Host stimuli and operator binding sites controlling protein interactions between virulence master regulator ToxR and ToxS in
            Vibrio cholerae

Background: Cholera, a diarrheal illness causes millions of deaths worldwide due to large outbreaks. Monoclonal antibody used as therapeutic purposes of cholera are prone to be unstable due to various factors including self-aggregation. Objectives: In this bioinformatic analysis, we identified the aggregation prone regions (APRs) of different immunogens of antibody sequences (i.e., CTB, ZnM-CTB, ZnP-CTB, TcpA-CT-CTB, ZnM-TcpA-CT-CTB, ZnP-TcpA-CT-CTB, ZnM-TcpA, ZnP-TcpA, TcpA-CT-TcpA, ZnM-TcpA-CT-TcpA, ZnP-TcpA-CT-TcpA, Ogawa, Inaba and ZnM-Inaba) raised against Vibrio cholerae. Methods: To determine APRs in antibody sequences that were generated after immunizing Vibrio cholerae immunogens on Mus musculus, a total of 94 sequences were downloaded as FASTA format from a protein database and the algorithms such as Tango, Waltz, PASTA 2.0, and AGGRESCAN were followed to analyze probable APRs in all of the sequences. Results: A remarkably high number of regions in the monoclonal antibodies were identified to be APRs which could explain a cause of instability/short term protection of anticholera vaccine. Conclusion: To increase the stability, it would be interesting to eliminate the APR residues from the therapeutic antibodies in a such way that the antigen binding sites or the complementarity determining region loops involved in antigen recognition are not disrupted.

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The Phosphorylated Estrogen Receptor α (ER) Cistrome Identifies a Subset of Active Enhancers Enriched for Direct ER-DNA Binding and the Transcription Factor GRHL2

Molecular and Cellular Biology ◽

10.1128/mcb.00417-18 ◽

2018 ◽

Vol 39 (3) ◽

Cited By ~ 5

Author(s):

Kyle T. Helzer ◽

Mary Szatkowski Ozers ◽

Mark B. Meyer ◽

Nancy A. Benkusky ◽

Natalia Solodin ◽

...

Keyword(s):

Estrogen Receptor ◽

Transcription Factor ◽

Dna Binding ◽

Protein Interactions ◽

Posttranslational Modifications ◽

Binding Sites ◽

Protein Function ◽

Estrogen Receptor Α ◽

Binding Activity

ABSTRACT Posttranslational modifications are key regulators of protein function, providing cues that can alter protein interactions and cellular location. Phosphorylation of estrogen receptor α (ER) at serine 118 (pS118-ER) occurs in response to multiple stimuli and is involved in modulating ER-dependent gene transcription. While the cistrome of ER is well established, surprisingly little is understood about how phosphorylation impacts ER-DNA binding activity. To define the pS118-ER cistrome, chromatin immunoprecipitation sequencing was performed on pS118-ER and ER in MCF-7 cells treated with estrogen. pS118-ER occupied a subset of ER binding sites which were associated with an active enhancer mark, acetylated H3K27. Unlike ER, pS118-ER sites were enriched in GRHL2 DNA binding motifs, and estrogen treatment increased GRHL2 recruitment to sites occupied by pS118-ER. Additionally, pS118-ER occupancy sites showed greater enrichment of full-length estrogen response elements relative to ER sites. In an in vitro DNA binding array of genomic binding sites, pS118-ER was more commonly associated with direct DNA binding events than indirect binding events. These results indicate that phosphorylation of ER at serine 118 promotes direct DNA binding at active enhancers and is a distinguishing mark for associated transcription factor complexes on chromatin.

