Hemagglutinating and fusogenic activities of Newcastle disease virus: studies on receptor binding specificity and pH-induced conformational changes

Vaccinal and wild strains of Newcastle Disease virus (NDV) were analyzed for cell receptor binding and fusogenic biological properties associated with their HN (hemagglutinin-neuraminidase) and F (fusion protein) surface structures respectively. The evaluation of the biological activities of HN and F was carried out respectively by determination of hemagglutinating titers and hemolysis percentages, using erythrocytes from various animal origins at different pH values. Significant differences in hemagglutination titers for some strains of NDV were detected, when interacting with goose, sheep, guinea-pip and human "O" group erythrocytes at neutral pH. Diversity of hemolysis percentagens was observed between different NDV strains at acid pH. These analysis were developed to evaluate particular aspects of the actual influence of the receptor specifity and pH on the receptor binding and fusogenic processes of Newcastle Disease viruses.

DNA binding specificity of different STAT proteins. Comparison of in vitro specificity with natural target sites.

STAT transcription factors are expressed in many cell types and bind to similar sequences. However, different STAT gene knock-outs show very distinct phenotypes. To determine whether differences between the binding specificities of STAT proteins account for these effects, we compared the sequences bound by STAT1, STAT5A, STAT5B, and STAT6. One sequence set was selected from random oligonucleotides by recombinant STAT1, STAT5A, or STAT6. For another set including many weak binding sites, we quantified the relative affinities to STAT1, STAT5A, STAT5B, and STAT6. We compared the results to the binding sites in natural STAT target genes identified by others. The experiments confirmed the similar specificity of different STAT proteins. Detailed analysis indicated that STAT5A specificity is more similar to that of STAT6 than that of STAT1, as expected from the evolutionary relationships. The preference of STAT6 for sites in which the half-palindromes (TTC) are separated by four nucleotides (N(4)) was confirmed, but analysis of weak binding sites showed that STAT6 binds fairly well to N(3) sites. As previously reported, STAT1 and STAT5 prefer N(3) sites; however, STAT5A, but not STAT1, weakly binds N(4) sites. None of the STATs bound to half-palindromes. There were no specificity differences between STAT5A and STAT5B.

The carbohydrate-binding specificity and molecular modelling of Canavalia maritima and Dioclea grandiflora lectins

The carbohydrate-binding specificity of lectins from the seeds of Canavalia maritima and Dioclea grandiflora was studied by hapten-inhibition of haemagglutination using various sugars and sugar derivatives as inhibitors, including N-acetylneuraminic acid and N-acetylmuramic acid. Despite some discrepancies, both lectins exhibited a very similar carbohydrate-binding specificity as previously reported for other lectins from Diocleinae (tribe Phaseoleae, sub-tribe Diocleinae). Accordingly, both lectins exhibited almost identical hydropathic profiles and their three-dimensional models built up from the atomic coordinates of ConA looked very similar. However, docking experiments of glucose and mannose in their monosaccharide-binding sites, by comparison with the ConA-mannose complex used as a model, revealed conformational changes in side chains of the amino acid residues involved in the binding of monosaccharides. These results fully agree with crystallographic data showing that binding of specific ligands to ConA requires conformational chances of its monosaccharide-binding site.

Mutants of common bean alpha-amylase inhibitor-2 as an approach to investigate binding specificity to alpha-amylases

Despite the presence of a family of defense proteins, Phaseolus vulgaris can be attacked by bruchid insects resulting in serious damage to stored grains. The two distinct active forms of a-amylase inhibitors, a-AI1 and a-AI2, in P. vulgaris show different specificity toward a-amylases. Zabrotes subfasciatus a-amylase is inhibited by a-AI2 but not by a-AI1. In contrast, porcine a-amylase is inhibited by a-AI1 but not by a-AI2. The objective of this work was to understand the molecular basis of the specificity of two inhibitors in P. vulgaris (a-AI1 and a-AI2) in relation to a-amylases. Mutants of a-AI2 were made and expressed in tobacco plants. The results showed that all the a-AI2 mutant inhibitors lost their activity against the insect a-amylases but none exhibited activity toward the mammalian a-amylase. The replacement of His33 of a-AI2 with the a-AI1-like sequence Ser-Tyr-Asn abolished inhibition of Z. subfasciatus a-amylase. From structural modeling, the conclusion is that the size and complexity of the amylase-inhibitor interface explain why mutation of the N-terminal loop and resultant abolition of Z. subfasciatus a-amylase inhibition are not accompanied by gain of inhibitory activity against porcine a-amylase.

