Karyopherin β2 mediates nuclear import of a mRNA binding protein

We have cloned and sequenced cDNA for human karyopherin β2, also known as transportin. In a solution binding assay, recombinant β2 bound directly to recombinant nuclear mRNA-binding protein A1. Binding was inhibited by a peptide representing A1’s previously characterized M9 nuclear localization sequence (NLS), but not by a peptide representing a classical NLS. As previously shown for karyopherin β1, karyopherin β2 bound to several nucleoporins containing characteristic peptide repeat motifs. In a solution binding assay, both β1 and β2 competed with each other for binding to immobilized repeat nucleoporin Nup98. In digitonin-permeabilized cells, β2 was able to dock A1 at the nuclear rim and to import it into the nucleoplasm. At low concentrations of β2, there was no stimulation of import by the exogenous addition of the GTPase Ran. However, at higher concentrations of β2 there was marked stimulation of import by Ran. Import was inhibited by the nonhydrolyzable GTP analog guanylyl imidodiphosphate by a Ran mutant that is unable to hydrolyze GTP and also by wheat germ agglutinin. Consistent with the solution binding results, karyopherin β2 inhibited karyopherin α/β1-mediated import of a classical NLS containing substrate and, vice versa, β1 inhibited β2-mediated import of A1 substrate, suggesting that the two import pathways merge at the level of docking of β1 and β2 to repeat nucleoporins.

Results of MDR-1 vector modification trial indicate that granulocyte/macrophage colony-forming unit cells do not contribute to posttransplant hematopoietic recovery following intensive systemic therapy

To formally test the hypothesis that the granulocyte/macrophage colony-forming unit (GM-CFU) cells can contribute to early hematopoietic reconstitution immediately after transplant, the frequency of genetically modified GM-CFU after retroviral vector transduction was measured by a quantitative in situ polymerase chain reaction (PCR), which is specific for the multidrug resistance-1 (MDR-1) vector, and by a quantitative GM-CFU methylcellulose plating assay. The results of this analysis showed no difference between the transduction frequency in the products of two different transduction protocols: “suspension transduction” and “stromal growth factor transduction.” However, when an analysis of the frequency of cells positive for the retroviral MDR-1 vector posttransplantation was carried out, 0 of 10 patients transplanted with cells transduced by the suspension method were positive for the vector MDR-1 posttransplant, whereas 5 of 8 patients transplanted with the cells transduced by the stromal growth factor method were positive for the MDR-1 vector transcription unit by in situ or in solution PCR assay (a difference that is significant at the P = 0.0065 level by the Fisher exact test). These data suggest that only very small subsets of the GM-CFU fraction of myeloid cells, if any, contribute to the repopulation of the hematopoietic tissues that occurs following intensive systemic therapy and transplantation of autologous hematopoietic cells.

A novel assay for drug-DNA binding mode, affinity, and exclusion number: scanning force microscopy.

Determining the mode-of-binding of a DNA ligand is not always straightforward. Here, we establish a scanning force microscopic assay for mode-of-binding that is (i) direct: lengths of individual DNA-ligand complexes are directly measured; (ii) rapid: there are no requirements for staining or elaborate sample preparation; and (iii) unambiguous: an observed increase in DNA length upon addition of a ligand is definitive evidence for an intercalative mode-of-binding. Mode-of-binding, binding affinity, and site-exclusion number are readily determined from scanning force microscopy measurements of the changes in length of individual drug-DNA complexes as a function of drug concentration. With this assay, we resolve the ambiguity surrounding the mode of binding of 2,5-bis(4-amidinophenyl) furan (APF) to DNA and show that it binds to DNA by nonintercalative modes. APF is a member of an important class of aromatic dicationic drugs that show significant activity in the treatment of Pneumocystis carinii pneumonia, an opportunistic infection that is the leading cause of death in AIDS patients.