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A network of phosphatidylinositol 4,5-bisphosphate binding sites regulates gating of the Ca2+-activated Cl− channel ANO1 (TMEM16A)

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1904012116 ◽

2019 ◽

Vol 116 (40) ◽

pp. 19952-19962 ◽

Cited By ~ 11

Author(s):

Kuai Yu ◽

Tao Jiang ◽

YuanYuan Cui ◽

Emad Tajkhorshid ◽

H. Criss Hartzell

Keyword(s):

Protein Interactions ◽

Binding Sites ◽

Neuronal Excitability ◽

Computational Study ◽

Channel Gating ◽

Md Simulations ◽

Fluid Secretion ◽

Hydroxyl Groups ◽

Binding Modes ◽

Cytoplasmic Extension

ANO1 (TMEM16A) is a Ca2+-activated Cl− channel that regulates diverse cellular functions including fluid secretion, neuronal excitability, and smooth muscle contraction. ANO1 is activated by elevation of cytosolic Ca2+ and modulated by phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Here, we describe a closely concerted experimental and computational study, including electrophysiology, mutagenesis, functional assays, and extended sampling of lipid–protein interactions with molecular dynamics (MD) to characterize PI(4,5)P2 binding modes and sites on ANO1. ANO1 currents in excised inside-out patches activated by 270 nM Ca2+ at +100 mV are increased by exogenous PI(4,5)P2 with an EC50 = 1.24 µM. The effect of PI(4,5)P2 is dependent on membrane voltage and Ca2+ and is explained by a stabilization of the ANO1 Ca2+-bound open state. Unbiased atomistic MD simulations with 1.4 mol% PI(4,5)P2 in a phosphatidylcholine bilayer identified 8 binding sites with significant probability of binding PI(4,5)P2. Three of these sites captured 85% of all ANO1–PI(4,5)P2 interactions. Mutagenesis of basic amino acids near the membrane–cytosol interface found 3 regions of ANO1 critical for PI(4,5)P2 regulation that correspond to the same 3 sites identified by MD. PI(4,5)P2 is stabilized by hydrogen bonding between amino acid side chains and phosphate/hydroxyl groups on PI(4,5)P2. Binding of PI(4,5)P2 alters the position of the cytoplasmic extension of TM6, which plays a crucial role in ANO1 channel gating, and increases the accessibility of the inner vestibule to Cl− ions. We propose a model consisting of a network of 3 PI(4,5)P2 binding sites at the cytoplasmic face of the membrane allosterically regulating ANO1 channel gating.

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NMR for the design of functional mimetics of protein-protein interactions: one key is in the building of bridges

Biochemistry and Cell Biology ◽

10.1139/o98-046 ◽

1998 ◽

Vol 76 (2-3) ◽

pp. 177-188 ◽

Cited By ~ 18

Author(s):

Jianxing Song ◽

Feng Ni

Keyword(s):

Protein Interactions ◽

Binding Sites ◽

Experimental Approach ◽

Structural Information ◽

Peptide Ligands ◽

Protein Protein Interactions ◽

Binding Residues ◽

Related Proteins ◽

Binding Inhibitors

Using the design of bivalent and bridge-binding inhibitors of thrombin as an example, we review an NMR-based experimental approach for the design of functional mimetics of protein-protein interactions. The strategy includes: (i) identification of binding residues in peptide ligands by differential resonance perturbation, (ii) determination of protein-bound structures of peptide ligands by use of transferred NOEs, (iii) minimization of larger protein and peptide ligands on the basis of NMR structural information, and (iv) linkage of two weakly binding mimetics to produce an inhibitor with enhanced affinity and specificity. This approach can be especially effective for the design of potent and selective functional mimetics of protein-protein interactions because it is less likely that the surfaces of two related proteins or enzymes share two identical binding sites or regions.Key words: NMR, protein-protein interactions, functional mimetics, bridge-binding inhibitors, thrombin.

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Elucidating the druggable interface of protein−protein interactions using fragment docking and coevolutionary analysis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1615932113 ◽

2016 ◽

Vol 113 (50) ◽

pp. E8051-E8058 ◽

Cited By ~ 36

Author(s):

Fang Bai ◽

Faruck Morcos ◽

Ryan R. Cheng ◽

Hualiang Jiang ◽

José N. Onuchic

Keyword(s):

