scholarly journals Protein-Protein Interactions in the Complex between the Enhancer Binding Protein NIFA and the Sensor NIFL fromAzotobacter vinelandii

2001 ◽  
Vol 183 (4) ◽  
pp. 1359-1368 ◽  
Author(s):  
Tracy Money ◽  
Jason Barrett ◽  
Ray Dixon ◽  
Sara Austin
Keyword(s):  
Complex Formation ◽  
Protein Interactions ◽  
Azotobacter Vinelandii ◽  
Protein Complexes ◽  
Binding Protein ◽  
Limited Proteolysis ◽  
Regulatory System ◽  
Terminal Domain ◽  
Enhancer Binding Protein

ABSTRACT The enhancer binding protein NIFA and the sensor protein NIFL fromAzotobacter vinelandii comprise an atypical two-component regulatory system in which signal transduction occurs via complex formation between the two proteins rather than by the phosphotransfer mechanism, which is characteristic of orthodox systems. The inhibitory activity of NIFL towards NIFA is stimulated by ADP binding to the C-terminal domain of NIFL, which bears significant homology to the histidine protein kinase transmitter domains. Adenosine nucleotides, particularly MgADP, also stimulate complex formation between NIFL and NIFA in vitro, allowing isolation of the complex by cochromatography. Using limited proteolysis of the purified proteins, we show here that changes in protease sensitivity of the Q linker regions of both NIFA and NIFL occurred when the complex was formed in the presence of MgADP. The N-terminal domain of NIFA adjacent to the Q linker was also protected by NIFL. Experiments with truncated versions of NIFA demonstrate that the central domain of NIFA is sufficient to cause protection of the Q linker of NIFL, although in this case, stable protein complexes are not detectable by cochromatography.

scholarly journals Isolation and Properties of the Complex between the Enhancer Binding Protein NIFA and the Sensor NIFL

1999 ◽  
Vol 181 (15) ◽  
pp. 4461-4468 ◽  
Author(s):  
Tracy Money ◽  
Tamera Jones ◽  
Ray Dixon ◽  
Sara Austin
Keyword(s):  
Complex Formation ◽  
Azotobacter Vinelandii ◽  
Binding Protein ◽  
Protein Protein Interaction ◽  
Amino Terminal ◽  
Cell Extracts ◽  
Enhancer Binding Protein ◽  
Adenosine Nucleotides

ABSTRACT In Azotobacter vinelandii, activation ofnif gene expression by the transcriptional regulatory enhancer binding protein NIFA is controlled by the sensor protein NIFL in response to changes in levels of oxygen and fixed nitrogen in vivo. NIFL is a novel redox-sensing flavoprotein which is also responsive to adenosine nucleotides in vitro. Inhibition of NIFA activity by NIFL requires stoichiometric amounts of the two proteins, implying that the mechanism of inhibition is by direct protein-protein interaction rather than by catalytic modification of the NIFA protein. The formation of the inhibitory complex between NIFL and NIFA may be regulated by the intracellular ATP/ADP ratio. We show that adenosine nucleotides promote complex formation between purified NIFA and NIFL in vitro, allowing isolation of the NIFL-NIFA complex. The complex can also be isolated from cell extracts containing coexpressed NIFL and NIFA in the presence of MgADP. Removal of the nucleotide causes dissociation of the complex. Experiments with truncated proteins demonstrate that the amino-terminal domain of NIFA and the C-terminal region of NIFL potentiate the ADP-dependent stimulation of NIFL-NIFA complex formation.

scholarly journals Insight into the Dual Functions of Bacterial Enhancer-Binding Protein Rrp2 of Borrelia burgdorferi

2016 ◽  
Vol 198 (10) ◽  
pp. 1543-1552 ◽  
Author(s):  
Yanping Yin ◽  
Youyun Yang ◽  
Xuwu Xiang ◽  
Qian Wang ◽  
Zhang-Nv Yang ◽  
...  
Keyword(s):  
Dna Binding ◽  
Borrelia Burgdorferi ◽  
Binding Protein ◽  
Phosphorylation Site ◽  
Binding Motif ◽  
Cell Replication ◽  
Additional Function ◽  
Content Type ◽  
Terminal Domain ◽  
Enhancer Binding Protein

