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PubMed2026

Library-based, multiplexed strategy for mapping protein interaction networks via crosslinking.

BACKGROUND: Protein-protein interactions are fundamental to cellular function, yet resolving their interaction interfaces and dynamic behaviors in native biological contexts remains challenging, particularly for weak or transient interactions. Crosslinking strategies based on noncanonical amino acids offer an effective means to capture such interactions; however, traditional single-site incorporation provides limited coverage and may overlook critical interaction hotspots. RESULTS: By employing a mutagenesis library, multiple interaction partners and cross-linking sites of a target protein can be simultaneously screened in a single experiment, without prior knowledge of its precise structural or functional features, enabling effective and unbiased analysis of its interaction network. In this study, we constructed an amber codon-scanning mutagenesis library of PSMD10, facilitating independent incorporation of the photocrosslinking ncAA p-azido-phenylalanine at multiple distinct residues. This approach allowed us to systematically interrogate and precisely map potential interaction regions across the protein surface. Coupled with crosslinking mass spectrometry, we identified multiple residues involved in intermolecular interactions, as well as previously unreported interaction partners, including T2FA, TBA1C, and ATRIP. CONCLUSIONS: These findings expand our understanding of PSMD10-associated proteasome interactome, demonstrate a multiplexed strategy for in situ mapping of protein interaction interfaces with broad coverage, and offer a valuable platform for developing therapeutics that target protein-protein interactions.

Protein Interaction Mapping↗
PubMed1993

Affinity maturation of human growth hormone by monovalent phage display.

We describe a selection procedure for construction of very high affinity variants of human growth hormone (hGH) for binding to the extra cellular domain of its receptor (called the hGHbp). Five different libraries of mutated hGH genes (each containing approximately 2 x 10(5) protein variants) were created by randomly mutating four different codons at residues that were shown to be important for receptor binding by structural or functional criteria. Mutated proteins were displayed as single copies from their respective filamentous phagemid particles and sorted in vitro for binding to the immobilized hGHbp. Phagemid particles that bound the immobilized hGHbp were eluted and propagated. After three to seven rounds of binding enrichments, hGH variants were isolated that contained 2 to 4 mutations and exhibited three- to sixfold improvements in binding affinity. Because of the limits of DNA transfection efficiency in creating the library we could not sample thoroughly mutations at more than four codons at once. Nonetheless, the free energy effects for these mutations acted cumulatively. Thus, by combining affinity enhanced mutants from libraries independently sorted we created an hGH variant with 15 substitutions that bound approximately 400-fold more tightly to the hGHbp than wild-type hGH. The affinity enhancements occurred predominantly by slowing the off-rate of the hormone (> 60-fold), and partly through increasing the on-rate (up to 4-fold). Residues that were shown to be important for binding by alanine-scanning were most highly conserved after binding selection, and interestingly many of them could be further improved. Thus, we found it most effective to randomly mutate the residues that were shown to modulate affinity by alanine-scanning, and to combine the selectants from separate libraries that exhibit the highest affinities. The selection procedure and mutagenesis strategy provides a framework for affinity maturation of protein-protein complexes.

Amino Acid Sequence↗
PubMed1993

Antibody engineering by parsimonious mutagenesis.

The human monoclonal antibody (humAb) problem has largely been solved with the aid of the polymerase chain reaction (PCR) [Larrick et al., Bio/Technology 7 (1989a) 934-938; Larrick et al., Biochem. Biophys. Res. Commun. 160 (1989b) 1250-1256; Chiang et al., BioTechniques 7 (1989) 360-366]. Phage display has now made it possible to recover humAb with primary response level affinities (approx. 10(6) M-1) for virtually any antigen (including self antigens) from comprehensive libraries of B-cell repertoires from non-immunized humans [Marks et al., J. Mol. Biol. 222 (1991) 581-597; Marks et al., Bio/Technology 10 (1992) 779-783; Griffiths et al., EMBO J. 12 (1993) 725-734]. This means that the goal of therapeutic humAb without immunization is within reach. However, in order to achieve the affinities generally required for therapeutic use (> or = 10(9) M-1), reliable methods will be needed to complete the affinity maturation process in vitro. Available X-ray crystallographic data and energy calculations indicate that only a fraction of the substantial contact surface between the Ab and protein antigens contribute significantly to affinity. Thus, the remaining contact surface presents multiple opportunities to develop additional high-affinity contacts, needing only a means to identify them. To this end, we have developed a computer-assisted method for oligodeoxyribonucleotide-directed scanning mutagenesis, called parsimonious mutagenesis (PM), whereby all three complementarity-determining regions (CDR) of a variable region (V-region) gene can be simultaneously and thoroughly searched for improved variants in libraries of manageable size. These libraries are made with low-redundancy 'doping' codons and biased nucleotide (nt) mixtures designed to maximize the abundance of combining sites with predetermined proportions of preselected sets of alternative amino acids (aa). This allows the library to 'probe' the surface of the antigen one or a few aa residues at a time with a wide selection of aa side chains to search out and identify new high-affinity contacts. In addition to affinity maturation in vitro, PM can also be used to remove unwanted cross-reactivities and to 'reshape' rodent mAb for human therapeutic use.

