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PubMed1997

Subunit interactions in the Na,K-ATPase explored with the yeast two-hybrid system.

Subunit interactions of the alpha1- and beta1-subunits of the chicken Na,K-ATPase were explored with the yeast two-hybrid system. Gal4-fusion proteins containing domains of the alpha1- and beta1-subunits were designed for examining both intersubunit and intrasubunit protein-protein interactions. Regions of the alpha- and beta-subunits known to be involved in alpha-beta-subunit assembly were positive in two-hybrid assay, supporting the validity of the assays. A library of beta-subunit ectodomains with C-terminal truncations was screened to find the maximal truncation retaining an interaction with the alpha-subunit extracellular H7H8 loop (where H7 refers to the seventh membrane span, and so on). The maximal truncation removed all the cysteines involved in disulfide bridges, leaving only 63 amino acids of the beta-subunit ectodomain. Scanning alanine mutagenesis led to identification of an evolutionarily conserved sequence of four amino acids (SYGQ) in the extracellular H7H8 loop of the alpha-subunit that is crucial to alpha-beta-intersubunit interactions. Oligomerization studies with single domains failed to detect self-association of either of the two large cytosolic loops (H2H3 and H4H5) within the alpha-subunit. However, evidence was found for an interaction between these two cytoplasmic loops.

Amino Acid Sequence↗

PubMed2005

Characterization of T cell receptors engineered for high affinity against toxic shock syndrome toxin-1.

Superantigens, including bacterial enterotoxins, are a family of proteins that bind simultaneously to MHC class II molecules and the Vbeta regions of T cell receptors. This cross-linking results in the activation of a large population of T cells that release massive amounts of inflammatory cytokines, ultimately causing a condition known as toxic shock syndrome. The staphylococcal superantigen toxic shock syndrome toxin-1 (TSST-1) is a causative agent of this disease, but its structure in complex with the cognate T cell receptor (human Vbeta2.1) has not been determined. To understand the molecular details of the interaction and to develop high affinity antagonists to TSST-1, we used directed evolution to generate a panel of high affinity receptors for TSST-1. Yeast display libraries of random and site-directed hVbeta2.1 mutants were selected for improved domain stability and for higher affinity binding to TSST-1. Stability mutations allowed the individual Vbeta domains to be expressed in a bacterial expression system. Affinity mutations were generated in CDR2 and FR3 residues, yielding improvements in affinity of greater than 10,000-fold (a K(D) value of 180 pmol). Alanine scanning mutagenesis of hVbeta2.1 wild-type and mutated residues allowed us to generate a map of the binding site for TSST-1 and to construct a docking model for the hVbeta2.1-TSST-1 complex. Our experiments suggest that the energetic importance of a single hVbeta2.1 wild-type residue likely accounts for the restriction of TSST-1 specificity to only this human Vbeta region. The high affinity mutants described here thus provide critical insight into the molecular basis of TSST-1 specificity and serve as potential leads toward the development of therapeutic agents for superantigen-mediated disease.

Alanine↗

PubMed2004

Nonmycorrhizal (myc-) mutants of Hebeloma cylindrosporum obtained through insertional mutagenesis.

Polyethylene glycol-mediated transformation of protoplasts was used as a method for insertional mutagenesis to obtain mutants of the ectomycorrhizal fungus Hebeloma cylindrosporum impaired in symbiotic ability. Following restriction enzyme-mediated integration or conventional plasmid insertion, a library of 1,725 hygromycin-resistant monokaryotic transformants was generated and screened for the symbiotic defect, using Pinus pinaster seedlings as host plants. A total of 51 transformants displaying a dramatically reduced mycorrhizal ability were identified. Among them, 29 were nonmycorrhizal (myc-), but only 10 of them had integrated one or several copies of the transforming plasmid in their genome. Light and scanning electron microscopy observations of pine roots inoculated with myc- mutants suggested that we selected mutants blocked at early stages of interaction between partners or at the stage of Hartig net formation. Myc- mutants with plasmid insertions were crossed with a compatible wild-type monokaryon and allowed to fruit. Monokaryotic progenies were obtained in three independent crosses and were analyzed for symbiotic activity and plasmid insertion. In all three progenies, a 1:1 myc-:myc+ segregation ratio was observed, suggesting that each myc- phenotype resulted from a single gene mutation. However, for none of the three mutants, the myc- phenotype segregated with any of the plasmid insertions. Our results support the idea that master genes, the products of which are essential for symbiosis establishment, do exist in ectomycorrhizal fungi.