ABSTRACTIt is well established that the RpoN-RpoS sigma factor (σ54-σS) cascade plays an essential role in differential gene expression during the enzootic cycle ofBorrelia burgdorferi, the causative agent of Lyme disease. The RpoN-RpoS pathway is activated by the response regulator/σ54-dependent activator (also called bacterial enhancer-binding protein [bEBP]) Rrp2. One unique feature of Rrp2 is that this activator is essential for cell replication, whereas RpoN-RpoS is dispensable for bacterial growth. How Rrp2 controls cell replication, a function that is independent of RpoN-RpoS, remains to be elucidated. In this study, by generating a series of conditionalrrp2mutant strains, we demonstrated that the N-terminal receiver domain of Rrp2 is required for spirochetal growth. Furthermore, a D52A point mutation at the phosphorylation site within the N terminus of Rrp2 abolished cell replication. Mutation of the ATPase motif within the central domain of Rrp2 did not affect spirochetal replication, indicating that phosphorylation-dependent ATPase activity of Rrp2 for σ54activation is not required for cell growth. However, deletion of the C-terminal domain or a 16-amino-acid truncation of the helix-turn-helix (HTH) DNA-binding motif within the C-terminal domain of Rrp2 abolished spirochetal replication. It was shown that constitutive expression ofrpoSis deleterious to borrelial growth. We showed that the essential nature of Rrp2 is not due to an effect onrpoS. These data suggest that phosphorylation-dependent oligomerization and DNA binding of Rrp2 likely function as a repressor, independently of the activation of σ54, controlling an essential step of cell replication inB. burgdorferi.IMPORTANCEBacterial enhancer-binding proteins (bEBPs) are a unique group of transcriptional activators specifically required for σ54-dependent gene transcription. This work demonstrates that theB. burgdorferibEBP, Rrp2, has an additional function that is independent of σ54, that of its essentiality for spirochetal growth, and such a function is dependent on its N-terminal signal domain and C-terminal DNA-binding domain. These findings expand our knowledge on bEBP and provide a foundation to further study the underlying mechanism of this new function of bEBP.

c-Fos-induced activation of a TATA-box-containing promoter involves direct contact with TATA-box-binding protein

1994 ◽  
Vol 14 (9) ◽  
pp. 6021-6029
Author(s):  
R Metz ◽  
A J Bannister ◽  
J A Sutherland ◽  
C Hagemeier ◽  
E C O'Rourke ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Transcriptional Activation ◽  
Binding Protein ◽  
Tata Box ◽  
Binding Motif ◽  
Protein Protein Interactions ◽  
High Concentrations ◽  
Tata Box Binding Protein

Transcriptional activation in eukaryotes involves protein-protein interactions between regulatory transcription factors and components of the basal transcription machinery. Here we show that c-Fos, but not a related protein, Fra-1, can bind the TATA-box-binding protein (TBP) both in vitro and in vivo and that c-Fos can also interact with the transcription factor IID complex. High-affinity binding to TBP requires c-Fos activation modules which cooperate to activate transcription. One of these activation modules contains a TBP-binding motif (TBM) which was identified through its homology to TBP-binding viral activators. This motif is required for transcriptional activation, as well as TBP binding. Domain swap experiments indicate that a domain containing the TBM can confer TBP binding on Fra-1 both in vitro and in vivo. In vivo activation experiments indicate that a GAL4-Fos fusion can activate a promoter bearing a GAL4 site linked to a TATA box but that this activity does not occur at high concentrations of GAL4-Fos. This inhibition (squelching) of c-Fos activity is relieved by the presence of excess TBP, indicating that TBP is a direct functional target of c-Fos. Removing the TBM from c-Fos severely abrogates activation of a promoter containing a TATA box but does not affect activation of a promoter driven only by an initiator element. Collectively, these results suggest that c-Fos is able to activate via two distinct mechanisms, only one of which requires contact with TBP. Since TBP binding is not exhibited by Fra-1, TBP-mediated activation may be one characteristic that discriminates the function of Fos-related proteins.

scholarly journals Interfacial Peptides as Affinity Modulating Agents of Protein-Protein Interactions

Biomolecules ◽  
2022 ◽  
Vol 12 (1) ◽  
pp. 106
Author(s):  
Pavel V. Ershov ◽  
Yuri V. Mezentsev ◽  
Alexis S. Ivanov
Keyword(s):  
Protein Interactions ◽  
Targeted Delivery ◽  
Protein Complexes ◽  
Protein Protein Interactions ◽  
Good Efficiency ◽  
Cellular Targets ◽  
Proteolytic Stability ◽  
Clinically Significant

