Characterization of Dominantly Negative Mutant ClyA Cytotoxin Proteins in Escherichia coli

ABSTRACT We report studies of the subcellular localization of the ClyA cytotoxic protein and of mutations causing defective translocation to the periplasm in Escherichia coli. The ability of ClyA to translocate to the periplasm was abolished in deletion mutants lacking the last 23 or 11 amino acid residues of the C-terminal region. A naturally occurring ClyA variant lacking four residues (183 to 186) in a hydrophobic subdomain was retained mainly in the cytosolic fraction. These mutant proteins displayed an inhibiting effect on the expression of the hemolytic phenotype of wild-type ClyA. Studies in vitro with purified mutant ClyA proteins revealed that they were defective in formation of pore assemblies and that their activity in hemolysis assays and in single-channel conductance tests was at least 10-fold lower than that of the wild-type ClyA. Tests with combinations of the purified proteins indicated that mutant and wild-type ClyA interacted and that formation of heteromeric assemblies affected the pore-forming activity of the wild-type protein. The observed protein-protein interactions were consistent with, and provided a molecular explanation for, the dominant negative feature of the mutant ClyA variants.

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Analysis of ftsQ Mutant Alleles in Escherichia coli: Complementation, Septal Localization, and Recruitment of Downstream Cell Division Proteins

Journal of Bacteriology ◽

10.1128/jb.184.3.695-705.2002 ◽

2002 ◽

Vol 184 (3) ◽

pp. 695-705 ◽

Cited By ~ 34

Author(s):

Joseph C. Chen ◽

Michael Minev ◽

Jon Beckwith

Keyword(s):

Escherichia Coli ◽

Cell Division ◽

Random Mutagenesis ◽

Dominant Negative ◽

Restrictive Temperature ◽

Permissive Temperature ◽

Wild Type ◽

Negative Effects ◽

Mutant Proteins ◽

Division Site

ABSTRACT FtsQ, a 276-amino-acid, bitopic membrane protein, is one of the nine proteins known to be essential for cell division in gram-negative bacterium Escherichia coli. To define residues in FtsQ critical for function, we performed random mutagenesis on the ftsQ gene and identified four alleles (ftsQ2, ftsQ6, ftsQ15, and ftsQ65) that fail to complement the ftsQ1(Ts) mutation at the restrictive temperature. Two of the mutant proteins, FtsQ6 and FtsQ15, are functional at lower temperatures but are unable to localize to the division site unless wild-type FtsQ is depleted, suggesting that they compete poorly with the wild-type protein for septal targeting. The other two mutants, FtsQ2 and FtsQ65, are nonfunctional at all temperatures tested and have dominant-negative effects when expressed in an ftsQ1(Ts) strain at the permissive temperature. FtsQ2 and FtsQ65 localize to the division site in the presence or absence of endogenous FtsQ, but they cannot recruit downstream cell division proteins, such as FtsL, to the septum. These results suggest that FtsQ2 and FtsQ65 compete efficiently for septal targeting but fail to promote the further assembly of the cell division machinery. Thus, we have separated the localization ability of FtsQ from its other functions, including recruitment of downstream cell division proteins, and are beginning to define regions of the protein responsible for these distinct capabilities.

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Activities and regulation of peptidoglycan synthases

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2015.0031 ◽

2015 ◽

Vol 370 (1679) ◽

pp. 20150031 ◽

Cited By ~ 92

Author(s):

Alexander J. F. Egan ◽

Jacob Biboy ◽

Inge van't Veer ◽

Eefjan Breukink ◽

Waldemar Vollmer

Keyword(s):