Basidiomycota↗

PubMed2004

Identification and characterization of peptides that bind to cyanovirin-N, a potent human immunodeficiency virus-inactivating protein.

Cyanovirin-N (CV-N) exerts a potent human immunodeficiency virus (HIV)-inactivating activity against diverse strains of HIV by binding to the viral surface envelope glycoprotein gp120 and blocking its essential interactions with cellular receptors. Based on previous thermodynamic analyses, it has been speculated that discrete protein-protein interactions might play an important ancillary role in the CV-N/gp120 binding event, in addition to the interactions of CV-N with specific oligosaccharides present on gp120. Here, we report the identification and characterization of CV-N-binding peptides, which were isolated by screening of M13 phage-displayed peptide libraries. After performing three rounds of biopanning of the libraries against biotinylated CV-N, a CV-N-binding motif, X3CX6(W/F)(Y/F)CX2(Y/F), was evident. A vector was designed to express CV-N-binding peptides as a fusion with thioredoxin (Trx) containing a penta-His affinity tag. The CV-N-binding peptides fused with His-tagged Trx inhibited binding of the corresponding peptide-bearing phages to CV-N, confirming that the peptides possessed CV-N-binding activity. Optical biosensor binding studies showed that the one of the CV-N-binding peptide, TN10-1, bound to CV-N with a KD value of 1.9 microM. The results of alanine scanning mutagenesis of the peptide showed that aromatic residues at positions 11, 12, and 16, as well as the conformational structure of the peptide secured by a disulfide bond, were important for the binding interactions. A series of competitive binding assays confirmed that gp120 inhibited CV-N binding of the corresponding peptide-bearing phages, and suggested that TN10-1 peptides were mimicking the protein component of gp120 rather than mimicking specific oligosaccharides present on gp120.

Amino Acid Motifs↗

PubMed2002

A comprehensive alanine scanning mutagenesis of the Escherichia coli transcriptional activator SoxS: identifying amino acids important for DNA binding and transcription activation.

SoxS is the direct transcriptional activator of the superoxide regulon. SoxS recognizes a highly degenerate "soxbox" DNA sequence, and activates transcription from class I and class II promoters. SoxS is the smallest member of the AraC/XylS family of transcription regulators whose hallmark is dual helix-turn-helix (HTH) DNA-binding motifs. Evidence suggests that the N-terminal HTH motif of SoxS interacts with a highly conserved region of the soxbox termed recognition element 1 (RE1), while the C-terminal HTH motif interacts with the less conserved recognition element 2 (RE2). In the work described here, we prepared a complete library of 101 SoxS mutants containing single alanine substitutions of SoxS, and we characterized the mutant proteins in vivo and in vitro. With SoxS being closely related to MarA, we analyzed the effects of the SoxS mutations in the context of the MarA-mar crystal structure and with respect to the NMR study of MarA-DNA complexes in solution. From the properties of the alanine substitutions, we conclude the following. (1) Surface-exposed residues of helix 3 and helix 6, the recognition helices of the dual HTH motifs, are important to DNA binding and transcription activation; however, substitutions of residues predicted from the MarA-mar crystal structure to make contact with the sugar-phosphate backbone are more detrimental to DNA binding than mutations predicted to make base-specific contacts. (2) Substitution of several residues within the recognition helix predicted to make base-specific contacts with RE2 have relatively little effect on DNA-binding, suggesting the possibility of alternative protein-DNA interactions than those inferred from the MarA-mar crystal structure. (3) DNA binding and transcription activation were reduced by substitution of conserved amino acid residues comprising the hydrophobic core, presumably because they disrupt the structural integrity of SoxS. (4) Mutant K30A appears to be a positive control mutant defective in a protein-protein interaction with RNA polymerase that is required for transcription activation at all SoxS-dependent promoters because it binds and bends DNA normally but fails to activate transcription from both classes of promoters. Alanine substitutions of surface-exposed residues H3, K5, D9, S31, and V45 confer a similar phenotype. Since these residues are near K30 on the surface of the protein, the surface formed by the six residues may be used to make protein-protein interactions with RNA polymerase that are required for transcription activation at both class I and class II SoxS-dependent promoters. (5) Mutants F74A, D75A, M78A, D79A and Q85A appear to define a surface required for protein-protein interaction with RNA polymerase specifically at class II promoters because these positive control mutants bind and bend DNA normally but are defective in activation of class II promoters but not class I promoters. These SoxS mutants that bind and bend DNA normally but are defective in transcription activation represent the first positive control mutants with putative defects in protein-protein interactions with RNA polymerase among the SoxS/MarA/Rob subset of the AraC/XylS family of transcription regulators.

