Detecting protein–protein interactions by Xe-129 NMR

The phage-shock-protein (Psp) response maintains the proton-motive force (pmf) under extracytoplasmic stress conditions that impair the inner membrane (IM) in bacterial cells. In Escherichia coli transcription of the pspABCDE and pspG genes requires activation of σ 54-RNA polymerase by the enhancer-binding protein PspF. A regulatory network comprising PspF–A–C–B–ArcB controls psp expression. One key regulatory point is the negative control of PspF imposed by its binding to PspA. It has been proposed that under stress conditions, the IM-bound sensors PspB and PspC receive and transduce the signal(s) to PspA via protein–protein interactions, resulting in the release of the PspA–PspF inhibitory complex and the consequent induction of psp. In this work we demonstrate that PspB self-associates and interacts with PspC via putative IM regions. We present evidence suggesting that PspC has two topologies and that conserved residue G48 and the putative leucine zipper motif are determinants required for PspA interaction and signal transduction upon stress. We also establish that PspC directly interacts with the effector PspG, and show that PspG self-associates. These results are discussed in the context of formation and function of the Psp regulatory complex.

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Roles of the 14-3-3 gene family in cotton flowering

BMC Plant Biology ◽

10.1186/s12870-021-02923-9 ◽

2021 ◽

Vol 21 (1) ◽

Author(s):

Na Sang ◽

Hui Liu ◽

Bin Ma ◽

Xianzhong Huang ◽

Lu Zhuo ◽

...

Keyword(s):

Gene Family ◽

Protein Interactions ◽

Leucine Zipper ◽

Expression Patterns ◽

Bimolecular Fluorescence Complementation ◽

Structural Motif ◽

Virus Induced Gene Silencing ◽

Protein Protein Interactions ◽

Basic Leucine Zipper ◽

Genome Wide

Abstract Background In plants, 14-3-3 proteins, also called GENERAL REGULATORY FACTORs (GRFs), encoded by a large multigene family, are involved in protein–protein interactions and play crucial roles in various physiological processes. No genome-wide analysis of the GRF gene family has been performed in cotton, and their functions in flowering are largely unknown. Results In this study, 17, 17, 31, and 17 GRF genes were identified in Gossypium herbaceum, G. arboreum, G. hirsutum, and G. raimondii, respectively, by genome-wide analyses and were designated as GheGRFs, GaGRFs, GhGRFs, and GrGRFs, respectively. A phylogenetic analysis revealed that these proteins were divided into ε and non-ε groups. Gene structural, motif composition, synteny, and duplicated gene analyses of the identified GRF genes provided insights into the evolution of this family in cotton. GhGRF genes exhibited diverse expression patterns in different tissues. Yeast two-hybrid and bimolecular fluorescence complementation assays showed that the GhGRFs interacted with the cotton FLOWERING LOCUS T homologue GhFT in the cytoplasm and nucleus, while they interacted with the basic leucine zipper transcription factor GhFD only in the nucleus. Virus-induced gene silencing in G. hirsutum and transgenic studies in Arabidopsis demonstrated that GhGRF3/6/9/15 repressed flowering and that GhGRF14 promoted flowering. Conclusions Here, 82 GRF genes were identified in cotton, and their gene and protein features, classification, evolution, and expression patterns were comprehensively and systematically investigated. The GhGRF3/6/9/15 interacted with GhFT and GhFD to form florigen activation complexs that inhibited flowering. However, GhGRF14 interacted with GhFT and GhFD to form florigen activation complex that promoted flowering. The results provide a foundation for further studies on the regulatory mechanisms of flowering.

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Leucine periodicity of U2 small nuclear ribonucleoprotein particle (snRNP) A' protein is implicated in snRNP assembly via protein-protein interactions

Molecular and Cellular Biology ◽

10.1128/mcb.11.3.1578-1589.1991 ◽

1991 ◽

Vol 11 (3) ◽

pp. 1578-1589

Author(s):

L D Fresco ◽

D S Harper ◽

J D Keene

Keyword(s):