Drug Design ◽

Protein Interactions ◽

Binding Sites ◽

Hot Spots ◽

Therapeutic Agents ◽

Molecular Probes ◽

Therapeutic Drug ◽

Protein Protein Interactions ◽

Protein Surface ◽

Fragment Docking

Protein−protein interactions play a central role in cellular function. Improving the understanding of complex formation has many practical applications, including the rational design of new therapeutic agents and the mechanisms governing signal transduction networks. The generally large, flat, and relatively featureless binding sites of protein complexes pose many challenges for drug design. Fragment docking and direct coupling analysis are used in an integrated computational method to estimate druggable protein−protein interfaces. (i) This method explores the binding of fragment-sized molecular probes on the protein surface using a molecular docking-based screen. (ii) The energetically favorable binding sites of the probes, called hot spots, are spatially clustered to map out candidate binding sites on the protein surface. (iii) A coevolution-based interface interaction score is used to discriminate between different candidate binding sites, yielding potential interfacial targets for therapeutic drug design. This approach is validated for important, well-studied disease-related proteins with known pharmaceutical targets, and also identifies targets that have yet to be studied. Moreover, therapeutic agents are proposed by chemically connecting the fragments that are strongly bound to the hot spots.

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Fused protein domains inhibit DNA binding by LexA.

Molecular and Cellular Biology ◽

10.1128/mcb.12.7.3006 ◽

1992 ◽

Vol 12 (7) ◽

pp. 3006-3014 ◽

Cited By ~ 105

Author(s):

E A Golemis ◽

R Brent

Keyword(s):

Dna Binding ◽

Protein Interactions ◽

In Vitro Assays ◽

Dimerization Domain ◽

Activation Domains ◽

Dna Binding Domains ◽

Binding Domains ◽

Operator Binding

Many studies of transcription activation employ fusions of activation domains to DNA binding domains derived from the bacterial repressor LexA and the yeast activator GAL4. Such studies often implicitly assume that DNA binding by the chimeric proteins is equivalent to that of the protein donating the DNA binding moiety. To directly investigate this issue, we compared operator binding by a series of LexA-derivative proteins to operator binding by native LexA, by using both in vivo and in vitro assays. We show that operator binding by many proteins such as LexA-Myc, LexA-Fos, and LexA-Bicoid is severely impaired, while binding of other LexA-derivative proteins, such as those that carry bacterially encoded acidic sequences ("acid blobs"), is not. Our results also show that DNA binding by LexA derivatives that contain the LexA carboxy-terminal dimerization domain (amino acids 88 to 202) is considerably stronger than binding by fusions that lack it and that heterologous dimerization motifs cannot substitute for the LexA88-202 function. These results suggest the need to reevaluate some previous studies of activation that employed LexA derivatives and modifications to recent experimental approaches that use LexA and GAL4 derivatives to detect and study protein-protein interactions.

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Repression of hsp70 heat shock gene transcription by the suppressor of hairy-wing protein of Drosophila melanogaster.

Molecular and Cellular Biology ◽

10.1128/mcb.11.4.1894 ◽

1991 ◽

Vol 11 (4) ◽

pp. 1894-1900 ◽

Cited By ~ 91

Author(s):

C Holdridge ◽

D Dorsett

Keyword(s):

Drosophila Melanogaster ◽

Heat Shock ◽

Protein Binding ◽

Protein Interactions ◽

Binding Sites ◽

Zinc Finger Protein ◽

Gene Promoter ◽

Heat Shock Gene ◽

Protein Binding Sites ◽

Shock Gene

The suppressor of hairy-wing [su(Hw)] locus of Drosophila melanogaster encodes a zinc finger protein that binds a repeated motif in the gypsy retroposon. Mutations of su(Hw) suppress the phenotypes associated with mutations caused by gypsy insertions. To examine the mechanisms by which su(Hw) alters gene expression, a fragment of gypsy containing multiple su(Hw) protein-binding sites was inserted into various locations in the well-characterized Drosophila hsp70 heat shock gene promoter. We found no evidence for activation of basal hsp70 transcription by su(Hw) protein in cultured Drosophila cells but observed that it can repress heat shock-induced transcription. Repression occurred only when su(Hw) protein-binding sites were positioned between binding sites for proteins required for heat shock transcription. We propose that su(Hw) protein interferes nonspecifically with protein-protein interactions required for heat shock transcription, perhaps sterically, or by altering the ability of DNA to bend or twist.