Alanine↗

PubMed2003

Complex with a phage display-derived peptide provides insight into the function of insulin-like growth factor I.

The dramatic improvement in the NMR spectra of insulin-like growth factor I (IGF-I) in the presence of a peptide identified from a phage display library has allowed for the first time the determination of a high-resolution solution structure for much of IGF-I. The three helices of IGF-I in this complex have an arrangement similar to that seen in high-resolution crystal structures of IGF-I and insulin, although there are differences in the conformation and precise location of helix 3. A cluster of hydrophobic and basic side chains within the turn-helix motif of the peptide contact a hydrophobic patch on helices 1 and 3 of IGF-I. The importance of this patch for tight binding was verified using alanine scanning mutagenesis of the peptide in two different phage display formats. Consistent with its antagonistic activity, the peptide binds to a region implicated by mutagenesis studies to be important for association with IGF binding proteins (IGFBPs). The ability of the peptide to also inhibit signaling has important implications for the manner in which IGF-I interacts with its receptor. Interestingly, the peptide uses the same binding site as detergent and a fragment of IGFBP-5 identified in other IGF-I complexes. The ligand-induced structural variability of helix 3 in these complexes suggests that exchange between such conformations may be the source of the dynamic nature of free IGF-I and likely has functional significance for the ability of IGF-I to recognize two signaling receptors and six binding proteins with high affinity.

Alanine↗

PubMed1998

Analysis of BR96 binding sites for antigen and anti-idiotype by codon-based scanning mutagenesis.

We performed a scanning mutagenesis study of heavy chain complementarity-determining region (CDR) residues to identify how mutations affected binding of the anti-carcinoma mAb BR96 to Ag, Lewis Y, and to an anti-Id Ab (anti-Id). By ELISA, we demonstrated that the anti-Id bound close to the Ag binding site of BR96, but the anti-Id and Ag sites were not identical. Immunoblot analysis and screening of light and heavy chain CDR libraries with multiple mutations in each CDR suggested that the heavy chain had greater involvement in anti-Id binding. We then analyzed contributions of individual residues in the heavy chain CDRs to binding of Ag and anti-Id. In a filamentous phage vector containing BR96 V region sequences, mutations were introduced by codon-based mutagenesis at single positions within the three heavy chain CDRs. The resulting libraries of Fab fragments had all amino acids represented at a CDR position. We evaluated the expressed Fabs for binding to Ag and anti-Id by plaque lift assay. We identified the positions with mutations that had the greatest negative effect on binding to the anti-Id and to Ag and analyzed them on the basis of the BR96 x-ray structure. The residues most important for binding to the anti-Id were located in heavy chain CDR1 and CDR2 and were peripheral to the residues within the Lewis Y binding pocket.

Animals↗

PubMed1996

hymA (hypha-like metulae), a new developmental mutant of Aspergillus nidulans.

Asexual fruiting body development in Aspergillus nidulans requires a precise spatial and temporal coordinated expression of many genes. Insertional mutagenesis was used to isolate and characterize a new mutant of A. nidulans in which hyphal growth was slightly reduced and conidiophore development was specifically blocked at the metula stage. In contrast to the uninucleate metulae of the wild-type, in the mutant these structures were elongated, multinucleate and septate. Further differentiation and production of phialides by a budding-like process was not observed. The mutant metulae thus resembled hyphae rather than metulae and the gene was therefore named hypha-like metulae (hymA). The hymA gene was mapped to linkage group VI. The integrated vector was rescued with border sequences from the integration site. The border sequences were used to isolate a cosmid from a wild-type library which was subcloned to a 5 kb fragment able to complement the mutation in trans. This fragment encoded a 1.8 kb transcript expressed in hyphae and throughout development. It is proposed that hymA is involved in budding processes, and is required for the formation of metulae and for their further differentiation.

