Identification of Antisense RNA Stem-Loops That Inhibit RNA–Protein Interactions Using a Bacterial Reporter System

Crystallization of recombinant proteins has been fundamental to our understanding of protein function, dysfunction, and molecular recognition. However, this information has often been gleaned under extremely nonphysiological protein, salt, and H+ concentrations. Here, we describe the development of a robust Inka1-Box (iBox)–PAK4cat system that spontaneously crystallizes in several mammalian cell types. The semi-quantitative assay described here allows the measurement of in vivo protein-protein interactions using a novel GFP-linked reporter system that produces fluorescent readouts from protein crystals. We combined this assay with in vitro X-ray crystallography and molecular dynamics studies to characterize the molecular determinants of the interaction between the PDZ2 domain of Na+/H+ exchange regulatory cofactor NHE-RF1 (NHERF1) and cystic fibrosis transmembrane conductance regulator (CFTR), a protein complex pertinent to the genetic disease cystic fibrosis. These experiments revealed the crystal structure of the extended PDZ domain of NHERF1 and indicated, contrary to what has been previously reported, that residue selection at positions −1 and −3 of the PDZ-binding motif influences the affinity and specificity of the NHERF1 PDZ2-CFTR interaction. Our results suggest that this system could be utilized to screen additional protein-protein interactions, provided they can be accommodated within the spacious iBox-PAK4cat lattice.

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A multi-reporter bacterial 2-hybrid assay for the fast, simple, and dynamic assay of PDZ domain – peptide interactions

10.1101/512566 ◽

2019 ◽

Author(s):

David M. Ichikawa ◽

Carles Corbi-Verge ◽

Michael J. Shen ◽

Jamie Snider ◽

Victoria Wong ◽

...

Keyword(s):

Protein Interactions ◽

Accurate Determination ◽

Pdz Domain ◽

Relative Strength ◽

Protein Protein Interactions ◽

Reporter System ◽

Protein Motif ◽

Wide Range ◽

Domain Peptide

AbstractThe accurate determination of protein-protein interactions is an important goal of molecular biology and much progress has been made with modern methods emerging in the past decade. However, current methods have limitations, including scale and restriction to high affinity interactions. This has limited our understanding of the large subset of protein-motif interactions. Here we describe a modified bacterial-hybrid assay that employs a combined selectable and scalable reporter system that allows the screen of large protein-peptide libraries and their sort by relative strength. We have applied this tool to characterize a set of human and E. coli PDZ domains. Our results are consistent with prior characterization of these proteins and the improved sensitivity increases our ability to predict known and novel in vivo binding partners. This approach allows for the recovery of a wide range of affinities with a high throughput method that does not sacrifice the scale of the screen.

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Screening for Novel Small-Molecule Inhibitors Targeting the Assembly of Influenza Virus Polymerase Complex by a Bimolecular Luminescence Complementation-Based Reporter System

Journal of Virology ◽

10.1128/jvi.02282-16 ◽

2016 ◽

Vol 91 (5) ◽

Cited By ~ 7

Author(s):

Chunfeng Li ◽

Zining Wang ◽

Yang Cao ◽

Lulan Wang ◽

Jingyun Ji ◽

...

Keyword(s):