Protein Interactions ◽

Leucine Zipper ◽

Tandem Repeats ◽

Mutational Analysis ◽

Particle Density ◽

Protein Protein Interactions ◽

Small Nuclear Ribonucleoprotein ◽

Cell Extracts ◽

Associated Proteins ◽

Nuclear Ribonucleoprotein

Recombinant A' protein could be reconstituted into U2 small nuclear ribonucleoprotein particles (snRNPs) upon addition to HeLa cell extracts as determined by coimmunoprecipitation and particle density; however, direct binding to U2 RNA could not be demonstrated except in the presence of the U2 snRNP B" protein. Mutational analysis indicated that a central core region of A' was required for particle reconstitution. This region consists of five tandem repeats of approximately 24 amino acids each that exhibit a periodicity of leucine and asparagine residues that is distinct from the leucine zipper. Similar leucine-rich (Leu-Leu motif) repeats are characteristic of a diverse array of soluble and membrane-associated proteins from yeasts to humans but have not been reported previously to reside in nuclear proteins. Several of these proteins, including Toll, chaoptin, RNase/angiogenin inhibitors, lutropin-choriogonadotropin receptor, carboxypeptidase N, adenylyl cyclase, CD14, and human immunodeficiency virus type 1 Rev, may be involved in protein-protein interactions. Our findings suggest that in cell extracts the Leu-Leu motif of A' is required for reconstitution with U2 snRNPs and perhaps with other components involved in splicing through protein-protein interactions.

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Structural Classification of Bacterial Response Regulators: Diversity of Output Domains and Domain Combinations

Journal of Bacteriology ◽

10.1128/jb.01887-05 ◽

2006 ◽

Vol 188 (12) ◽

pp. 4169-4182 ◽

Cited By ~ 315

Author(s):

Michael Y. Galperin

Keyword(s):

Protein Interactions ◽

Rna Binding ◽

Transcriptional Regulators ◽

Bacterial Cells ◽

Protein Protein Interactions ◽

Response Regulators ◽

Dna Binding Domains ◽

Binding Domains ◽

Archaeal Species

ABSTRACT CheY-like phosphoacceptor (or receiver [REC]) domain is a common module in a variety of response regulators of the bacterial signal transduction systems. In this work, 4,610 response regulators, encoded in complete genomes of 200 bacterial and archaeal species, were identified and classified by their domain architectures. Previously uncharacterized output domains were analyzed and, in some cases, assigned to known domain families. Transcriptional regulators of the OmpR, NarL, and NtrC families were found to comprise almost 60% of all response regulators; transcriptional regulators with other DNA-binding domains (LytTR, AraC, Spo0A, Fis, YcbB, RpoE, and MerR) account for an additional 6%. The remaining one-third is represented by the stand-alone REC domain (∼14%) and its combinations with a variety of enzymatic (GGDEF, EAL, HD-GYP, CheB, CheC, PP2C, and HisK), RNA-binding (ANTAR and CsrA), protein- or ligand-binding (PAS, GAF, TPR, CAP_ED, and HPt) domains, or newly described domains of unknown function. The diversity of domain architectures and the abundance of alternative domain combinations suggest that fusions between the REC domain and various output domains is a widespread evolutionary mechanism that allows bacterial cells to regulate transcription, enzyme activity, and/or protein-protein interactions in response to environmental challenges. The complete list of response regulators encoded in each of the 200 analyzed genomes is available online at http://www.ncbi.nlm.nih.gov/Complete_Genomes/RRcensus.html .

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Domain swapping reveals the modular nature of Fos, Jun, and CREB proteins.

Molecular and Cellular Biology ◽

10.1128/mcb.10.9.4565 ◽

1990 ◽

Vol 10 (9) ◽

pp. 4565-4573 ◽

Cited By ~ 21

Author(s):

L J Ransone ◽

P Wamsley ◽

K L Morley ◽

I M Verma

Keyword(s):

Dna Binding ◽

Protein Interactions ◽

Leucine Zipper ◽

Chimeric Protein ◽

Domain Swapping ◽

Amino Terminus ◽

Protein Protein Interactions ◽

Amino Terminal ◽

Nih 3T3 ◽

Basic Region

The products of the Jun and Fos proto-oncogenes form a heterodimer that binds to and activates transcription from 12-O-tetradecanoylphorbol-13-acetate-responsive promoter elements (TGACTCA) and AP-1-binding sites (TGACATCA). These two proteins belong to a family of related transcription factors which contain similar domains required for protein dimerization and DNA binding but display different protein and DNA binding specificities. The basic region, required for DNA binding, is followed by a leucine zipper structure, a domain that mediates protein-protein interactions. To assess the role of these two domains in three related proteins, Fos, Jun, and CREB, we carried out extensive domain-swapping analysis. We found that (i) dimers formed by two Jun leucine zipper-containing proteins were unable to bind DNA as efficiently as a Fos-Jun combination, regardless of the source of the basic region; (ii) the Fos leucine zipper was unable to form either homo- or heterodimers with a chimeric protein containing a Fos leucine zipper; (iii) the Fos basic region was capable of binding to an AP-1 site; (iv) replacement of the Jun amino terminus with that of CREB had little effect on dimerization, whereas replacement with the amino terminus of Fos disrupted both protein-protein and protein-DNA interactions; (v) changes in relative affinities of the Fos and Jun basic regions for the AP-1 element were dependent on the secondary contributions of amino-terminal residues; and (vi) the Fos-Jun chimeric constructs cooperated in transcriptional transactivation of the Jun promoter in NIH 3T3 cells.