Aspergillus nidulans↗

PubMed1993

Identification of a peptide which binds to the carbohydrate-specific monoclonal antibody B3.

The monoclonal antibody (mAb) B3 recognizes an antigen found on the surface of many adenocarcinoma cells. While the structure of the cellular antigen is unknown, epitope mapping using neoglycoproteins with known carbohydrate moieties indicates that the mAb B3 reacts with the LewisY (LeY) antigen [Pastan et al., Cancer Res. 51 (1991) 3781-3787]. We have used mAb B3 to select for peptides that mimic the carbohydrate structure using libraries of filamentous phage displaying random peptides on their surface. Phage that were selected coded for the sequence APWLYGPA. The corresponding peptide was synthesized and tested for its ability to bind to mAb B3. The peptide was found to inhibit specifically the binding of 111In-labeled mAb B3 to A431 adenocarcinoma cells, as well as to inhibit killing of these cells by a B3 immunotoxin. In addition, the LeY carbohydrate, lactodifucotetraose, was able to compete with the phage displaying this peptide for binding to mAb B3. Alanine-scanning mutagenesis of the sequence coding for this peptide indicates that four residues, PWLY, were critical for binding to the mAb. The sequence is similar to other sequences known to mimic carbohydrate structures.

Adenocarcinoma↗

PubMed2000

Rapid mapping of protein functional epitopes by combinatorial alanine scanning.

A combinatorial alanine-scanning strategy was used to determine simultaneously the functional contributions of 19 side chains buried at the interface between human growth hormone and the extracellular domain of its receptor. A phage-displayed protein library was constructed in which the 19 side chains were preferentially allowed to vary only as the wild type or alanine. The library pool was subjected to binding selections to isolate functional clones, and DNA sequencing was used to determine the alanine/wild-type ratio at each varied position. This ratio was used to calculate the effect of each alanine substitution as a change in free energy relative to that of wild type. Only seven side chains contribute significantly to the binding interaction, and these conserved residues form a compact cluster in the human growth hormone tertiary structure. The results were in excellent agreement with free energy data previously determined by conventional alanine-scanning mutagenesis and suggest that this technology should be useful for analyzing functional epitopes in proteins.

Alanine↗

PubMed2005

Fine tuning of the specificity of an anti-progesterone antibody by first and second sphere residue engineering.

The specificity of anti-progesterone P15G12C12G11 antibody was improved by combination of in vitro scanning saturation mutagenesis and error-prone PCR. The most evolved mutant is able to discriminate against 5beta- or 5alpha-dihydroprogesterone, 23 and 15 times better than the starting antibody, while maintaining the affinity for progesterone that remains in the picomolar range. The high level of homology with anti-progesterone monoclonal antibody DB3 allowed the construction of three-dimensional models of P15G12C12G11 based on the structures of DB3 in complex with various steroids. These models together with binding data, derived from site-directed mutagenesis, were used to build a phage library in which five first sphere positions in complementarity-determining regions 2H and 3L were varied. Variants selected by an initial screening in competition against a large excess of 5beta- or 5alpha-dihydroprogesterone were characterized by a convergent amino acid signature different from that of the wild-type antibody and had lower cross-reactivity. Binding properties of this first set of mutants were further improved by the addition of second sphere mutations selected independently from an error-prone library. The three-dimensional models of the best variant show changes in the antigen binding site that explain well the increase in selectivity. The improvements are partly linked to a change in the canonical class of the light chain third hypervariable loop.

5-alpha-Dihydroprogesterone↗

PubMed2025

Mutational scanning of TnpB reveals latent activity for genome editing.