Amino Acid Sequence↗
PubMed1995

Activating transcription factor 1 and cyclic AMP response element modulator can modulate the activity of the immunoglobulin kappa 3' enhancer.

Previously we determined that the immunoglobulin kappa 3' enhancer (kappa E3') contains at least two functional DNA sequences (PU.1/NF-EM5 and E2A) within its 132-base pair active core. We have determined that the activities of these two sequences are insufficient to account for the entire activity of the 132-base pair core. Using site-directed linker scan mutagenesis across the core fragment we identified several additional functional sequences. We used one of these functional sequences to screen a lambda gt11 cDNA expression library resulting in the isolation of cDNA clones encoding the transcription factors ATF-1 (activating transcription factor) and CREM (cyclic AMP response element modulator). Because ATF-1 and CREM are known to bind to cAMP response elements (CRE), this functional sequence was named the kappa E3'-CRE. We show that dibutyryl cAMP can increase kappa E3' enhancer activity, and in transient expression assays ATF-1 caused a 4-5-fold increase in the activity of the core enhancer while CREM-alpha expression resulted in repression of enhancer activity. RNA analyses showed increased levels of ATF-1 mRNA during B cell development and some changes in CREM transcript processing. By joining various fragments of the kappa E3' enhancer to the kappa E3'-CRE, we observed that the kappa E3'-CRE can synergistically increase transcription in association with the PU.1/NF-EM5 binding sites, suggesting a functional interaction between the proteins that bind to these DNA sequences. Consistent with this possibility, we found that ATF-1 and CREM can physically interact with PU.1. The isolation of activator and repressor proteins that bind to the kappa E3'-CRE may relate to previous conflicting results concerning the role of the cAMP signal transduction pathway in kappa gene transcription.

Activating Transcription Factor 1↗
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↗
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↗
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↗
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↗
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↗
PubMed1988

Random oligonucleotide mutagenesis: application to a large protein coding sequence of a major histocompatibility complex class I gene, H-2DP.

We have used random oligonucleotide mutagenesis (or saturation mutagenesis) to create a library of point mutations in the alpha 1 protein domain of a Major Histocompatibility Complex (MHC) molecule. This protein domain is critical for T cell and B cell recognition. We altered the MHC class I H-2DP gene sequence such that synthetic mutant alpha 1 exons (270 bp of coding sequence), which contain mutations identified by sequence analysis, can replace the wild type alpha 1 exon. The synthetic exons were constructed from twelve overlapping oligonucleotides which contained an average of 1.3 random point mutations per intact exon. DNA sequence analysis of mutant alpha 1 exons has shown a point mutant distribution that fits a Poisson distribution, and thus emphasizes the utility of this mutagenesis technique to "scan" a large protein sequence for important mutations. We report our use of saturation mutagenesis to scan an entire exon of the H-2DP gene, a cassette strategy to replace the wild type alpha 1 exon with individual mutant alpha 1 exons, and analysis of mutant molecules expressed on the surface of transfected mouse L cells.

Amino Acid Sequence↗
PubMed1993

Minimal replication origin of the 200-kilobase Halobacterium plasmid pNRC100.