The design, computer modeling, solution structure, and biological evaluation of synthetic analogs of bryostatin 1

The bryostatins are a unique family of emerging cancer chemotherapeutic candidates isolated from marine bryozoa. Although the biochemical basis for their therapeutic activity is not known, these macrolactones exhibit high affinities for protein kinase C (PKC) isozymes, compete for the phorbol ester binding site on PKC, and stimulate kinase activity in vitro and in vivo. Unlike the phorbol esters, they are not first-stage tumor promoters. The design, computer modeling, NMR solution structure, PKC binding, and functional assays of a unique class of synthetic bryostatin analogs are described. These analogs (7b, 7c, and 8) retain the putative recognition domain of the bryostatins but are simplified through deletions and modifications in the C4-C14 spacer domain. Computer modeling of an analog prototype (7a) indicates that it exists preferentially in two distinct conformational classes, one in close agreement with the crystal structure of bryostatin 1. The solution structure of synthetic analog 7c was determined by NMR spectroscopy and found to be very similar to the previously reported structures of bryostatins 1 and 10. Analogs 7b, 7c, and 8 bound strongly to PKC isozymes with Ki = 297, 3.4, and 8.3 nM, respectively. Control 7d, like the corresponding bryostatin derivative, exhibited weak PKC affinity, as did the derivative, 9, lacking the spacer domain. Like bryostatin, acetal 7c exhibited significant levels of in vitro growth inhibitory activity (1.8–170 ng/ml) against several human cancer cell lines, providing an important step toward the development of simplified, synthetically accessible analogs of the bryostatins.

High-affinity binding of the Drosophila Numb phosphotyrosine-binding domain to peptides containing a Gly-Pro-(p)Tyr motif

The phosphotyrosine-binding (PTB) domain is a recently identified protein module that has been characterized as binding to phosphopeptides containing an NPXpY motif (X = any amino acid). We describe here a novel peptide sequence recognized by the PTB domain from Drosophila Numb (dNumb), a protein involved in cell fate determination and asymmetric cell division during the development of the Drosophila nervous system. Using a Tyr-oriented peptide library to screen for ligands, the dNumb PTB domain was found to bind selectively to peptides containing a YIGPYφ motif (φ represents a hydrophobic residue). A synthetic peptide containing this sequence bound specifically to the isolated dNumb PTB domain in solution with a dissociation constant (Kd) of 5.78 ± 0.74 μM. Interestingly, the affinity of this peptide for the dNumb PTB domain was increased (Kd = 1.41 ± 0.10 μM) when the second tyrosine in the sequence was phosphorylated. Amino acid substitution studies of the phosphopeptide demonstrated that a core motif of sequence GP(p)Y is required for high-affinity binding to the dNumb PTB domain. Nuclear magnetic resonance experiments performed on isotopically labeled protein complexed with either Tyr- or pTyr-containing peptides suggest that the same set of amino acids in the dNumb PTB domain is involved in binding both phosphorylated and nonphosphorylated forms of the peptide. The in vitro selectivity of the dNumb PTB domain is therefore markedly different from those of the Shc and IRS-1 PTB domains, in that it interacts preferentially with a GP(p)Y motif, rather than NPXpY, and does not absolutely require ligand phosphorylation for binding. Our results suggest that the PTB domain is a versatile protein module, capable of exhibiting varied binding specificities.

Ribosome display efficiently selects and evolves high-affinity antibodies in vitro from immune libraries

Ribosome display was applied for affinity selection of antibody single-chain fragments (scFv) from a diverse library generated from mice immunized with a variant peptide of the transcription factor GCN4 dimerization domain. After three rounds of ribosome display, positive scFvs were isolated and characterized. Several different scFvs were selected, but those in the largest group were closely related to each other and differed in 0 to 5 amino acid residues with respect to their consensus sequence, the likely common progenitor. The best scFv had a dissociation constant of (4 ± 1) × 10−11 M, measured in solution. One amino acid residue in complementarity determining region L1 was found to be responsible for a 65-fold higher affinity than the likely progenitor. It appears that this high-affinity scFv was selected from the mutations occurring during ribosome display in vitro, and that this constitutes an affinity maturation inherent in this method. The in vitro-selected scFvs could be functionally expressed in the Escherichia coli periplasm with good yields or prepared by in vitro refolding. Thus, ribosome display can be a powerful methodology for in vitro library screening and simultaneous sequence evolution.