TnpB is a diverse family of RNA-guided endonucleases associated with prokaryotic transposons. Due to their small size and putative evolutionary relationship to CRISPR-Cas12, TnpB enzymes hold significant potential for genome editing. However, most TnpBs lack robust gene editing activity, and unbiased profiling of mutational effects on editing activity has not been explored. Here, we mapped comprehensive sequence-function landscapes of a TnpB ribonucleoprotein and discovered many activating mutations in both the protein and RNA. One- and two-position RNA mutants outperform existing variants, highlighting the utility of systematic RNA scaffold mutagenesis. Leveraging the protein's mutational landscape, we identified enhanced TnpB variants from a combinatorial library of activating mutations. These variants enhanced editing in human cells, N. benthamiana, pepper, and rice, with up to a fifty-fold increase compared to wild-type TnpB. These findings highlight previously unknown elements critical for regulating TnpB endonuclease activity and reveal surprising latent activity accessible through mutation.

Journal Article↗

PubMed2002

Random peptide bacteriophage display as a probe for urokinase receptor ligands.

The urokinase receptor is a multi-functional protein that plays a central role in cell surface plasminogen activation, cell migration, and cell adhesion. We previously demonstrated that high affinity peptide ligands for the urokinase receptor, which are urokinase competitors, can be obtained from a 15mer peptide library (Goodson et al., 1994). In order to probe for additional urokinase receptor binding sites we affinity selected the same bacteriophage library on complexes of soluble urokinase receptor (suPAR) and the receptor binding domain of urokinase, residues 1-48 (uPA1-48). Bacteriophage were isolated which bound to suPAR and suPAR:uPA1-48 complexes with high yield. The peptide sequences encoded by these bacteriophage were distinct from those obtained previously on urokinase receptor expressing cells, and comprise two groups based upon effects on su-PAR:1-anilino-8-napthalene sulfonate (ANS) fluorescence, and vitronectin binding competition. Alanine scanning mutagensis of the soluble peptides was used to define minimal regions and key residues for suPAR binding by competition with the parent bacteriophage. A comparison of these results with sequences of domains of both vitronectin and integrin alpha-chains, which have been reported to be important for urokinase receptor binding, suggests that the homology with the peptide sequences selected is functionally significant.

Alanine↗

PubMed1991

Selecting high-affinity binding proteins by monovalent phage display.

Variants of human growth hormone (hGH) with increased affinity and specificity for the hGH receptor were isolated using an improved phage display system. Nearly one million random mutants of hGH were generated at 12 sites previously shown to modulate binding to the hGH receptor or human prolactin (hPRL) receptor. The mutant hormones were displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. After three to six cycles of enrichment for hGH-phage particles that bound to hGH receptor beads, we isolated hGH mutants that exhibited consensus binding sequences for the hGH receptor. Residues previously identified as important for hGH receptor binding by alanine-scanning mutagenesis were more highly conserved by this selection method. However, other residues nearby were not optimal, and by mutating them, hormone variants having greater affinity and selectivity for the hGH receptor were isolated. This approach should be useful for those who wish to modify and understand the energetics of protein-ligand interfaces.

Amino Acid Sequence↗

PubMed1994

SIS/PDGF-B promoter isolation and characterization of regulatory elements necessary for basal expression of the SIS/PDGF-B gene in U2-OS osteosarcoma cells.

Platelet-derived growth factor BB, encoded by the SIS/PDGF-B gene, is a potent mitogen for cells of mesenchymal origin, and the SIS/PDGF-B gene is expressed in a large percentage of human mesenchymal tumor cells establishing a growth-promoting, autocrine growth circuit. A 4-kb fragment, containing the SIS/PDGF-B promoter, was isolated from a human genomic library, and a series of 5'-nested deletions and linker-scanning mutants were used to identify positive regulatory elements that are necessary for the constitutive expression of this gene in human U2-OS osteosarcoma cells. A 250-bp fragment, lying immediately 5' to the SIS/PDGF-B mRNA initiation site (+1), retained full promoter activity, and positive regulatory elements at -228 to -219, -97 to -88 (SIS distal element) and -58 to -39 (SIS proximal element, SPE) were identified. Insertion of the 20-bp SPE into a heterologous, minimal promoter resulted in >5-fold transcriptional activation which was ablated by mutations to the SPE. High resolution mutagenesis within the 20-bp SPE, indicated the necessity of a CACCC motif for activity. Gel shift analysis of SPE-binding proteins in U2-OS nuclear extracts identified Sp1 and two additional binding factors that could be competed away from SPE binding by adding excess consensus Sp1 or CACC oligonucleotides. The individual and aggregate roles of the SPE and two weaker positive regulatory elements in regulating SIS/PDGF-B transcription in these tumor cells is considered.