Escherichia Coli ◽

Protein Interactions ◽

Cytoplasmic Membrane ◽

Current Knowledge ◽

Protein Protein Interactions ◽

Penicillin Binding Proteins ◽

Multiple Protein ◽

Cell Division Protein ◽

The Impact

Peptidoglycan (PG) is an essential component in the cell wall of nearly all bacteria, forming a continuous, mesh-like structure, called the sacculus, around the cytoplasmic membrane to protect the cell from bursting by its turgor. Although PG synthases, the penicillin-binding proteins (PBPs), have been studied for 70 years, useful in vitro assays for measuring their activities were established only recently, and these provided the first insights into the regulation of these enzymes. Here, we review the current knowledge on the glycosyltransferase and transpeptidase activities of PG synthases. We provide new data showing that the bifunctional PBP1A and PBP1B from Escherichia coli are active upon reconstitution into the membrane environment of proteoliposomes, and that these enzymes also exhibit DD-carboxypeptidase activity in certain conditions. Both novel features are relevant for their functioning within the cell. We also review recent data on the impact of protein–protein interactions and other factors on the activities of PBPs. As an example, we demonstrate a synergistic effect of multiple protein–protein interactions on the glycosyltransferase activity of PBP1B, by its cognate lipoprotein activator LpoB and the essential cell division protein FtsN.

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Cross-subunit catalysis and a new phenomenon of recessive resurrection in Escherichia coli RNase E

Nucleic Acids Research ◽

10.1093/nar/gkz1152 ◽

2019 ◽

Vol 48 (2) ◽

pp. 847-861 ◽

Cited By ~ 2

Author(s):

Nida Ali ◽

Jayaraman Gowrishankar

Keyword(s):

Escherichia Coli ◽

Active Site ◽

Rna Binding ◽

Initial Step ◽

Dominant Negative ◽

Rnase E ◽

Wild Type ◽

Catalytic Active Site

Abstract RNase E is a 472-kDa homo-tetrameric essential endoribonuclease involved in RNA processing and turnover in Escherichia coli. In its N-terminal half (NTH) is the catalytic active site, as also a substrate 5′-sensor pocket that renders enzyme activity maximal on 5′-monophosphorylated RNAs. The protein's non-catalytic C-terminal half (CTH) harbours RNA-binding motifs and serves as scaffold for a multiprotein degradosome complex, but is dispensable for viability. Here, we provide evidence that a full-length hetero-tetramer, composed of a mixture of wild-type and (recessive lethal) active-site mutant subunits, exhibits identical activity in vivo as the wild-type homo-tetramer itself (‘recessive resurrection’). When all of the cognate polypeptides lacked the CTH, the active-site mutant subunits were dominant negative. A pair of C-terminally truncated polypeptides, which were individually inactive because of additional mutations in their active site and 5′-sensor pocket respectively, exhibited catalytic function in combination, both in vivo and in vitro (i.e. intragenic or allelic complementation). Our results indicate that adjacent subunits within an oligomer are separately responsible for 5′-sensing and cleavage, and that RNA binding facilitates oligomerization. We propose also that the CTH mediates a rate-determining initial step for enzyme function, which is likely the binding and channelling of substrate for NTH’s endonucleolytic action.

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Subunit Oligomerization and Substrate Recognition of the Escherichia coli ClpYQ (HslUV) Protease Implicated by In Vivo Protein-Protein Interactions in the Yeast Two-Hybrid System

Journal of Bacteriology ◽

10.1128/jb.185.8.2393-2401.2003 ◽

2003 ◽

Vol 185 (8) ◽

pp. 2393-2401 ◽

Cited By ~ 11

Author(s):

Yi-Ying Lee ◽

Chiung-Fang Chang ◽

Chueh-Ling Kuo ◽

Meng-Ching Chen ◽

Chien Hung Yu ◽

...

