scholarly journals Mapping functional regions of essential bacterial proteins with dominant-negative protein fragments

2022 ◽  
Author(s):  
Andrew Savinov ◽  
Andres Fernandez ◽  
Stanley Fields
Keyword(s):  
Protein Interactions ◽  
Inhibitory Activity ◽  
Dominant Negative ◽  
Single Amino Acid ◽  
Generic Properties ◽  
Inhibitory Protein ◽  
Protein Fragments ◽  
Interaction Sites ◽  
Trade Offs

Massively-parallel measurements of dominant negative inhibition by protein fragments have been used to map protein interaction sites and discover peptide inhibitors. However, the underlying principles governing fragment-based inhibition have thus far remained unclear. Here, we adapt a high-throughput inhibitory fragment assay for use in Escherichia coli, applying it to a set of ten essential proteins. This approach yielded single amino acid resolution maps of inhibitory activity, with peaks localized to functionally important interaction sites, including oligomerization interfaces and folding contacts. Leveraging these data, we perform a systematic analysis to uncover principles of fragment-based inhibition. We determine a robust negative correlation between susceptibility to inhibition and cellular protein concentration, demonstrating that inhibitory fragments likely act primarily by titrating native protein interactions. We also characterize a series of trade-offs related to fragment length, showing that shorter peptides allow higher-resolution mapping but suffer from lower inhibitory activity. We employ an unsupervised statistical analysis to show that the inhibitory activities of protein fragments are largely driven not by generic properties such as charge, hydrophobicity, and secondary structure, but by the more specific characteristics of their bespoke macromolecular interactions. AlphaFold computational modeling of peptide complexes with one protein shows that the inhibitory activity of peptides is associated with their predicted ability to form native-like interactions. Overall, this work demonstrates fundamental characteristics of inhibitory protein fragment function and provides a foundation for understanding and controlling protein interactions in vivo.

scholarly journals Gic2p May Link Activated Cdc42p to Components Involved in Actin Polarization, Including Bni1p and Bud6p (Aip3p)

2000 ◽  
Vol 20 (17) ◽  
pp. 6244-6258 ◽  
Author(s):  
Malika Jaquenoud ◽  
Matthias Peter
Keyword(s):  
Actin Cytoskeleton ◽  
Dominant Negative ◽  
Single Amino Acid ◽  
Polarized Growth ◽  
Hybrid Analysis ◽  
Actin Organization ◽  
Bud Emergence ◽  
Bud Site ◽  
Two Hybrid

ABSTRACT Gic2p is a Cdc42p effector which functions during cytoskeletal organization at bud emergence and in response to pheromones, but it is not understood how Gic2p interacts with the actin cytoskeleton. Here we show that Gic2p displayed multiple genetic interactions with Bni1p, Bud6p (Aip3p), and Spa2p, suggesting that Gic2p may regulate their function in vivo. In support of this idea, Gic2p cofractionated with Bud6p and Spa2p and interacted with Bud6p by coimmunoprecipitation and two-hybrid analysis. Importantly, localization of Bni1p and Bud6p to the incipient bud site was dependent on active Cdc42p and the Gic proteins but did not require an intact actin cytoskeleton. We identified a conserved domain in Gic2p which was necessary for its polarization function but dispensable for binding to Cdc42p-GTP and its localization to the site of polarization. Expression of a mutant Gic2p harboring a single-amino-acid substitution in this domain (Gic2pW23A) interfered with polarized growth in a dominant-negative manner and prevented recruitment of Bni1p and Bud6p to the incipient bud site. We propose that at bud emergence, Gic2p functions as an adaptor which may link activated Cdc42p to components involved in actin organization and polarized growth, including Bni1p, Spa2p, and Bud6p.

scholarly journals Positive and Negative Regulation of Poly(A) Nuclease

2004 ◽  
Vol 24 (12) ◽  
pp. 5521-5533 ◽  
Author(s):  
David A. Mangus ◽  
Matthew C. Evans ◽  
Nathan S. Agrin ◽  
Mandy Smith ◽  
Preetam Gongidi ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Protein Interactions ◽  
Binding Pocket ◽  
Negative Regulation ◽  
Single Amino Acid ◽  
Carboxy Terminus ◽  
Protein Protein Interactions ◽  
C Terminus

