Multipronged ESI–MS Approach for Studying Glycan-Binding Protein Interactions with Glycoproteins

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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Reconstitution of receptor/GTP-binding protein interactions

Biochimica et Biophysica Acta (BBA) - Reviews on Biomembranes ◽

10.1016/0304-4157(91)90007-j ◽

1991 ◽

Vol 1071 (4) ◽

pp. 473-501 ◽

Cited By ~ 19

Author(s):

Richard A. Cerione

Keyword(s):

Protein Interactions ◽

Binding Protein ◽

Gtp Binding Protein ◽

Gtp Binding

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p300/cAMP-responsive Element-binding Protein Interactions with Ets-1 and Ets-2 in the Transcriptional Activation of the Human Stromelysin Promoter

Journal of Biological Chemistry ◽

10.1074/jbc.274.24.17342 ◽

1999 ◽

Vol 274 (24) ◽

pp. 17342-17352 ◽

Cited By ~ 101

Author(s):

Gopalswamy Jayaraman ◽

Rampalli Srinivas ◽

Catherine Duggan ◽

Elisabeth Ferreira ◽

Sathyamangalam Swaminathan ◽

...

Keyword(s):

Protein Interactions ◽

Transcriptional Activation ◽

Binding Protein ◽

Element Binding Protein ◽

Responsive Element ◽

Camp Responsive Element Binding

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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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Do electrospray mass spectra reflect the ligand binding state of proteins in solution?

Canadian Journal of Chemistry ◽

10.1139/v05-194 ◽

2005 ◽

Vol 83 (11) ◽

pp. 1953-1960 ◽

Cited By ~ 13

Author(s):

Belal M Hossain ◽

Douglas A Simmons ◽

Lars Konermann

Keyword(s):

Mass Spectrometry ◽

Gas Phase ◽

Protein Interactions ◽

Noncovalent Complex ◽

Deuterium Exchange ◽

Solution Phase ◽

Hydrogen Deuterium Exchange ◽

Esi Ms ◽

Protein Ions ◽

Hydrogen Deuterium

Electrospray ionization (ESI) mass spectrometry (MS) has become a popular tool for monitoring ligandprotein and proteinprotein interactions. Due to the "gentle" nature of the ionization process, it is often possible to transfer weakly bound complexes into the gas phase, thus making them amenable to MS detection. One problem with this technique is the potential occurrence of fragmentation events during ESI. Also, some analytes tend to cluster together during ionization, thus forming nonspecific gas-phase assemblies that do not represent solution-phase complexes. In this work, we implemented a hydrogendeuterium exchange (HDX) approach that can reveal whether or not the free and (or) bound constituents of a complex observed in ESI-MS reflect the binding situation in solution. Proteins are subjected to ESI immediately following an isotopic labeling pulse; only ligand-free and ligand-bound protein ions that were formed directly from the corresponding solution-phase species showed different HDX levels. Using myoglobin as a model system, it is demonstrated that this approach can readily distinguish scenarios where the hemeprotein interactions were disrupted in solution from those where dissociation of the complex occurred in the gas phase. Experiments on cytochrome c strongly suggest that dimeric protein ions observed in ESI-MS reflect aggregates that were formed in solution.Key words: electrospray mass spectrometry, ligandprotein interaction, noncovalent complex, hydrogendeuterium exchange, protein folding.

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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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