancer binding protein alpha (C/EBP alpha) during preadipocyte differentiation, expression of the mouse obese (ob) gene is immediately preceded by the expression of C/EBP alpha. While the 5' flanking region of the mouse ob gene contains several consensus C/EBP binding sites, only one of these sites appears to be functional. DNase I cleavage inhibition patterns (footprinting) of the ob gene promoter revealed that recombinant C/EBP alpha, as well as a nuclear factor present in fully differentiated 3T3-L1 adipocytes, but present at a much lower level in preadipocytes, protects the same region between nucleotides -58 and -42 relative to the transcriptional start site. Electrophoretic mobility-shift analysis using nuclear extracts from adipose tissue or 3T3-L1 adipocytes and an oligonucleotide probe corresponding to a consensus C/EBP binding site at nucleotides -55 to -47 generated a specific protein-oligonucleotide complex that was supershifted by antibody against C/EBP alpha. Probes corresponding to two upstream consensus C/EBP binding sites failed to generate protein-oligonucleotide complexes. Cotransfection of a C/EBP alpha expression vector into 3T3-L1 cells with a series of 5' truncated ob gene promoter constructs activated reporter gene expression with all constructs containing the proximal C/EBP binding site (nucleotides -55 to -47). Mutation of this site blocked transactivation by C/EBP alpha. Taken together, these findings implicate C/EBP alpha as a transcriptional activator of the ob gene promoter and identify the functional C/EBP binding site in the promoter.