Elucidation of a PTS–Carbohydrate Chemotactic Signal Pathway in Escherichia coli Using a Time-resolved Behavioral Assay

Chemotaxis of Escherichia coli toward phosphotransferase systems (PTSs)–carbohydrates requires phosphoenolpyruvate-dependent PTSs as well as the chemotaxis response regulator CheY and its kinase, CheA. Responses initiated by flash photorelease of a PTS substrates d-glucose and its nonmetabolizable analog methyl α-d-glucopyranoside were measured with 33-ms time resolution using computer-assisted motion analysis. This, together with chemotactic mutants, has allowed us to map out and characterize the PTS chemotactic signal pathway. The responses were absent in mutants lacking the general PTS enzymes EI or HPr, elevated in PTS transport mutants, retarded in mutants lacking CheZ, a catalyst of CheY autodephosphorylation, and severely reduced in mutants with impaired methyl-accepting chemotaxis protein (MCP) signaling activity. Response kinetics were comparable to those triggered by MCP attractant ligands over most of the response range, the most rapid being 11.7 ± 3.1 s−1. The response threshold was <10 nM for glucose. Responses to methyl α-d-glucopyranoside had a higher threshold, commensurate with a lower PTS affinity, but were otherwise kinetically indistinguishable. These facts provide evidence for a single pathway in which the PTS chemotactic signal is relayed rapidly to MCP–CheW–CheA signaling complexes that effect subsequent amplification and slower CheY dephosphorylation. The high sensitivity indicates that this signal is generated by transport-induced dephosphorylation of the PTS rather than phosphoenolpyruvate consumption.

Stepwise in vitro affinity maturation of Vitaxin, an αvβ3-specific humanized mAb

A protein engineering strategy based on efficient and focused mutagenesis implemented by codon-based mutagenesis was developed. Vitaxin, a humanized version of the antiangiogenic antibody LM609 directed against a conformational epitope of the αvβ3 integrin complex, was used as a model system. Specifically, focused mutagenesis was used in a stepwise fashion to rapidly improve the affinity of the antigen binding fragment by greater than 90-fold. In the complete absence of structural information about the Vitaxin-αvβ3 interaction, phage-expressed antibody libraries for all six Ig heavy and light chain complementarity-determining regions were expressed and screened by a quantitative assay to identify variants with improved binding to αvβ3. The Vitaxin variants in these libraries each contained a single mutation, and all 20 amino acids were introduced at each complementarity-determining region residue, resulting in the expression of 2,336 unique clones. Multiple clones displaying 2- to 13-fold improved affinity were identified. Subsequent expression and screening of a library of 256 combinatorial variants of the optimal mutations identified from the primary libraries resulted in the identification of multiple clones displaying greater than 50-fold enhanced affinity. These variants inhibited ligand binding to receptor more potently as demonstrated by inhibition of cell adhesion and ligand competition assays. Because of the limited mutagenesis and combinatorial approach, Vitaxin variants with enhanced affinity were identified rapidly and required the synthesis of only 2,592 unique variants. The use of such small focused libraries obviates the need for phage affinity selection approaches typically used, permitting the use of functional assays and the engineering of proteins expressed in mammalian cell culture.

A general assay for antibody catalysis using acridone as a fluorescent tag.

A simple and highly sensitive catalysis assay is demonstrated based on analyzing reactions with acridonetagged compounds by thin-layer chromatography. As little as 1 pmol of product is readily visualized by its blue fluorescence under UV illumination and identified by its retention factor (Rf). Each assay requires only 10 microliters of solution. The method is reliable, inexpensive, versatile, and immediately applicable in repetitive format for screening catalytic antibody libraries. Three examples are presented: (i) the epoxidation of acridone labeled (S)-citronellol. The pair of stereoisomeric epoxides formed is resolved on the plate, which provides a direct selection method for enantioselective epoxidation catalysts. (ii) Oxidation of acridone-labeled 1-hexanol to 1-hexanal. The activity of horse liver alcohol dehydrogenase is detected. (iii) Indirect product labeling of released aldehyde groups by hydrazone formation with an acridone-labeled hydrazide. Activity of catalytic antibodies for hydrolysis of enol ethers is detected.

Isolation of HLA-DR1.(staphylococcal enterotoxin A)2 trimers in solution.