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Detection of Specific Protein–Protein Interactions in Nanocages by Engineering Bipartite FlAsH Binding Sites

Journal of the American Chemical Society ◽

10.1021/ja4085034 ◽

2013 ◽

Vol 135 (44) ◽

pp. 16618-16624 ◽

Cited By ~ 17

Author(s):

Thomas A. Cornell ◽

Jing Fu ◽

Stephanie H. Newland ◽

Brendan P. Orner

Keyword(s):

Protein Interactions ◽

Binding Sites ◽

Specific Protein ◽

Protein Protein Interactions

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Isolation and characterization of the rat huntingtin promoter

Biochemical Journal ◽

10.1042/bj3360227 ◽

1998 ◽

Vol 336 (1) ◽

pp. 227-234 ◽

Cited By ~ 17

Author(s):

Carsten HOLZMANN ◽

Winfried MÄUELER ◽

Dirk PETERSOHN ◽

Thorsten SCHMIDT ◽

Gerald THIEL ◽

...

Keyword(s):

Protein Interactions ◽

Binding Sites ◽

Neurodegenerative Disorder ◽

Core Promoter ◽

Chinese Hamster ◽

Gc Content ◽

Ovary Cell ◽

Mobility Shift ◽

Isolation And Characterization ◽

Hd Gene

Huntington's disease (HD) is a neurodegenerative disorder caused by a (CAG)>37 repeat expansion in a novel gene of unknown function. Although the huntingtin gene is expressed in neuronal and non-neuronal tissues, the disease affects nerve cells of selected regional areas of the central nervous system. To gain insight into the regulation of the HD gene we analysed 1348 bp of the rat huntingtin promoter region. This region lacks a TATA and a CAAT box, is rich in GC content and has several consensus sequences for binding sites for SP1, PEA3, Sif and H2A. The stretch between nucleotides -56 and -206 relative to the first ATG is highly conserved between human and rodents and it harbours several potential binding sites for transcription factors. We analysed deletion mutants fused with the chloramphenicol acetyltransferase reporter gene in transfected, HD-expressing neuronal (NS20Y, NG108-15) and non-neuronal Chinese hamster ovary cell lines. Hence these cells should contain the required trans-acting factors necessary for HD gene expression. Partial deletion of the evolutionarily conserved part of the promoter significantly decreases the activity in both neuronal and non-neuronal cells, indicating that the core promoter activity is located between nucleotides -332 and -15. DNase I footprinting and electrophoretic mobility-shift assays were used to define the nucleotide positions and binding affinity of DNA–protein interactions.

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Docking and Assembly of the Type II Secretion Complex of Vibrio cholerae

Journal of Bacteriology ◽

10.1128/jb.01701-08 ◽

2009 ◽

Vol 191 (9) ◽

pp. 3149-3161 ◽

Cited By ~ 56

Author(s):

Suzanne R. Lybarger ◽

Tanya L. Johnson ◽

Miranda D. Gray ◽

Aleksandra E. Sikora ◽

Maria Sandkvist

Keyword(s):

Vibrio Cholerae ◽

Protein Interactions ◽

Fluorescent Protein ◽

Outer Membrane Proteins ◽

Subcellular Location ◽

Type Ii Secretion ◽

Type Ii ◽

Protein Fusion ◽

Green Fluorescent

ABSTRACT Secretion of cholera toxin and other virulence factors from Vibrio cholerae is mediated by the type II secretion (T2S) apparatus, a multiprotein complex composed of both inner and outer membrane proteins. To better understand the mechanism by which the T2S complex coordinates translocation of its substrates, we are examining the protein-protein interactions of its components, encoded by the extracellular protein secretion (eps) genes. In this study, we took a cell biological approach, observing the dynamics of fluorescently tagged EpsC and EpsM proteins in vivo. We report that the level and context of fluorescent protein fusion expression can have a bold effect on subcellular location and that chromosomal, intraoperon expression conditions are optimal for determining the intracellular locations of fusion proteins. Fluorescently tagged, chromosomally expressed EpsC and EpsM form discrete foci along the lengths of the cells, different from the polar localization for green fluorescent protein (GFP)-EpsM previously described, as the fusions are balanced with all their interacting partner proteins within the T2S complex. Additionally, we observed that fluorescent foci in both chromosomal GFP-EpsC- and GFP-EpsM-expressing strains disperse upon deletion of epsD, suggesting that EpsD is critical to the localization of EpsC and EpsM and perhaps their assembly into the T2S complex.

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