ABSTRACT PAN, a yeast poly(A) nuclease, plays an important nuclear role in the posttranscriptional maturation of mRNA poly(A) tails. The activity of this enzyme is dependent on its Pan2p and Pan3p subunits, as well as the presence of poly(A)-binding protein (Pab1p). We have identified and characterized the associated network of factors controlling the maturation of mRNA poly(A) tails in yeast and defined its relevant protein-protein interactions. Pan3p, a positive regulator of PAN activity, interacts with Pab1p, thus providing substrate specificity for this nuclease. Pab1p also regulates poly(A) tail trimming by interacting with Pbp1p, a factor that appears to negatively regulate PAN. Pan3p and Pbp1p both interact with themselves and with the C terminus of Pab1p. However, the domains required for Pan3p and Pbp1p binding on Pab1p are distinct. Single amino acid changes that disrupt Pan3p interaction with Pab1p have been identified and define a binding pocket in helices 2 and 3 of Pab1p's carboxy terminus. The importance of these amino acids for Pab1p-Pan3p interaction, and poly(A) tail regulation, is underscored by experiments demonstrating that strains harboring substitutions in these residues accumulate mRNAs with long poly(A) tails in vivo.

scholarly journals Interactions between Hepatitis Delta Virus Proteins

2000 ◽  
Vol 74 (12) ◽  
pp. 5509-5515 ◽  
Author(s):  
Gloria Moraleda ◽  
Kate Dingle ◽  
Preetha Biswas ◽  
Jinhong Chang ◽  
Harmon Zuccola ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Hepatitis Delta Virus ◽  
Biological Activities ◽  
Dominant Negative ◽  
Genome Replication ◽  
Protein Protein Interactions ◽  
Particle Assembly ◽  
Hepatitis Delta ◽  
Delta Virus

ABSTRACT The 195- and 214-amino-acid (aa) forms of the delta protein (δAg-S and δAg-L, respectively) of hepatitis delta virus (HDV) differ only in the 19-aa C-terminal extension unique to δAg-L. δAg-S is needed for genome replication, while δAg-L is needed for particle assembly. These proteins share a region at aa 12 to 60, which mediates protein-protein interactions essential for HDV replication. H. Zuccola et al. (Structure 6:821–830, 1998) reported a crystal structure for a peptide spanning this region which demonstrates an antiparallel coiled-coil dimer interaction with the potential to form tetramers of dimers. Our studies tested whether predictions based on this structure could be extrapolated to conditions where the peptide was replaced by full-length δAg-S or δAg-L, and when the assays were not in vitro but in vivo. Nine amino acids that are conserved between several isolates of HDV and predicted to be important in multimerization were mutated to alanine on both δAg-S and δAg-L. We found that the predicted hierarchy of importance of these nine mutations correlated to a significant extent with the observed in vivo effects on the ability of these proteins to (i) support intrans the replication of the HDV genome when expressed on δAg-S and (ii) act as dominant-negative inhibitors of replication when expressed on δAg-L. We thus infer that these biological activities of δAg depend on ordered protein-protein interactions.

scholarly journals Unusual duplication mutation in a surface loop of human transthyretin leads to an aggressive drug-resistant amyloid disease

2018 ◽  
Vol 115 (28) ◽  
pp. E6428-E6436 ◽  
Author(s):  
Elena S. Klimtchuk ◽  
Tatiana Prokaeva ◽  
Nicholas M. Frame ◽  
Hassan A. Abdullahi ◽  
Brian Spencer ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Amyloid Fibrils ◽  
Tertiary Structure ◽  
Full Length ◽  
Single Amino Acid ◽  
Surface Loop ◽  
Amyloid Disease ◽  
Poor Treatment ◽  
The Stability