Systematic structural studies of iron superoxide dismutases from human parasites and a statistical coupling analysis of metal binding specificity

Superoxide dismutases (SODs) are a crucial class of enzymes in the combat against intracellular free radical damage. They eliminate superoxide radicals by converting them into hydrogen peroxide and oxygen. In spite of their very different life cycles and infection strategies, the human parasites Plasmodium falciparum, Trypanosoma cruzi and Trypanosoma brucei are known to be sensitive to oxidative stress. Thus the parasite Fe-SODs have become attractive targets for novel drug development. Here we report the crystal structures of FeSODs from the trypanosomes T. brucei at 2.0 angstrom and T. cruzi at 1.9 angstrom resolution, and that from P. falciparum at a higher resolution (2.0 angstrom) to that previously reported. The homodimeric enzymes are compared to the related human MnSOD with particular attention to structural aspects which are relevant for drug design. Although the structures possess a very similar overall fold, differences between the enzymes at the entrance to the channel which leads to the active site could be identified. These lead to a slightly broader and more positively charged cavity in the parasite enzymes. Furthermore, a statistical coupling analysis (SCA) for the whole Fe/MnSOD family reveals different patterns of residue coupling for Mn and Fe SODs, as well as for the dimeric and tetrameric states. In both cases, the statistically coupled residues lie adjacent to the conserved core surrounding the metal center and may be expected to be responsible for its fine tuning, leading to metal ion specificity.

Molecular characterization of Arabidopsis thaliana PUF proteins - binding specificity and target candidates

PUF proteins regulate both stability and translation through sequence-specific binding to the 3` UTR of target mRNA transcripts. Binding is mediated by a conserved PUF domain, which contains eight repeats of approximately 36 amino acids each. Found in all eukaryotes, they have been related to several developmental processes. Analysis of the 25 Arabidopsis Pumilio (APUM) proteins presenting PUF repeats reveals that 12 (APUM-1 to APUM-12) have a PUF domain with 50-75% similarity to the Drosophila PUF domain. Through three-hybrid assays, we show that APUM-1 to APUM-6 can bind specifically to the Nanos response element sequence recognized by Drosophila Pumilio. Using an Arabidopsis RNA library in a three-hybrid screening, we were able to identify an APUM-binding consensus sequence. Computational analysis allowed us to identify the APUM-binding element within the 3` UTR in many Arabidopsis transcripts, even in important mRNAs related to shoot stem cell maintenance. We demonstrate that APUM-1 to APUM-6 are able to bind specifically to APUM-binding elements in the 3` UTR of WUSCHEL, CLAVATA-1, PINHEAD/ZWILLE and FASCIATA-2 transcripts. The results obtained in the present study indicate that the APUM proteins may act as regulators in Arabidopsis through an evolutionarily conserved mechanism, which may open up a new approach for investigating mRNA regulation in plants.

A streptavidin mutant with altered ligand-binding specificity

The biotin-binding site of streptavidin was modified to alter its ligand-binding specificity. In natural streptavidin, the side chains of N23 and S27 make two of the three hydrogen bonds with the ureido oxygen of biotin. These two residues were mutated to severely weaken biotin binding while attempting to maintain the affinity for two biotin analogs, 2-iminobiotin and diaminobiotin. Redesigning of the biotin-binding site used the difference in local electrostatic charge distribution between biotin and these biotin analogs. Free energy calculations predicted that the introduction of a negative charge at the position of S27 plus the mutation N23A should disrupt two of the three hydrogen bonds between natural streptavidin and the ureido oxygen of biotin. In contrast, the imino hydrogen of 2-iminobiotin should form a hydrogen bond with the side chain of an acidic amino acid at position 27. This should reduce the biotin-binding affinity by approximately eight orders of magnitude, while leaving the affinities for these biotin analogs virtually unaffected. In good agreement with these predictions, a streptavidin mutant with the N23A and S27D substitutions binds 2-iminobiotin with an affinity (Ka) of 1 × 106 M−1, two orders of magnitude higher than that for biotin (1 × 104 M−1). In contrast, the binding affinity of this streptavidin mutant for diaminobiotin (2.7 × 104 M−1) was lower than predicted (2.9 × 105 M−1), suggesting the position of the diaminobiotin in the biotin-binding site was not accurately determined by modeling.

OBA/Ku86: DNA Binding Specificity and Involvement in Mammalian DNA Replication

Ors-binding activity (OBA) was previously semipurified from HeLa cells through its ability to interact specifically with the 186-basepair (bp) minimal replication origin of ors8 and support ors8 replication in vitro. Here, through competition band-shift analyses, using as competitors various subfragments of the 186-bp minimal ori, we identified an internal region of 59 bp that competed for OBA binding as efficiently as the full 186-bp fragment. The 59-bp fragment has homology to a 36-bp sequence (A3/4) generated by comparing various mammalian replication origins, including the ors. A3/4 is, by itself, capable of competing most efficiently for OBA binding to the 186-bp fragment. Band-shift elution of the A3/4–OBA complex, followed by Southwestern analysis using the A3/4 sequence as probe, revealed a major band of ∼92 kDa involved in the DNA binding activity of OBA. Microsequencing analysis revealed that the 92-kDa polypeptide is identical to the 86-kDa subunit of human Ku antigen. The affinity-purified OBA fraction obtained using an A3/4 affinity column also contained the 70-kDa subunit of Ku and the DNA-dependent protein kinase catalytic subunit. In vitro DNA replication experiments in the presence of A3/4 oligonucleotide or anti-Ku70 and anti-Ku86 antibodies implicate Ku in mammalian DNA replication.