Mutational studies indicate that the superantigen staphylococcal enterotoxin A (SEA) has two separate binding sites for major histocompatibility complex (MHC) class II molecules. Direct evidence is provided here for the formation of SEA-MHC class II trimers in solution. Isoelectric focusing separated SEA-HLA-DR1 complexes into both dimers and HLA-DR1.SEA2 trimers. The molar ratio of components was determined by dual isotope labeling. The SEA mutant SEA-F47S, L48S, Y92A, which is deficient in MHC class II alpha-chain binding, formed only dimers with HLA-DR1, whereas a second SEA mutant, SEA-H225A, which lacks high-affinity MHC class II beta-chain binding was incapable of forming any complexes. Thus SEA binding to its MHC receptor is a two-step process involving initial beta-chain binding followed by cooperative binding of a second SEA molecule to the class II alpha chain.

Retinoid X receptor alpha forms tetramers in solution.

Protein-protein interactions allow the retinoid X receptor (RXR) to bind to cognate DNA as a homo- or a heterodimer and to participate in mediating the effects of a variety of hormones on gene transcription. Here we report a systematic study of the oligomeric state of RXR in the absence of a DNA template. We have used electrophoresis under nondenaturing conditions and chemical crosslinking to show that in solution, RXR alpha forms homodimers as well as homotetramers. The dissociation constants governing dimer and tetramer formation were estimated by fluorescence anisotropy studies. The results indicate that RXR tetramers are formed with a high affinity and that at protein concentrations higher than about 70 nM, tetramers will constitute the predominant species. Tetramer formation may provide an additional level of the regulation of gene transcription mediated by RXRs.

Interferon gamma signals via a high-affinity multisubunit receptor complex that contains two types of polypeptide chain.

Signaling by interferon gamma (IFN-gamma) requires two structurally related cell surface proteins: a ligand-binding polypeptide, known as the IFN-gamma receptor (IFN-gamma R), and an accessory factor. However, it is not known whether IFN-gamma forms a ternary complex with the IFN-gamma R and accessory factor to initiate signaling. Here we demonstrate complex formation between IFN-gamma and the two proteins, both in solution and at the cell surface. We observe complexes containing ligand, two molecules of IFN-gamma R (designated the IFN-gamma R alpha chain), and one or two molecules of accessory factor (designated the IFN-gamma R beta chain). Transfected cells expressing both IFN-gamma R chains bind IFN-gamma with higher affinity than do cells expressing alpha chain alone. Anti-beta-chain antibodies prevent the beta chain from participating in the ligand-receptor complex, reduce the affinity for IFN-gamma, and block signaling. Soluble alpha- or beta-chain extracellular domains also inhibit function. These results demonstrate that IFN-gamma signals via a high-affinity multisubunit complex that contains two types of receptor chain and suggest a potential approach to inhibiting specific actions of IFN-gamma by blocking the association of receptor subunits.

Mutational analysis of the conserved region 2 site of adenovirus E1A and its effect on binding to the retinoblastoma gene product: use of the "double-tagging" assay.

We have explored the feasibility of using a "double-tagging" assay for assessing which amino acids of a protein are responsible for its binding to another protein. We have chosen the adenovirus E1A-retinoblastoma gene product (pRB) proteins for a model system, and we focused on the high-affinity conserved region 2 of adenovirus E1A (CR2). We used site-specific mutagenesis to generate a mutant E1A gene with a lysine instead of an aspartic acid at position 121 within the CR2 site. We demonstrated that this mutant exhibited little binding to pRB by the double-tagging assay. We also have shown that this lack of binding is not due to any significant decrease in the level of expression of the beta-galactosidase-E1A fusion protein. We then created a "library" of phage expressing beta-galactosidase-E1A fusion proteins with a variety of different mutations within CR2. This library of E1A mutations was used in a double-tagging screening to identify mutant clones that bound to pRB. Three classes of phage were identified: the vast majority of clones were negative and exhibited no binding to pRB. Approximately 1 in 10,000 bound to pRB but not to E1A ("true positives"). A variable number of clones appeared to bind equally well to both pRB and E1A ("false positives"). The DNA sequence of 10 true positive clones yielded the following consensus sequence: DLTCXEX, where X = any amino acid. The recovery of positive clones with only one of several allowed amino acids at each position suggests that most, if not all, of the conserved residues play an important role in binding to pRB. On the other hand, the DNA sequence of the negative clones appeared random. These results are consistent with those obtained from other sources. These data suggest that a double-tagging assay can be employed for determining which amino acids of a protein are important for specifying its interaction with another protein if the complex forms within bacteria. This assay is rapid and up to 1 x 10(6) mutations can be screened at one time.