Base Sequence↗

PubMed2006

An ordered, nonredundant library of Pseudomonas aeruginosa strain PA14 transposon insertion mutants.

Random transposon insertion libraries have proven invaluable in studying bacterial genomes. Libraries that approach saturation must be large, with multiple insertions per gene, making comprehensive genome-wide scanning difficult. To facilitate genome-scale study of the opportunistic human pathogen Pseudomonas aeruginosa strain PA14, we constructed a nonredundant library of PA14 transposon mutants (the PA14NR Set) in which nonessential PA14 genes are represented by a single transposon insertion chosen from a comprehensive library of insertion mutants. The parental library of PA14 transposon insertion mutants was generated by using MAR2xT7, a transposon compatible with transposon-site hybridization and based on mariner. The transposon-site hybridization genetic footprinting feature broadens the utility of the library by allowing pooled MAR2xT7 mutants to be individually tracked under different experimental conditions. A public, internet-accessible database (the PA14 Transposon Insertion Mutant Database, http://ausubellab.mgh.harvard.edu/cgi-bin/pa14/home.cgi) was developed to facilitate construction, distribution, and use of the PA14NR Set. The usefulness of the PA14NR Set in genome-wide scanning for phenotypic mutants was validated in a screen for attachment to abiotic surfaces. Comparison of the genes disrupted in the PA14 transposon insertion library with an independently constructed insertion library in P. aeruginosa strain PAO1 provides an estimate of the number of P. aeruginosa essential genes.

DNA Transposable Elements↗

PubMed2001

Structure-function analysis of a phage display-derived peptide that binds to insulin-like growth factor binding protein 1.

Highly structured, peptide antagonists of the interaction between insulin-like growth factor 1 (IGF-I) and IGF binding protein 1 (IGFBP-1) have recently been discovered by phage display of naïve peptide libraries [Lowman, H. B., et al. (1998) Biochemistry 37, 8870--8878]. We now report a detailed analysis of the features of this turn-helix peptide motif that are necessary for IGFBP-1 binding and structural integrity. Further rounds of phage randomization indicate the importance of residues contributing to a hydrophobic patch on one face of the helix. Alanine-scanning substitutions confirm that the hydrophobic residues are necessary for binding. However, structural analysis by NMR spectroscopy indicates that some of these analogues are less well folded. Structured, high-affinity analogues that lack the disulfide bond were prepared by introducing a covalent constraint between side chains at positions i and i + 7 or i + 8 within the helix. Analogues based on this scaffold demonstrate that a helical conformation is present in the bound state, and that hydrophobic side chains in this helix, and residues immediately preceding it, interact with IGFBP-1. By comparison of alanine scanning data for IGF-I and the turn-helix peptide, we propose a model for common surface features of these molecules that recognize IGFBP-1.

Alanine↗

PubMed1995

Codon-based combinatorial alanine scanning site-directed mutagenesis: design, implementation, and polymerase chain reaction screening.

Combinatorial alanine scanning mutagenesis is a powerful tool for the exploration of protein structure-function relationships. Unfortunately, combinatorial alanine replacement of multiple residues using standard site-directed mutagenesis is restricted to a subset of amino acids. To circumvent this limitation, an efficient procedure for combinatorial site-specific replacement by alanine of any residue in a given protein sequence has been established. The method, which involves simple procedures and commonly used materials, is based upon the use of codon-based mutagenesis. A defined ratio of alanine to wild-type codon was introduced at each predetermined triplet using the "column-splitting" technique during oligonucleotide synthesis. High-throughout genetic screening of mutant libraries was facilitated by the incorporation of diagnostic restriction sites at targeted codons followed by a PCR-based screening procedure. The method was tested on a set of 13 residues located at the interface between the variable domains of a Fab fragment of an antibody. The occurrence of alanine substitution was found to be comparable to the statistically predicted distribution.

Alanine↗