Improved DNA binding specificity from polyzinc finger peptides by using strings of two-finger units

Multizinc finger peptides are likely to reach increased prominence in the search for the “ideal” designer transcription factor for in vivo applications such as gene therapy. However, for these treatments to be effective and safe, the peptides must bind with high affinity and, more importantly, with great specificity. Our previous research has shown that zinc finger arrays can be made to bind 18 bp of DNA with picomolar affinity, but also has suggested that arrays of fingers also may bind tightly to related sequences. This work addresses the question of zinc finger DNA binding specificity. We show that by changing the way in which zinc finger arrays are constructed—by linking three two-finger domains rather than two three-finger units—far greater target specificity can be achieved through increased discrimination against mutated or closely related sequences. These new peptides have the added capability of being able to span two short gaps of unbound DNA, although still binding with picomolar affinity to their target sites. We believe that this new method of constructing zinc finger arrays will offer greater efficacy in the fields of gene therapy and in the production of transgenic organisms than previously reported zinc finger arrays.

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.

DNA binding specificity of two homeodomain proteins in vitro and in Drosophila embryos.

In previous experiments, the homeodomain proteins even-skipped and fushi-tarazu were found to UV cross-link to a surprisingly wide array of DNA sites in living Drosophila embryos. We now show that UV cross-linking gives a highly accurate measure of DNA binding by these proteins. In addition, the binding of even-skipped and fushi-tarazu proteins has been measured in vitro to the same DNA fragments that were examined in vivo. This analysis shows that these proteins have broad DNA recognition properties in vitro that are likely to be important determinants of their distribution on DNA in vivo, but it also shows that in vitro DNA binding specificity alone is not sufficient to explain the distribution of these proteins in embryos.

Phage display selection of ligand residues important for Src homology 3 domain binding specificity.

The Src homology 3 (SH3) domain is a 50-aa modular unit present in many cellular proteins involved in intracellular signal transduction. It functions to direct protein-protein interactions through the recognition of proline-rich motifs on associated proteins. SH3 domains are important regulatory elements that have been demonstrated to specify distinct regulatory pathways important for cell growth, migration, differentiation, and responses to the external milieu. By the use of synthetic peptides, ligands have been shown to consist of a minimum core sequence and to bind to SH3 domains in one of two pseudosymmetrical orientations, class I and class II. The class I sites have the consensus sequence ZP(L/P)PP psi P whereas the class II consensus is PP psi PPZ (where psi is a hydrophobic residue and Z is a SH3 domain-specific residue). We previously showed by M13 phage display that the Src, Fyn, Lyn, and phosphatidylinositol 3-kinase (PI3K) SH3 domains preferred the same class I-type core binding sequence, RPLPP psi P. These results failed to explain the specificity for cellular proteins displayed by SH3 domains in cells. In the current study, class I and class II core ligand sequences were displayed on the surface of bacteriophage M13 with five random residues placed either N- or C-terminal of core ligand residues. These libraries were screened for binding to the Src, Fyn, Lyn, Yes, and PI3K SH3 domains. By this approach, additional ligand residue preferences were identified that can increase the affinity of SH3 peptide ligands at least 20-fold compared with core peptides. The amino acids selected in the flanking sequences were similar for Src, Fyn, and Yes SH3 domains; however, Lyn and PI3K SH3 domains showed distinct binding specificities. These results indicate that residues that flank the core binding sequences shared by many SH3 domains are important determinants of SH3 binding affinity and selectivity.

Protein-Binding Microarray Analysis of Tumor Suppressor AP2α Target Gene Specificity.

Cheap and massively parallel methods to assess the DNA-binding specificity of transcription factors are actively sought, given their prominent regulatory role in cellular processes and diseases. Here we evaluated the use of protein-binding microarrays (PBM) to probe the association of the tumor suppressor AP2α with 6000 human genomic DNA regulatory sequences. We show that the PBM provides accurate relative binding affinities when compared to quantitative surface plasmon resonance assays. A PBM-based study of human healthy and breast tumor tissue extracts allowed the identification of previously unknown AP2α target genes and it revealed genes whose direct or indirect interactions with AP2α are affected in the diseased tissues. AP2α binding and regulation was confirmed experimentally in human carcinoma cells for novel target genes involved in tumor progression and resistance to chemotherapeutics, providing a molecular interpretation of AP2α role in cancer chemoresistance. Overall, we conclude that this approach provides quantitative and accurate assays of the specificity and activity of tumor suppressor and oncogenic proteins in clinical samples, interfacing genomic and proteomic assays.