Use of periplasmic target protein capture for phage display engineering of tight-binding protein–protein interactions

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.

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A crosslinked and ribosylated actin trimer does not interact productively with myosin

Biochemistry and Cell Biology ◽

10.1139/bcb-2018-0082 ◽

2019 ◽

Vol 97 (2) ◽

pp. 140-147 ◽

Cited By ~ 1

Author(s):

Navneet Sidhu ◽

John F. Dawson

Keyword(s):

Binding Site ◽

Protein Interactions ◽

Binding Proteins ◽

Binding Protein ◽

Dnase I ◽

Protein Protein Interactions ◽

Adp Ribosylation ◽

Myosin Binding ◽

Pointed End

A purified F-actin-derived actin trimer that interacts with end-binding proteins did not activate or bind the side-binding protein myosin under rigor conditions. Remodeling of the actin trimer by the binding of gelsolin did not rescue myosin binding, nor did the use of different means of inhibiting the polymerization of the trimer. Our results demonstrate that ADP-ribosylation on all actin subunits of an F-actin-derived trimer inhibits myosin binding and that the binding of DNase-I to the pointed end subunits of a crosslinked trimer also remodels the myosin binding site. Taken together, this work highlights the need for a careful balance between modification of actin subunits and maintaining protein–protein interactions to produce a physiologically relevant short F-actin complex.

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Predicting virus-receptor mutant binding by molecular dynamics simulation

10.7287/peerj.preprints.138v1 ◽

2013 ◽

Author(s):

Austin G Meyer ◽

Sara L Sawyer ◽

Andrew D Ellington ◽

Claus O Wilke

Keyword(s):

Protein Interactions ◽

Tight Binding ◽

Dynamics Simulation ◽

Virus Protein ◽

Protein Protein Interactions ◽

Distance Curve ◽

Protein Protein Interaction ◽

Human Transferrin Receptor ◽

Machupo Virus ◽

Receptor Mutant

Existing computational methods to predict protein–protein interaction affinity often perform poorly in important test cases. In particular, the effects of multiple mutations, non-alanine substitutions, and flexible loops are difficult to predict with available tools and protocols. We present here a new method to interrogate affinity differences resulting from mutations in a host-virus protein–protein interface. Our method is based on extensive non-equilibrium all atom simulations: We computationally pull the machupo virus (MACV) spike glycoprotein (GP1) away from the human transferrin receptor (hTfR1) and estimate affinity using the max imum applied force during a pulling simulation and the area under the force-versus-distance curve. We find that these quantities can provide novel biophysical insight into the GP1/hTfR1 interaction. First, with no prior knowledge of the system we can differentiate among wild type and mutant complexes. Second, although the static co-crystal structure shows two large hydrogen-bonding networks in the GP1/hTfR1 interface, our simulations indicate that one of them may not be important for tight binding. Third, one viral site known to be critical for infection may mark an important evolutionary suppressor site for infection-resistant hTfR1 mutants. Finally, our method provides an elegant framework to compare the effects of multi ple mutations, individually and jointly, on protein–protein interactions.

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NMR as a Tool to Target Protein–Protein Interactions

Disruption of Protein-Protein Interfaces ◽

10.1007/978-3-642-37999-4_4 ◽

2013 ◽

pp. 83-111

Author(s):

Rebecca Del Conte ◽

Daniela Lalli ◽

Paola Turano

Keyword(s):

Protein Interactions ◽

Target Protein ◽

Protein Protein Interactions

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Structural Insights into the Interactions of Candidal Enolase with Human Vitronectin, Fibronectin and Plasminogen

International Journal of Molecular Sciences ◽

10.3390/ijms21217843 ◽

2020 ◽

Vol 21 (21) ◽

pp. 7843 ◽

Cited By ~ 1

Author(s):

Dorota Satala ◽

Grzegorz Satala ◽

Justyna Karkowska-Kuleta ◽

Michal Bukowski ◽

Anna Kluza ◽

...

Keyword(s):

Protein Interactions ◽

Dissociation Constants ◽

Tight Binding ◽

Protein Protein Interactions ◽

Glycolysis Pathway ◽

Human Proteins ◽

Activity Center ◽

Moonlighting Functions ◽

Structural Insights ◽

Internal Motif

Significant amounts of enolase—a cytosolic enzyme involved in the glycolysis pathway—are exposed on the cell surface of Candida yeast. It has been hypothesized that this exposed enolase form contributes to infection-related phenomena such as fungal adhesion to human tissues, and the activation of fibrinolysis and extracellular matrix degradation. The aim of the present study was to characterize, in structural terms, the protein-protein interactions underlying these moonlighting functions of enolase. The tight binding of human vitronectin, fibronectin and plasminogen by purified C. albicans and C. tropicalis enolases was quantitatively analyzed by surface plasmon resonance measurements, and the dissociation constants of the formed complexes were determined to be in the 10−7–10−8 M range. In contrast, the binding of human proteins by the S.cerevisiae enzyme was much weaker. The chemical cross-linking method was used to map the sites on enolase molecules that come into direct contact with human proteins. An internal motif 235DKAGYKGKVGIAMDVASSEFYKDGK259 in C. albicans enolase was suggested to contribute to the binding of all three human proteins tested. Models for these interactions were developed and revealed the sites on the enolase molecule that bind human proteins, extensively overlap for these ligands, and are well-separated from the catalytic activity center.