Keyword(s):

Escherichia Coli ◽

Hybrid System ◽

Protein Interactions ◽

Large Subunit ◽

Protein Protein Interactions ◽

Yeast Two Hybrid ◽

Yeast Two Hybrid System ◽

Two Hybrid

ABSTRACT The Escherichia coli ClpYQ (HslUV) is an ATP-dependent protease that consists of an ATPase large subunit with homology to other Clp family ATPases and a peptidase small subunit related to the proteasomal β-subunits of eukaryotes. Six identical subunits of both ClpY and ClpQ self-assemble into an oligomeric ring, and two rings of each subunit, two ClpQ rings surrounded by single ClpY rings, form a dumbbell shape complex. The ClpYQ protease degrades the cell division inhibitor, SulA, and a positive regulator of capsule transcription, RcsA, as well as RpoH, a heat shock sigma transcription factor. Using the yeast-two hybrid system, we explored the in vivo protein-protein interactions of the individual subunits of the ClpYQ protease involved in self-oligomerization, as well as in recognition of specific substrates. Interactions were detected with ClpQ/ClpQ, ClpQ/ClpY, and ClpY/SulA. No interactions were observed in experiments with ClpY/ClpY, ClpQ/RcsA, and ClpQ/SulA. However, ClpY, lacking domain I (ClpYΔI) was able to interact with itself and with intact ClpY. The C-terminal region of ClpY is important for interaction with other ClpY subunits. The previously defined PDZ-like domains at the C terminus of ClpY, including both D1 and D2, were determined to be indispensable for substrate binding. Various deletion and random point mutants of SulA were also made to verify significant interactions with ClpY. Thus, we demonstrated in vivo hetero- and homointeractions of ClpQ and ClpY molecules, as well as a direct association between ClpY and substrate SulA, thereby supporting previous in vitro biochemical findings.

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Mutation Effects on Remote Epitopes of An Autoantigen Decoded with Simulated B-Factors

10.1101/559963 ◽

2019 ◽

Author(s):

Yuan-Ping Pang ◽

Marta Casal Moura ◽

Gwen E. Thompson ◽

Amber M. Hummel ◽

Darlene R. Nelson ◽

...

Keyword(s):

Autoimmune Diseases ◽

Protein Interactions ◽

Granulomatosis With Polyangiitis ◽

Wild Type ◽

Protein Protein Interactions ◽

Triple Mutant ◽

Experimental Conditions ◽

Chain Flexibility

Proteinase 3 (PR3) is a neutrophil serine protease targeted by anti-neutrophil cytoplasmic antibodies (ANCAs) in the autoimmune disease granulomatosis with polyangiitis (GPA)1–5. PR3 mutants were developed to investigate how PR3 interacts with the ANCAs and whether the interactions can be intervened by therapeutics. One mutant with a Ser195Ala mutation (iPR3-Val103) recognized as many ANCAs as wild-type PR3 (PR3-Val103)6–9, indicating that PR3-Val103 and iPR3-Val103 have equivalent ANCA-binding capabilities. A triple mutant of the latter (iHm5-Val103) bound a monoclonal antibody (moANCA518) from a patient with GPA on an epitope remote from the mutation sites. Unexpectedly, the corresponding epitope of iPR3-Val103 was inaccessible to moANCA518 under the same experimental conditions10,11. These observations demonstrate that a latent epitope of PR3 can be activated surprisingly by remote mutations in PR311. Here we report a comparative analysis of simulated B-factors (i.e., measurements of local mobility) of PR3-Val103, iPR3-Val103, and iHm5-Val103, demonstrating that the binding of moANCA518 to iHm5-Val103 is enabled by an increase in main-chain flexibility in the latent epitope caused by the remote muta-tions in iHm5-Val103. This epitope activation—achieved in vitro by remote mutations as we demonstrated or in vivo conceivably by remote protein»protein interactions12 or remote po-lymorphisms—may be a fundamental feature of antibody-mediated autoimmune diseases. Rigidifying B-cell epitopes on an autoantigen with therapeutics designed using the B-factor analysis disclosed here may lead to effective treatments for these autoimmune diseases by making the autoantigen inaccessible to existing autoantibodies. This analysis may also be used to predict and characterize epitopes and remote mutation effects.