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Leucine periodicity of U2 small nuclear ribonucleoprotein particle (snRNP) A' protein is implicated in snRNP assembly via protein-protein interactions.

Molecular and Cellular Biology ◽

10.1128/mcb.11.3.1578 ◽

1991 ◽

Vol 11 (3) ◽

pp. 1578-1589 ◽

Cited By ~ 18

Author(s):

L D Fresco ◽

D S Harper ◽

J D Keene

Keyword(s):

Protein Interactions ◽

Leucine Zipper ◽

Tandem Repeats ◽

Mutational Analysis ◽

Particle Density ◽

Protein Protein Interactions ◽

Small Nuclear Ribonucleoprotein ◽

Cell Extracts ◽

Associated Proteins ◽

Nuclear Ribonucleoprotein

Recombinant A' protein could be reconstituted into U2 small nuclear ribonucleoprotein particles (snRNPs) upon addition to HeLa cell extracts as determined by coimmunoprecipitation and particle density; however, direct binding to U2 RNA could not be demonstrated except in the presence of the U2 snRNP B" protein. Mutational analysis indicated that a central core region of A' was required for particle reconstitution. This region consists of five tandem repeats of approximately 24 amino acids each that exhibit a periodicity of leucine and asparagine residues that is distinct from the leucine zipper. Similar leucine-rich (Leu-Leu motif) repeats are characteristic of a diverse array of soluble and membrane-associated proteins from yeasts to humans but have not been reported previously to reside in nuclear proteins. Several of these proteins, including Toll, chaoptin, RNase/angiogenin inhibitors, lutropin-choriogonadotropin receptor, carboxypeptidase N, adenylyl cyclase, CD14, and human immunodeficiency virus type 1 Rev, may be involved in protein-protein interactions. Our findings suggest that in cell extracts the Leu-Leu motif of A' is required for reconstitution with U2 snRNPs and perhaps with other components involved in splicing through protein-protein interactions.

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Applications of In-Cell NMR in Structural Biology and Drug Discovery

International Journal of Molecular Sciences ◽

10.3390/ijms20010139 ◽

2019 ◽

Vol 20 (1) ◽

pp. 139 ◽

Cited By ~ 9

Author(s):

CongBao Kang

Keyword(s):

Drug Discovery ◽

Conformational Changes ◽

Protein Interactions ◽

Mammalian Cells ◽

Structural Information ◽

Living Cells ◽

Bacterial Cells ◽

Protein Protein Interactions ◽

Nmr Studies ◽

Post Translational Modifications

In-cell nuclear magnetic resonance (NMR) is a method to provide the structural information of a target at an atomic level under physiological conditions and a full view of the conformational changes of a protein caused by ligand binding, post-translational modifications or protein–protein interactions in living cells. Previous in-cell NMR studies have focused on proteins that were overexpressed in bacterial cells and isotopically labeled proteins injected into oocytes of Xenopus laevis or delivered into human cells. Applications of in-cell NMR in probing protein modifications, conformational changes and ligand bindings have been carried out in mammalian cells by monitoring isotopically labeled proteins overexpressed in living cells. The available protocols and successful examples encourage wide applications of this technique in different fields such as drug discovery. Despite the challenges in this method, progress has been made in recent years. In this review, applications of in-cell NMR are summarized. The successful applications of this method in mammalian and bacterial cells make it feasible to play important roles in drug discovery, especially in the step of target engagement.