Influenza Virus ◽

Virus Replication ◽

Small Molecule ◽

Protein Interactions ◽

Critical Role ◽

Protein Protein Interactions ◽

Reporter System ◽

Polymerase Complex ◽

Virus Rna ◽

Influenza Virus Replication

ABSTRACT Influenza virus RNA-dependent RNA polymerase consists of three viral protein subunits: PA, PB1, and PB2. Protein-protein interactions (PPIs) of these subunits play pivotal roles in assembling the functional polymerase complex, which is essential for the replication and transcription of influenza virus RNA. Here we developed a highly specific and robust bimolecular luminescence complementation (BiLC) reporter system to facilitate the investigation of influenza virus polymerase complex formation. Furthermore, by combining computational modeling and the BiLC reporter assay, we identified several novel small-molecule compounds that selectively inhibited PB1-PB2 interaction. Function of one such lead compound was confirmed by its activity in suppressing influenza virus replication. In addition, our studies also revealed that PA plays a critical role in enhancing interactions between PB1 and PB2, which could be important in targeting sites for anti-influenza intervention. Collectively, these findings not only aid the development of novel inhibitors targeting the formation of influenza virus polymerase complex but also present a new tool to investigate the exquisite mechanism of PPIs. IMPORTANCE Formation of the functional influenza virus polymerase involves complex protein-protein interactions (PPIs) of PA, PB1, and PB2 subunits. In this work, we developed a novel BiLC assay system which is sensitive and specific to quantify both strong and weak PPIs between influenza virus polymerase subunits. More importantly, by combining in silico modeling and our BiLC assay, we identified a small molecule that can suppress influenza virus replication by disrupting the polymerase assembly. Thus, we developed an innovative method to investigate PPIs of multisubunit complexes effectively and to identify new molecules inhibiting influenza virus polymerase assembly.

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Genome-wide analysis of protein-protein interactions and involvement of viral proteins in SARS-CoV-2 replication

10.21203/rs.3.rs-337427/v1 ◽

2021 ◽

Author(s):

Yiling Jiang ◽

Kuijie Tong ◽

Roubin Yao ◽

Yuanze Zhou ◽

Hanwen Lin ◽

...

Keyword(s):

Protein Interactions ◽

Viral Protein ◽

Antiviral Drug ◽

Viral Proteins ◽

Protein Protein Interactions ◽

Reporter System ◽

N Protein ◽

Genome Wide ◽

N Terminus ◽

Protein Functions

Abstract Analysis of viral protein-protein interactions is an essential step to uncover the viral protein functions and the molecular mechanism for the assembly of a viral protein complex. We employed a mammalian two-hybrid system to screen all the viral proteins of SARS-CoV-2 for the protein-protein interactions. Our study detected 48 interactions, 14 of which were firstly reported here. Unlike Nsp1 of SARS-CoV, Nsp1 of SARS-CoV-2 has the most interacting partners among all the viral proteins and likely functions as a hub for the viral proteins. Five self-interactions were confirmed, and five interactions, Nsp1/Nsp3.1, Nsp3.1/N, Nsp3.2/Nsp12, Nsp10/Nsp14, and Nsp10/Nsp16, were determined to be positive bidirectionally. Using the replicon reporter system of SARS-CoV-2, we screened all viral proteins for their impacts on the viral replication and revealed Nsp3.1, the N-terminus of Nsp3, significantly inhibited the replicon reporter gene expression. We found Nsp3 interacted with N through its acidic region at N-terminus, while N interacted with Nsp3 through its NTD, which is rich in the basic amino acids. Furthermore, using purified truncated N and Nsp3 proteins, we determined the direct interactions between Nsp3 and N protein. In summary, our findings provided a basis for understanding the functions of coronavirus proteins and supported the potential of interactions as the target for antiviral drug development.

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In-vivo crystals reveal protein interactions

10.1101/778779 ◽

2019 ◽

Author(s):

Eleanor R. Martin ◽

Alessandro Barbieri ◽

Robert C. Ford ◽

Robert C. Robinson

Keyword(s):

Protein Interactions ◽

Protein Function ◽

Pdz Domain ◽

Cell Types ◽

Binding Motif ◽

Protein Protein Interactions ◽

Reporter System ◽

X Ray Crystallography

AbstractCrystallisation of recombinant proteins has been fundamental to our understanding of protein function, dysfunction, and molecular recognition. However, this information has often been gleaned under non-physiological extremes of protein, salt, and H+ concentrations. Here, we describe the development of the robust iBox-PAK4cat system that spontaneously crystallises in several mammalian cell types. The developments described here allow the quantitation of in-vivo protein-protein interactions using a novel GFP-linked reporter system. Here, we have combined this assay with in-vitro X-ray crystallography and molecular dynamics studies characterise the molecular determinants of the interaction between NHERF1 PDZ2 and CFTR, a protein complex pertinent to the genetic disease cystic fibrosis. These studies have revealed the crystal structure of the extended PDZ domain of NHERF1, and indicated, contrary to previous reports, that residue selection at −1 and −3 PDZ-binding motif positions influence the affinity and specificity of the interaction. The results presented here demonstrate that the iBox-PAK4cat assay could easily be utilised to screen other protein-protein interactions.