Transthyretin (TTR) is a globular tetrameric transport protein in plasma. Nearly 140 single amino acid substitutions in TTR cause life-threatening amyloid disease. We report a one-of-a-kind pathological variant featuring a Glu51, Ser52 duplication mutation (Glu51_Ser52dup). The proband, heterozygous for the mutation, exhibited an unusually aggressive amyloidosis that was refractory to treatment with the small-molecule drug diflunisal. To understand the poor treatment response and expand therapeutic options, we explored the structure and stability of recombinant Glu51_Ser52dup. The duplication did not alter the protein secondary or tertiary structure but decreased the stability of the TTR monomer and tetramer. Diflunisal, which bound with near-micromolar affinity, partially restored tetramer stability. The duplication had no significant effect on the free energy and enthalpy of diflunisal binding, and hence on the drug–protein interactions. However, the duplication induced tryptic digestion of TTR at near-physiological conditions, releasing a C-terminal fragment 49–129 that formed amyloid fibrils under conditions in which the full-length protein did not. Such C-terminal fragments, along with the full-length TTR, comprise amyloid deposits in vivo. Bioinformatics and structural analyses suggested that increased disorder in the surface loop, which contains the Glu51_Ser52dup duplication, not only helped generate amyloid-forming fragments but also decreased structural protection in the amyloidogenic residue segment 25–34, promoting misfolding of the full-length protein. Our studies of a unique duplication mutation explain its diflunisal-resistant nature, identify misfolding pathways for amyloidogenic TTR variants, and provide therapeutic targets to inhibit amyloid fibril formation by variant TTR.

scholarly journals Identification of RNA-associated peptides, iRAP, defines precise sites of protein-RNA interaction

2018 ◽  
Author(s):  
Lauri Peil ◽  
Sakharam Waghmare ◽  
Lutz Fischer ◽  
Michaela Spitzer ◽  
David Tollervey ◽  
...  
Keyword(s):  
Rna Binding ◽  
Protein Domain ◽  
Single Amino Acid ◽  
Sequence Features ◽  
Interaction Sites ◽  
Single Amino Acid Residue ◽  
Protein Domain Structure ◽  
Bona Fide ◽  
Rna Interaction

The identification of ever-increasing numbers of RNA species has underlined the importance of robust characterization of bona fide sites of protein-RNA interaction. UV crosslinking can be used to identify precise RNA targets for individual proteins, transcriptome-wide. Here we sought to generate reciprocal data, identifying precise sites of RNA-binding proteome-wide. The resulting technique, identification of RNA-associated peptides (iRAP), was used to locate 1331 unique RNA-interaction sites at single amino acid residue resolution in 324 S. cerevisiae proteins. Our identified RNA-interaction sites in characterized RNA-protein complex agree well with available high-resolution structures. In known RNA-interacting protiens, 317 sites fall into known and suspected RNA-interaction domains while only 21 sites fall into other annotated sequence features. Strikingly, 993 of the sites identified fall into protein regions that lack any recognizable protein domain structure or annotated sequence features. This suggests that, despite binding RNA in vivo, many of these proteins will not have defined functions in RNA metabolism.

scholarly journals Membrane-permeable C-terminal Dopamine Transporter Peptides Attenuate Amphetamine-evoked Dopamine Release

2013 ◽  
Vol 288 (38) ◽  
pp. 27534-27544 ◽  
Author(s):  
Mattias Rickhag ◽  
William A. Owens ◽  
Marie-Therese Winkler ◽  
Kristine Nørgaard Strandfelt ◽  
Mette Rathje ◽  
...  
Keyword(s):  
Dopamine Transporter ◽  
Protein Interactions ◽  
Dominant Negative ◽  
Tat Protein ◽  
Protein Protein Interactions ◽  
C Terminus ◽  
Discs Large ◽  
Dependent Protein ◽  
Binding Sequence

The dopamine transporter (DAT) is responsible for sequestration of extracellular dopamine (DA). The psychostimulant amphetamine (AMPH) is a DAT substrate, which is actively transported into the nerve terminal, eliciting vesicular depletion and reversal of DA transport via DAT. Here, we investigate the role of the DAT C terminus in AMPH-evoked DA efflux using cell-permeant dominant-negative peptides. A peptide, which corresponded to the last 24 C-terminal residues of DAT (TAT-C24 DAT) and thereby contained the Ca2+-calmodulin-dependent protein kinase IIα (CaMKIIα) binding domain and the PSD-95/Discs-large/ZO-1 (PDZ)-binding sequence of DAT, was made membrane-permeable by fusing it to the cell membrane transduction domain of the HIV-1 Tat protein (TAT-C24WT). The ability of TAT-C24WT but not a scrambled peptide (TAT-C24Scr) to block the CaMKIIα-DAT interaction was supported by co-immunoprecipitation experiments in heterologous cells. In heterologous cells, we also found that TAT-C24WT, but not TAT-C24Scr, decreased AMPH-evoked 1-methyl-4-phenylpyridinium efflux. Moreover, chronoamperometric recordings in striatum revealed diminished AMPH-evoked DA efflux in mice preinjected with TAT-C24WT. Both in heterologous cells and in striatum, the peptide did not further inhibit efflux upon KN-93-mediated inhibition of CaMKIIα activity, consistent with a dominant-negative action preventing binding of CaMKIIα to the DAT C terminus. This was further supported by the ability of a peptide with perturbed PDZ-binding sequence, but preserved CaMKIIα binding (TAT-C24AAA), to diminish AMPH-evoked DA efflux in vivo to the same extent as TAT-C24WT. Finally, AMPH-induced locomotor hyperactivity was attenuated following systemic administration of TAT-C24WT but not TAT-C24Scr. Summarized, our findings substantiate that DAT C-terminal protein-protein interactions are critical for AMPH-evoked DA efflux and suggest that it may be possible to target protein-protein interactions to modulate transporter function and interfere with psychostimulant effects.