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

Molecular and Cellular Biology ◽

10.1128/mcb.13.12.7874 ◽

1993 ◽

Vol 13 (12) ◽

pp. 7874-7880 ◽

Cited By ~ 61

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.

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

Infection and Immunity ◽

10.1128/iai.74.4.2093-2101.2006 ◽

2006 ◽

Vol 74 (4) ◽

pp. 2093-2101 ◽

Cited By ~ 9

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.

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Identification of amino acid residues required for Ras p21 target activation.

Molecular and Cellular Biology ◽

10.1128/mcb.11.8.3997 ◽

1991 ◽

Vol 11 (8) ◽

pp. 3997-4004 ◽

Cited By ~ 41

Author(s):

M S Marshall ◽

L J Davis ◽

R D Keys ◽

S D Mosser ◽

W S Hill ◽

...

Keyword(s):

Biological Activity ◽

Amino Acid ◽

Protein Interactions ◽

Amino Acid Residues ◽

Ras Proteins ◽

Protein Protein Interactions ◽

Biological Assays ◽

Ras P21

The Krev-1 gene has been shown to suppress ras-mediated transformation in vitro. Both ras and Krev-1 proteins have identical effector domains (ras residues 32 to 40), which are required for biological activity and for the interaction of Ras p21 with Ras GTPase-activating protein (GAP). In this study, five amino acid residues flanking the ras effector domain, which are not conserved with the Krev-1 protein, were shown to be required for normal protein-protein interactions and biological activity. The substitution of Krev-1 p21 residues 26, 27, 30, 31, and 45 with the corresponding amino acid residues from Ras p21 resulted in a Krev-1 protein which had ras function in both mammalian and yeast biological assays. Replacement of these residues in Ras p21 with the corresponding Krev-1 p21 amino acids resulted in ras proteins which were impaired biologically or reduced in their affinity for in vitro GAP binding. Evaluation of these mutant ras proteins have implications for Ras p21-GAP interactions in vivo.

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Novel trans-Acting Bacillus subtilis glnA Mutations That Derepress glnRA Expression

Journal of Bacteriology ◽

10.1128/jb.01734-08 ◽

2009 ◽

Vol 191 (8) ◽

pp. 2485-2492 ◽

Cited By ~ 14

Author(s):

Susan H. Fisher ◽

Lewis V. Wray

Keyword(s):

Bacillus Subtilis ◽

Transcription Factors ◽

Dna Binding ◽

Glutamine Synthetase ◽

Protein Interactions ◽

Feedback Inhibition ◽

Wild Type ◽

Protein Protein Interactions ◽

Enzymatic Assays

ABSTRACT Bacillus subtilis contains two nitrogen transcription factors, GlnR and TnrA. The activities of GlnR and TnrA are regulated by direct protein-protein interactions with the feedback-inhibited form of glutamine synthetase (GS). To look for other factors involved in regulating GlnR activity, we isolated mutants with constitutive glnRA expression (GlnC). The twenty-seven GlnC mutants isolated in this mutant screen all contained mutations tightly linked to the glnRA operon which encodes GlnR (glnR) and GS (glnA). Four GlnC mutants contained mutations in the glnR gene that most likely impair the ability of GlnR to bind DNA. Three other GlnC mutants contained novel glnA mutations (S55F, V173I, and L174F). GlnR regulation was completely relieved in the three glnA mutants, while only modest defects in TnrA regulation were observed. In vitro enzymatic assays showed that the purified S55F mutant enzyme was catalytically defective while the V173I and L174F enzymes were highly resistant to feedback inhibition. The V173I and L174F GS proteins were found to require higher glutamine concentrations than the wild-type GS to regulate the DNA-binding activities of GlnR and TnrA in vitro. These results are consistent with a model where feedback-inhibited GS is the only cellular factor involved in regulating the activity of GlnR in B. subtilis.

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