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Kinetic and subcellular analysis of PS-ASO/protein interactions with P54nrb and RNase H1

Nucleic Acids Research ◽

10.1093/nar/gkz771 ◽

2019 ◽

Vol 47 (20) ◽

pp. 10865-10880 ◽

Cited By ~ 7

Author(s):

Timothy A Vickers ◽

Meghdad Rahdar ◽

Thazha P Prakash ◽

Stanley T Crooke

Keyword(s):

Protein Interaction ◽

Protein Interactions ◽

Catalytic Domain ◽

Live Cells ◽

Early Event ◽

Reporter System ◽

Protein Protein Interaction ◽

Domain Interactions ◽

Specific Proteins

Abstract The rapid RNase H1-dependent mislocalization of heterodimer proteins P54nrb and PSF to nucleoli is an early event in the pathway that explains the effects of most toxic phosphorothioate ASOs (PS-ASOs). Using a recently developed NanoLuciferace (NLuc)-based structural complementation reporter system which allows us to observe ASO/protein interactions in real time in live cells, we have determined that safe and toxic PS-ASOs associate with these proteins with kinetics and impact on subcellular localization that differ. Toxic PS-ASOs interact in a complex that includes RNase H1, P54nrb and PSF; but RNase H1/P54nrb complexes were observed in only the cells treated with toxic, but not safe PS-ASOs. In addition, experiments performed in vitro suggest that RNA is also a required component of the complex. The protein–protein interaction between P54nrb and RNase H1 requires the spacer region of RNAse H1, while the P54nrb core domains are required for association with RNase H1. In addition, we have determined that PS-ASOs bind P54nrb via RRM1 and RRM2, while they bind RNase H1 primarily via the hybrid binding domain, however catalytic domain interactions also contribute to overall affinity. These ASO–protein interactions are highly influenced by the chemistry of the PS-ASO binding environment, however little correlation between affinity for specific proteins and PS-ASO toxicity was observed.

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Synthesis of fluorinated maltose derivatives for monitoring protein interaction by 19F NMR

Beilstein Journal of Organic Chemistry ◽

10.3762/bjoc.8.51 ◽

2012 ◽

Vol 8 ◽

pp. 448-455 ◽

Cited By ~ 19

Author(s):

Michaela Braitsch ◽

Hanspeter Kählig ◽

Georg Kontaxis ◽

Michael Fischer ◽

Toshinari Kawada ◽

...

Keyword(s):

Protein Interaction ◽

Protein Interactions ◽

Binding Protein ◽

Maltose Binding Protein ◽

Interaction Patterns ◽

Protein Protein Interactions ◽

Reporter System ◽

Perfect Agreement ◽

Ligand Interaction ◽

Chemical Shift Data

A novel reporter system, which is applicable to the 19F NMR investigation of protein interactions, is presented. This approach uses 2-F-labeled maltose as a spy ligand to indirectly probe protein–ligand or protein–protein interactions of proteins fused or tagged to the maltose-binding protein (MBP). The key feature is the simultaneous NMR observation of both 19F NMR signals of gluco/manno-type-2-F-maltose-isomers; one isomer (α-gluco-type) binds to MBP and senses the protein interaction, and the nonbinding isomers (β-gluco- and/or α/β-manno-type) are utilized as internal references. Moreover, this reporter system was used for relative affinity studies of fluorinated and nonfluorinated carbohydrates to the maltose-binding protein, which were found to be in perfect agreement with published X-ray data. The results of the NMR competition experiments together with the established correlation between 19F chemical shift data and molecular interaction patterns, suggest valuable applications for studies of protein–ligand interaction interfaces.

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