scholarly journals The loop region of the helix-loop-helix protein Id1 is critical for its dominant negative activity.

1993 ◽  
Vol 13 (12) ◽  
pp. 7874-7880 ◽  
Author(s):  
S Pesce ◽  
R Benezra
Keyword(s):  
Amino Acids ◽  
Protein Interactions ◽  
Dominant Negative ◽  
Site Directed Mutagenesis ◽  
Protein Protein Interactions ◽  
Helix Loop Helix ◽  
Loop Region ◽  
Dominant Negative Activity

Id1, a helix-loop-helix (HLH) protein which lacks a DNA binding domain, has been shown to negatively regulate other members of the HLH family by direct protein-protein interactions, both in vitro and in vivo. In this study, we report the results of site-directed mutagenesis experiments aimed at defining the regions of Id1 which are important for its activity. We have found that the HLH domain of Id1 is necessary and nearly sufficient for its activity. In addition, we show that two amino acid residues at the amino terminus of the Id1 loop are critical for its activity, perhaps by specifying the correct dimerization partners. In this regard, replacing the first four amino acids of the loops of the basic HLH proteins E12 and E47 with the corresponding amino acids of Id1 confers Id1 dimerization specificity. These studies point to the loop region as an important structural and functional element of the Id subfamily of HLH proteins.

scholarly journals Insights into the molecular mechanisms of action of bioportides: a strategy to target protein-protein interactions

2015 ◽  
Vol 17 ◽  
Author(s):  
John Howl ◽  
Sarah Jones
Keyword(s):  
Protein Interactions ◽  
Molecular Mechanisms ◽  
Solid Phase ◽  
Cell Penetrating Peptides ◽  
Dominant Negative ◽  
Protein Protein Interactions ◽  
Structural Modifications ◽  
Anti Cancer ◽  
The Many

Cell-penetrating peptides (CPPs) are reliable vehicles for the target-selective intracellular delivery of therapeutic agents. The identification and application of numerous intrinsically bioactive CPPs, now designated as bioportides, is further endorsement of the tremendous clinical potential of CPP technologies. The refinement of proteomimetic bioportides, particularly sequences that mimic cationic α-helical domains involved in protein-protein interactions (PPIs), provides tremendous opportunities to modulate this emergent drug modality in a clinical setting. Thus, a number of CPP-based constructs are currently undergoing clinical trials as human therapeutics, with a particular focus upon anti-cancer agents. A well-characterised array of synthetic modifications, compatible with modern solid-phase synthesis, can be utilised to improve the biophysical and pharmacological properties of bioportides and so achieve cell-and tissue-selective targeting in vivo. Moreover, considering the recent successful development of stapled α-helical peptides as anti-cancer agents, we hypothesise that similar structural modifications are applicable to the design of bioportides that more effectively modulate the many interactomes known to underlie human diseases. Thus, we propose that stapled-helical bioportides could satisfy all of the clinical requirements for metabolically stable, intrinsically cell-permeable agents capable of regulating discrete PPIs by a dominant negative mode of action with minimal toxicity.

scholarly journals Mapping of a Domain Required for Protein-Protein Interactions and Inhibitory Activity of a Helicobacter pylori Dominant-Negative VacA Mutant Protein

2006 ◽  
Vol 74 (4) ◽  
pp. 2093-2101 ◽  
Author(s):  
Victor J. Torres ◽  
Mark S. McClain ◽  
Timothy L. Cover
Keyword(s):  
Amino Acids ◽  
Helicobacter Pylori ◽  
Protein Interactions ◽  
Inhibitory Activity ◽  
Mammalian Cells ◽  
Mutational Analysis ◽  
Dominant Negative ◽  
Mutant Protein ◽  
Wild Type ◽  
Protein Protein Interactions