The identification of disease-related protein-protein interactions (PPIs) creates objective conditions for their pharmacological modulation. The contact area (interfaces) of the vast majority of PPIs has some features, such as geometrical and biochemical complementarities, “hot spots”, as well as an extremely low mutation rate that give us key knowledge to influence these PPIs. Exogenous regulation of PPIs is aimed at both inhibiting the assembly and/or destabilization of protein complexes. Often, the design of such modulators is associated with some specific problems in targeted delivery, cell penetration and proteolytic stability, as well as selective binding to cellular targets. Recent progress in interfacial peptide design has been achieved in solving all these difficulties and has provided a good efficiency in preclinical models (in vitro and in vivo). The most promising peptide-containing therapeutic formulations are under investigation in clinical trials. In this review, we update the current state-of-the-art in the field of interfacial peptides as potent modulators of a number of disease-related PPIs. Over the past years, the scientific interest has been focused on following clinically significant heterodimeric PPIs MDM2/p53, PD-1/PD-L1, HIF/HIF, NRF2/KEAP1, RbAp48/MTA1, HSP90/CDC37, BIRC5/CRM1, BIRC5/XIAP, YAP/TAZ–TEAD, TWEAK/FN14, Bcl-2/Bax, YY1/AKT, CD40/CD40L and MINT2/APP.

Purification of complexes of nuclear oncogene p53 with rat and Escherichia coli heat shock proteins: in vitro dissociation of hsc70 and dnaK from murine p53 by ATP

1988 ◽  
Vol 8 (3) ◽  
pp. 1206-1215
Author(s):  
C F Clarke ◽  
K Cheng ◽  
A B Frey ◽  
R Stein ◽  
P W Hinds ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Protein Interactions ◽  
Protein Complexes ◽  
P53 Protein ◽  
Immunoaffinity Column ◽  
E Coli ◽  
Shock Proteins

Oligomeric protein complexes containing the nuclear oncogene p53 and the simian virus 40 large tumor antigen (D. I. H. Linzer and A. J. Levine, Cell 17:43-51, 1979), the adenovirus E1B 55-kilodalton (kDa) tumor antigen, and the heat shock protein hsc70 (P. Hinds, C. Finlay, A. Frey, and A. J. Levine, Mol. Cell. Biol. 7:2863-2869, 1987) have all been previously described. To begin isolating, purifying, and testing these complexes for functional activities, we have developed a rapid immunoaffinity column purification. p53-protein complexes are eluted from the immunoaffinity column by using a molar excess of a peptide comprising the epitope recognized by the p53 monoclonal antibody. This mild and specific elution condition allows p53-protein interactions to be maintained. The hsc70-p53 complex from rat cells is heterogeneous in size, with some forms of this complex associated with a 110-kDa protein. The maximum apparent molecular mass of such complexes is 660,000 daltons. Incubation with micromolar levels of ATP dissociates this complex in vitro into p53 and hsc70 110-kDa components. Nonhydrolyzable substrates of ATP fail to promote this dissociation of the complex. Murine p53 synthesized in Escherichia coli has been purified 660-fold on the same antibody affinity column and was found to be associated with an E. coli protein of 70 kDa. Immunoblot analysis with specific antisera demonstrated that this E. coli protein was the heat shock protein dnaK, which has extensive sequence homology with the rat hsc70 protein. Incubation of the immunopurified p53-dnaK complex with ATP resulted in the dissociation of the p53-dnaK complex as it did with the p53-hsc70 complex. This remarkable conservation of p53-heat shock protein interactions and the specificity of dissociation reactions suggest a functionally important role for heat shock proteins in their interactions with oncogene proteins.

scholarly journals A single domain of yeast poly(A)-binding protein is necessary and sufficient for RNA binding and cell viability.

1987 ◽  
Vol 7 (9) ◽  
pp. 3268-3276 ◽  
Author(s):  
A B Sachs ◽  
R W Davis ◽  
R D Kornberg
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Association Constant ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Binding Protein ◽  
High Affinity ◽  
Terminal Domain ◽  
Necessary And Sufficient

The poly(A)-binding protein (PAB) gene of Saccharomyces cerevisiae is essential for cell growth. A 66-amino acid polypeptide containing half of a repeated N-terminal domain can replace the entire protein in vivo. Neither an octapeptide sequence conserved among eucaryotic RNA-binding proteins nor the C-terminal domain of PAB is required for function in vivo. A single N-terminal domain is nearly identical to the entire protein in the number of high-affinity sites for poly(A) binding in vitro (one site with an association constant of approximately 2 X 10(7) M-1) and in the size of the binding site (12 A residues). Multiple N-terminal domains afford a mechanism of PAB transfer between poly(A) strands.