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Domain swapping reveals the modular nature of Fos, Jun, and CREB proteins

Molecular and Cellular Biology ◽

10.1128/mcb.10.9.4565-4573.1990 ◽

1990 ◽

Vol 10 (9) ◽

pp. 4565-4573

Author(s):

L J Ransone ◽

P Wamsley ◽

K L Morley ◽

I M Verma

Keyword(s):

Dna Binding ◽

Protein Interactions ◽

Leucine Zipper ◽

Chimeric Protein ◽

Domain Swapping ◽

Amino Terminus ◽

Protein Protein Interactions ◽

Amino Terminal ◽

Nih 3T3 ◽

Basic Region

The products of the Jun and Fos proto-oncogenes form a heterodimer that binds to and activates transcription from 12-O-tetradecanoylphorbol-13-acetate-responsive promoter elements (TGACTCA) and AP-1-binding sites (TGACATCA). These two proteins belong to a family of related transcription factors which contain similar domains required for protein dimerization and DNA binding but display different protein and DNA binding specificities. The basic region, required for DNA binding, is followed by a leucine zipper structure, a domain that mediates protein-protein interactions. To assess the role of these two domains in three related proteins, Fos, Jun, and CREB, we carried out extensive domain-swapping analysis. We found that (i) dimers formed by two Jun leucine zipper-containing proteins were unable to bind DNA as efficiently as a Fos-Jun combination, regardless of the source of the basic region; (ii) the Fos leucine zipper was unable to form either homo- or heterodimers with a chimeric protein containing a Fos leucine zipper; (iii) the Fos basic region was capable of binding to an AP-1 site; (iv) replacement of the Jun amino terminus with that of CREB had little effect on dimerization, whereas replacement with the amino terminus of Fos disrupted both protein-protein and protein-DNA interactions; (v) changes in relative affinities of the Fos and Jun basic regions for the AP-1 element were dependent on the secondary contributions of amino-terminal residues; and (vi) the Fos-Jun chimeric constructs cooperated in transcriptional transactivation of the Jun promoter in NIH 3T3 cells.

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APPL1 is a multifunctional endosomal signaling adaptor protein

Biochemical Society Transactions ◽

10.1042/bst20160191 ◽

2017 ◽

Vol 45 (3) ◽

pp. 771-779 ◽

Cited By ~ 19

Author(s):

Nicole L. Diggins ◽

Donna J. Webb

Keyword(s):

Signaling Pathways ◽

Protein Interactions ◽

Leucine Zipper ◽

Adaptor Protein ◽

Adaptor Proteins ◽

Ph Domain ◽

Protein Protein Interactions ◽

Multiple Protein ◽

Signaling Receptors ◽

Ptb Domain

Endosomal adaptor proteins are important regulators of signaling pathways underlying many biological processes. These adaptors can integrate signals from multiple pathways via localization to specific endosomal compartments, as well as through multiple protein–protein interactions. One such adaptor protein that has been implicated in regulating signaling pathways is the adaptor protein containing a pleckstrin homology (PH) domain, phosphotyrosine-binding (PTB) domain, and leucine zipper motif 1 (APPL1). APPL1 localizes to a subset of Rab5-positive endosomes through its Bin–Amphiphysin–Rvs and PH domains, and it coordinates signaling pathways through its interaction with many signaling receptors and proteins through its PTB domain. This review discusses our current understanding of the role of APPL1 in signaling and trafficking, as well as highlights recent work into the function of APPL1 in cell migration and adhesion.

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A PAR domain transcription factor is involved in the expression from a hematopoietic-specific promoter for the human LMO2 gene

Blood ◽

10.1182/blood-2002-09-2702 ◽

2003 ◽

Vol 101 (12) ◽

pp. 4757-4764 ◽

Cited By ~ 24

Author(s):

Scott C. Crable ◽

Kathleen P. Anderson

Keyword(s):

Transcription Factor ◽

Protein Interactions ◽

Leucine Zipper ◽

Cell Lineage ◽

Erythroid Cell ◽

Protein Protein Interactions ◽

Consensus Binding Site ◽

Basic Leucine Zipper ◽

Specific Expression ◽

Dna Binding Factors

AbstractThe transcription factor LMO2 is believed to exert its effect through the formation of protein-protein interactions with other DNA-binding factors such as GATA-1 and TAL1. Although LMO2 has been shown to be critical for the formation of the erythroid cell lineage, the gene is also expressed in a number of nonerythroid tissues. In this report, we demonstrate that the more distal of the 2 promoters for the LMO2 gene is highly restricted in its pattern of expression, directing the hematopoietic-specific expression of this gene. Deletion and mutation analyses have identified a critical cis element in the first untranslated exon of the gene. This element is a consensus-binding site for a small family of basic leucine zipper proteins containing a proline and acidic amino acid–rich (PAR) domain. Although all 3 members of this family are produced in erythroid cells, only 2 of these proteins, thyrotroph embryonic factor and hepatic leukemia factor, can activate transcription from this LMO2 promoter element. These findings represent a novel mechanism in erythroid gene regulation because PAR proteins have not previously been implicated in this process.

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