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Small-Molecule Inhibitors That Target Protein-Protein Interactions in the RAD51 Family of Recombinases

ChemMedChem ◽

10.1002/cmdc.201402428 ◽

2014 ◽

Vol 10 (2) ◽

pp. 296-303 ◽

Cited By ~ 26

Author(s):

Duncan E. Scott ◽

Anthony G. Coyne ◽

Ashok Venkitaraman ◽

Tom L. Blundell ◽

Chris Abell ◽

...

Keyword(s):

Small Molecule ◽

Protein Interactions ◽

Small Molecule Inhibitors ◽

Target Protein ◽

Protein Protein Interactions

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Bcl-2 and FKBP12 bind to IP3 and ryanodine receptors at overlapping sites: the complexity of protein–protein interactions for channel regulation

Biochemical Society Transactions ◽

10.1042/bst20140298 ◽

2015 ◽

Vol 43 (3) ◽

pp. 396-404 ◽

Cited By ~ 10

Author(s):

Tim Vervliet ◽

Jan B. Parys ◽

Geert Bultynck

Keyword(s):

Protein Interactions ◽

Hippocampal Neurons ◽

Binding Protein ◽

Ryanodine Receptors ◽

B Cell Lymphoma ◽

Experimental Models ◽

Amino Acid Sequences ◽

Protein Protein Interactions ◽

Fk506 Binding Protein ◽

Fk506 Binding Proteins

The 12- and 12.6-kDa FK506-binding proteins, FKBP12 (12-kDa FK506-binding protein) and FKBP12.6 (12.6-kDa FK506-binding protein), have been implicated in the binding to and the regulation of ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP3Rs), both tetrameric intracellular Ca2+-release channels. Whereas the amino acid sequences responsible for FKBP12 binding to RyRs are conserved in IP3Rs, FKBP12 binding to IP3Rs has been questioned and could not be observed in various experimental models. Nevertheless, conservation of these residues in the different IP3R isoforms and during evolution suggested that they could harbour an important regulatory site critical for IP3R-channel function. Recently, it has become clear that in IP3Rs, this site was targeted by B-cell lymphoma 2 (Bcl-2) via its Bcl-2 homology (BH)4 domain, thereby dampening IP3R-mediated Ca2+ flux and preventing pro-apoptotic Ca2+ signalling. Furthermore, vice versa, the presence of the corresponding site in RyRs implied that Bcl-2 proteins could associate with and regulate RyR channels. Recently, the existence of endogenous RyR–Bcl-2 complexes has been identified in primary hippocampal neurons. Like for IP3Rs, binding of Bcl-2 to RyRs also involved its BH4 domain and suppressed RyR-mediated Ca2+ release. We therefore propose that the originally identified FKBP12-binding site in IP3Rs is a region critical for controlling IP3R-mediated Ca2+ flux by recruiting Bcl-2 rather than FKBP12. Although we hypothesize that anti-apoptotic Bcl-2 proteins, but not FKBP12, are the main physiological inhibitors of IP3Rs, we cannot exclude that Bcl-2 could help engaging FKBP12 (or other FKBP isoforms) to the IP3R, potentially via calcineurin.

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A phage display system designed to detect and study protein-protein interactions

Molecular Microbiology ◽

10.1111/j.1365-2958.2007.06077.x ◽

2008 ◽

Vol 67 (4) ◽

pp. 719-728 ◽

Cited By ~ 6

Author(s):

Catherine L. Bair ◽

Amos Oppenheim ◽

Andrei Trostel ◽

Gali Prag ◽

Sankar Adhya

Keyword(s):

Phage Display ◽

Protein Interactions ◽

Display System ◽

Protein Protein Interactions

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Synthesis of Epitope-Targeting Antibody Based on Native Protein–Protein Interactions for Ftsz Filamentation Suppressor

10.21203/rs.3.rs-757826/v1 ◽

2021 ◽

Author(s):

Hikaru Nakazawa ◽

Taiji Katsuki ◽

Takashi Matsui ◽

Atsushi Tsugita ◽

Takeshi Yokoyama ◽

...

Keyword(s):

Molecular Evolution ◽

Phage Display ◽

Protein Interactions ◽

Selection Process ◽

Evolutionary Engineering ◽

Temperature Sensitive ◽

Native Protein ◽

Protein Protein Interactions ◽

Division Rate ◽

Evolution Approach

Abstract Phage display and biopanning is a powerful tool for generating binding molecules for a specific target. However, the selection process based on binding affinity provides no assurance for the antibody’s affinity to the target epitope. In this study, we propose a molecular-evolution approach guided by native protein–protein interactions to generate epitope-targeting antibodies. The binding-site sequence in a native protein was grafted into a complementarity-determining region (CDR) in the antibody, and a nonrelated CDR loop (in the grafted antibody) was randomized by phage display techniques. In this construction of antibodies by integrating graft and evolution technology (CAnIGET method), suitable grafting of the functional sequence weakly functionalized the antibody, and the molecular-evolution approach enhanced the binding function to inhibit the native protein–protein interactions. Antibody fragments with an affinity for filamenting temperature-sensitive mutant Z (FtsZ) were constructed and completely inhibited the polymerization of FtsZ. Consequently, the expression of these fragments drastically decreased the cell division rate. We demonstrate the potential of the CAnIGET method with the use of native protein–protein interactions for steady epitope-specific evolutionary engineering.

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