scholarly journals In vitro evolution of an HIV integrase binding protein from a library of C-terminal domain γS-crystallin variants

2012 ◽  
Vol 22 (17) ◽  
pp. 5584-5589 ◽  
Author(s):  
Issa S. Moody ◽  
Shawn C. Verde ◽  
Cathie M. Overstreet ◽  
W. Edward Robinson ◽  
Gregory A. Weiss
Keyword(s):  
Binding Protein ◽  
Hiv Integrase ◽  
In Vitro Evolution ◽  
Terminal Domain

scholarly journals Integrated analysis of RNA-binding protein complexes using in vitro selection and high-throughput sequencing and sequence specificity landscapes (SEQRS)

Methods ◽  
2017 ◽  
Vol 118-119 ◽  
pp. 171-181 ◽  
Author(s):  
Tzu-Fang Lou ◽  
Chase A. Weidmann ◽  
Jordan Killingsworth ◽  
Traci M. Tanaka Hall ◽  
Aaron C. Goldstrohm ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Sequencing ◽  
In Vitro Selection ◽  
Rna Binding ◽  
Protein Complexes ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Integrated Analysis ◽  
Sequence Specificity

scholarly journals Evolutionary diversification of protein–protein interactions by interface add-ons

2017 ◽  
Vol 114 (40) ◽  
pp. E8333-E8342 ◽  
Author(s):  
Maximilian G. Plach ◽  
Florian Semmelmann ◽  
Florian Busch ◽  
Markus Busch ◽  
Leonhard Heizinger ◽  
...  
Keyword(s):  
Protein Interaction ◽  
Protein Interactions ◽  
Protein Complexes ◽  
Evolutionary Strategy ◽  
Structural Level ◽  
Protein Protein Interactions ◽  
Protein Protein Interaction ◽  
Evolutionary Diversification

Cells contain a multitude of protein complexes whose subunits interact with high specificity. However, the number of different protein folds and interface geometries found in nature is limited. This raises the question of how protein–protein interaction specificity is achieved on the structural level and how the formation of nonphysiological complexes is avoided. Here, we describe structural elements called interface add-ons that fulfill this function and elucidate their role for the diversification of protein–protein interactions during evolution. We identified interface add-ons in 10% of a representative set of bacterial, heteromeric protein complexes. The importance of interface add-ons for protein–protein interaction specificity is demonstrated by an exemplary experimental characterization of over 30 cognate and hybrid glutamine amidotransferase complexes in combination with comprehensive genetic profiling and protein design. Moreover, growth experiments showed that the lack of interface add-ons can lead to physiologically harmful cross-talk between essential biosynthetic pathways. In sum, our complementary in silico, in vitro, and in vivo analysis argues that interface add-ons are a practical and widespread evolutionary strategy to prevent the formation of nonphysiological complexes by specializing protein–protein interactions.

scholarly journals Interaction of the Antiactivator FleN with the Transcriptional Activator FleQ Regulates Flagellar Number inPseudomonas aeruginosa

2001 ◽  
Vol 183 (22) ◽  
pp. 6636-6644 ◽  
Author(s):  
Nandini Dasgupta ◽  
Reuben Ramphal
Keyword(s):  
Binding Protein ◽  
Direct Interaction ◽  
Transcriptional Activator ◽  
Binding Motif ◽  
Yeast Two Hybrid System ◽  
Gtp Binding ◽  
C Terminus ◽  
Enhancer Binding Protein ◽  
Two Hybrid

ABSTRACT Flagellar number in Pseudomonas aeruginosa is controlled by FleN, a putative ATP/GTP binding protein. Disruption offleN results in multiflagellation of the otherwise monoflagellate strains PAK and PAO1 and is associated with a chemotactic defect. We propose that flagellar number is maintained by the antiactivator FleN, which downregulates flagellar genes by binding to their transcriptional activator, FleQ, an enhancer binding protein belonging to the NifA subfamily. In this report we demonstrate direct interaction of FleN and FleQ in the yeast two-hybrid system. Mutagenesis of the putative ATP/GTP binding motif in FleN24K→Q and truncation of FleN at either the N or C terminus abrogates this interaction. FleN does not inhibit the DNA binding ability of FleQ in vitro, thus indicating that it probably utilizes another mechanism(s) to serve as a FleQ antiactivator.

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