ABSTRACT The Helicobacter pylori VacA toxin is an 88-kDa secreted protein that causes multiple alterations in mammalian cells and is considered an important virulence factor in the pathogenesis of peptic ulcer disease and gastric cancer. We have shown previously that a VacA mutant protein lacking amino acids 6 to 27 (Δ6-27p88 VacA) is able to inhibit many activities of wild-type VacA in a dominant-negative manner. Analysis of a panel of C-terminally truncated Δ6-27p88 VacA proteins indicated that a fragment containing amino acids 1 to 478 (Δ6-27p48) exhibited a dominant-negative phenotype similar to that of the full-length Δ6-27p88 VacA protein. In contrast, a shorter VacA fragment lacking amino acids 6 to 27 (Δ6-27p33) did not exhibit detectable inhibitory activity. The Δ6-27p48 protein physically interacted with wild-type p88 VacA, whereas the Δ6-27p33 protein did not. Mutational analysis indicated that amino acids 351 to 360 are required for VacA protein-protein interactions and for dominant-negative inhibitory activity. The C-terminal portion (p55 domain) of wild-type p88 VacA could complement either Δ6-27p33 or Δ(6-27/351-360)p48, reconstituting dominant-negative inhibitory activity. Collectively, our data provide strong evidence that the inhibitory properties of dominant-negative VacA mutant proteins are dependent on interactions between the mutant VacA proteins and wild-type VacA, and they allow mapping of a domain involved in the formation of oligomeric VacA complexes.

Insight into Structure-Function Relationships of β-Lactamase and BLIPs Interface Plasticity using Protein-Protein Interactions

2019 ◽  
Vol 25 (31) ◽  
pp. 3378-3389 ◽  
Author(s):  
Tara C. Yadav ◽  
Vidhu Agarwal ◽  
Amit K. Srivastava ◽  
Navdeep Raghuwanshi ◽  
Pritish Varadwaj ◽  
...  
Keyword(s):  
Amino Acid ◽  
Protein Interactions ◽  
Inhibitory Activity ◽  
Sequence Similarity ◽  
Classical Model ◽  
Sequencing Analysis ◽  
Beta Lactamase ◽  
Beta Sheet ◽  
Protein Protein Interactions ◽  
Inhibitory Protein

<P>Background: Mostly BLIPs are identified in soil bacteria Streptomyces and originally isolated from Streptomyces clavuligerus and can be utilized as a model system for biophysical, structural, mutagenic and computational studies. BLIP possess homology with two proteins viz., BLIP-I (Streptomyces exofoliatus) and BLP (beta-lactamase inhibitory protein like protein from S. clavuligerus). BLIP consists of 165 amino acid, possessing two homologues domains comprising helix-loop-helix motif packed against four stranded beta-sheet resulting into solvent exposed concave surface with extended four stranded beta-sheet. BLIP-I is a 157 amino acid long protein obtained from S. exofoliatus having 37% sequence identity to BLIP and inhibits beta-lactamase. </P><P> Methods: This review is intended to briefly illustrate the beta-lactamase inhibitory activity of BLIP via proteinprotein interaction and aims to open up a new avenue to combat antimicrobial resistance using peptide based inhibition. </P><P> Results: D49A mutation in BLIP-I results in a decrease in affinity for TEM-1 from 0.5 nM to 10 nM (Ki). It is capable of inhibiting TEM-1 and bactopenemase and differs from BLIP only in modulating cell wall synthesis enzyme. Whereas, BLP is a 154 amino acid long protein isolated from S. clavuligerus via DNA sequencing analysis of Cephamycin-Clavulanate gene bunch. It shares 32% sequence similarity with BLIP and 42% with BLIP-I. Its biological function is unclear and lacks beta-lactamase inhibitory activity. </P><P> Conclusion: Protein-protein interactions mediate a significant role in regulation and modulation of cellular developments and processes. Specific biological markers and geometric characteristics are manifested by active site binding clefts of protein surfaces which determines the specificity and affinity for their targets. TEM1.BLIP is a classical model to study protein-protein interaction. &#946;-Lactamase inhibitory proteins (BLIPs) interacts and inhibits various &#946;-lactamases with extensive range of affinities.</P>

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