We have identified the replication origin of pNRC100, a 200-kb plasmid of Halobacterium halobium, by assaying for replication ability of miniplasmids containing cloned fragments of pNRC100 and the mevinolin resistance selectable marker of Haloferax volcanii. First, we showed the replication ability of plasmid pNGHCMEV1, which contains the 19-kb HindIII-C fragment of pNRC100, by recovery of plasmid DNA from mevinolin-resistant transformants of H. halobium. The minimal replication origin of approximately 3.9 kb was defined by subcloning successively smaller regions of pNGHCMEV1 and assaying for plasmid replication in either H. halobium or H. volcanii. The same replication origin was also recovered after transformation of H. volcanii with a library of partial Sau3AI fragments of pNRC100. The nucleotide sequence of the minimal replication origin was determined and found to contain a long open reading frame, named repH, transcribed away from a highly A+T-rich region. The transcription start site was identified by primer extension analysis to be 17 to 18 nucleotides 5' to a putative repH start codon. The predicted product of the repH gene, an acidic protein with a molecular weight of 113,442, showed 24 to 27% identity with predicted gene products of H. volcanii plasmid pHV2 and H. halobium plasmid p phi HL, suggesting that each is involved in plasmid replication. One pNRC100 minireplicon, pNG11 delta 12, was analyzed by linker scanning mutagenesis, which showed the requirement of repH for replication. Restoration of the repH reading frame of one replication-defective pNG11 delta 12 derivative by introduction of a second small insertion resulted in reversion to replication proficiency. The replication ability of pNG11delta12 was lost when the entire A+T-rich region, about 550 bp long, was deleted but not when small insertions or deletions were introduced into this region. The presence of only 52 bp of the A+T-rich segment was sufficient to permit replication. The pNG11delta12 minireplicon was lost at high frequency from cells grown without mevinolin selection, suggesting that the plasmid partitioning locus of pNRC100 is absent in the minimal replication origin region. We discuss the possible roles of the repH gene and the A+T-rich region in replication of pNRC100.

Amino Acid Sequence↗
PubMed1991

Isolation and characterization of a dideoxyguanosine triphosphate-resistant mutant of human immunodeficiency virus reverse transcriptase.

The appearance of drug-resistant strains of viral pathogens is a major difficulty confounding current efforts to block viral infections. The identification and analysis of mutations responsible for drug resistance can provide important clues helpful in understanding the mechanisms of resistance and in the eventual development of better therapies. We have used a direct screening method to scan libraries of mutagenized genes encoding the reverse transcriptase of human immunodeficiency virus type 1, and have recovered a variant enzyme that is resistant to the chain-terminator inhibitor 2',3'-dideoxyguanosine triphosphate. The single substitution mutation in this variant conferred broad crossresistance to a variety of other antiviral compounds currently in clinical trials. Virus carrying the mutation was fully infectious in cultured human lymphocytes. The replication of the mutant virus was highly resistant to phosphonoformic acid but did not show increased resistance to the prodrug dideoxyguanosine.

Antiviral Agents↗
PubMed1991

Characterization of a novel liver-specific enhancer in the human prothrombin gene.

The 5'-flanking sequence of the human prothrombin gene was isolated by screening a human liver phage library with a human prothrombin cDNA as a hybridization probe. A phage was identified that contained 3 kilobase pairs of DNA upstream of the initiator methionine codon. Primer extension studies showed that the major transcription initiation sites were located 23 and 36 base pairs upstream of the initiator codon. DNA sequences in the 5'-flanking region of the human prothrombin gene were then analyzed for cis-activating transcriptional activity by a transient expression system using the human growth hormone gene as the reporter gene. The chimeric expression vector was introduced into HepG2 cells, and secreted human growth hormone was monitored by using a radio-immunoassay. These studies showed that the 3-kilo-base pair fragment contained sequences that were sufficient for the initiation of transcription in HepG2 cells. Subsequent deletion studies showed that the 3-kilobase pair fragment contained two elements: a weak promoter in the region immediately upstream of the mRNA coding sequence and an enhancer located between nucleotides -860 and -940. The enhancer element was active at a distance and in either orientation. In addition, the enhancer was liver cell-specific and acted on heterologous promoters including the herpes simplex virus thymidine kinase promoter and the mouse metallothionein I promoter. Comparison of the nucleotide sequence of the enhancer with a DNA sequence data base showed the enhancer sequence to be unique. The enhancer sequence is flanked by an inverted repeat 5' CCTCCC 3' and contains a putative binding site for hepatic nuclear factor 1. Deoxyribonuclease I footprint analysis and linker scanning mutagenesis showed that the enhancer contains multiple protein binding motifs. Mutagenesis of the 3' boundary CCTCCC sequence eliminated the enhancer activity. Comparison with other liver genes showed the presence of the CCTCCC sequence in the hepatitis B virus enhancer, the alpha 1-antitrypsin promoter, and the fibrinogen beta-chain promoter, suggesting a functional role for this motif.

Base Composition↗