Caught in the Act: Covalent Cross-Linking Captures Activator-Coactivator Interactions in Vivo

ACS Chemical Biology (Impact Factor: 5.33). 12/2011; 6(12):1321-6. DOI: 10.1021/cb200308e
Source: PubMed


Currently there are few methods suitable for the discovery and characterization of transient, moderate affinity protein-protein interactions in their native environment, despite their prominent role in a host of cellular functions including protein folding, signal transduction, and transcriptional activation. Here we demonstrate that a genetically encoded photoactivatable amino acid, p-benzoyl-l-phenylalanine, can be used to capture transient and/or low affinity binding partners in an in vivo setting. In this study, we focused on ensnaring the coactivator binding partners of the transcriptional activator VP16 in S. cerevisiae. The interactions between transcriptional activators and coactivators in eukaryotes are moderate in affinity and short-lived, and due in part to these characteristics, identification of the direct binding partners of activators in vivo has met with only limited success. We find through in vivo photo-cross-linking that VP16 contacts the Swi/Snf chromatin-remodeling complex through the ATPase Snf2(BRG1/BRM) and the subunit Snf5 with two distinct regions of the activation domain. An analogous experiment with Gal4 reveals that Snf2 is also a target of this activator. These results suggest that Snf2 may be a valuable target for small molecule probe discovery given the prominent role the Swi/Snf complex family plays in development and in disease. More significantly, the successful implementation of the in vivo cross-linking methodology in this setting demonstrates that it can be applied to the discovery and characterization of a broad range of transient and/or modest affinity protein-protein interactions.

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    • "Among dozens of different non-canonical amino acids, there are basically three different types of photoreactive groups, including azido-, diazirin- and benzoyl-groups, which can be incorporated into proteins using the expanded genetic code [14], [15]. For photo-cross-link experiments the non-canonical amino acid pBzpa is widely used to covalently capture the interaction of two proteins [13], [16], [17], [18], [19], [20], [21], [22]. The reason for this is that photolysis of the keto group within the benzophenone can be induced with a relative long wavelength (350–365 nm). "
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    ABSTRACT: Protein-protein interactions play an essential role in almost any biological processes. Therefore, there is a particular need for methods which describe the interactions of a defined target protein in its physiological context. Here we report a method to photo-cross-link interacting proteins in S. cerevisiae by using the non-canonical amino acid p-azido-L-phenylalanine (pAzpa). Based on the expanded genetic code the photoreactive non-canonical amino acid pAzpa was site-specifically incorporated at eight positions into a domain of Aha1 that was previously described to bind Hsp90 in vitro to function as a cochaperone of Hsp90 and activates its ATPase activity. In vivo photo-cross-linking to the cognate binding partner of Aha1 was carried out by irradiation of mutant strains with UV light (365 nm) to induce covalent intermolecular bonds. Surprisingly, an interaction between Aha1 and Hsp90 was not detected, although, we could confirm binding of suppressed pAzpa containing Aha1 to Hsp90 by native co-immunoprecipitation. However, a homodimer consisting of two covalently crosslinked Aha1 monomers was identified by mass spectrometry. This homodimer could also be confirmed using p-benzoyl-L-phenylalanine, another photoreactive non-canonical amino acid. Crosslinking was highly specific as it was dependent on irradiation using UV light, the exact position of the non-canonical amino acid in the protein sequence as well as on the addition of the non-canonical amino acid to the growth medium. Therefore it seems possible that an interaction of Aha1 with Hsp90 takes place at different positions than previously described in vitro highlighting the importance of in vivo techniques to study protein-protein interactions. Accordingly, the expanded genetic code can easily be applied to other S. cerevisiae proteins to study their interaction under physiological relevant conditions in vivo.
    PLoS ONE 03/2014; 9(3):e89436. DOI:10.1371/journal.pone.0089436 · 3.23 Impact Factor
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    • "This is of particular interest, since these interactions often are transient and have only moderate affinity, which makes them difficult targets for interaction studies (Melcher, 2000; Mapp and Ansari, 2007; Fuxreiter et al., 2008). Following proof-of-principle experiments to detect and characterize the interaction between Gal4 and its (high affinity) suppressor Gal80 in S. cerevisiae (Majmudar et al., 2009a), a series of studies was dedicated to uncover individual binding sites of several transcriptional activators with the co-activator Med15 and the chromatin-modifying coactivator complex Swi/Snf2 (Majmudar et al., 2009b; Krishnamurthy et al., 2011). Swi/Snf2 is a multimeric complex that uses a DNA-stimulated ATPase activity for nucleosome remodeling and plays important roles in tumor suppression (Wilson and Roberts, 2011). "
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    ABSTRACT: The expansion of the genetic code with non-canonical amino acids (ncAA) enables the chemical and biophysical properties of proteins to be tailored, inside cells, with a previously unattainable level of precision. A wide range of ncAA with functions not found in canonical amino acids have been genetically encoded in recent years and have delivered insights into biological processes that would be difficult to access with traditional approaches of molecular biology. A major field for the development and application of novel ncAA-functions has been transcription and its regulation. This is particularly attractive, since advanced DNA sequencing- and proteomics-techniques continue to deliver vast information on these processes on a global level, but complementing methodologies to study them on a detailed, molecular level and in living cells have been comparably scarce. In a growing number of studies, genetic code expansion has now been applied to precisely control the chemical properties of transcription factors, RNA polymerases and histones, and this has enabled new insights into their interactions, conformational changes, cellular localizations and the functional roles of posttranslational modifications.
    Frontiers in Chemistry 02/2014; 2:7. DOI:10.3389/fchem.2014.00007
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    ABSTRACT: Iron is an essential nutrient for the proliferation of Staphylococcus aureus during bacterial infections. The iron-regulated surface determinant (Isd) system of S. aureus transports and metabolizes iron porphyrin (heme) captured from the host organism. Transportation of heme across the thick cell wall of this bacterium requires multiple relay points. The mechanism by which heme is physically transferred between Isd transporters is largely unknown because of the transient nature of the interactions involved. Herein, we show that the IsdC transporter not only passes heme ligand to another class of Isd transporter, as previously known, but can also perform self-transfer reactions. IsdA shows a similar ability. A genetically encoded photoreactive probe was used to survey the regions of IsdC involved in self-dimerization. We propose an updated model that explicitly considers self-transfer reactions to explain heme delivery across the cell wall. An analogous photo-cross-linking strategy was employed to map transient interactions between IsdC and IsdE transporters. These experiments identified a key structural element involved in the rapid and specific transfer of heme from IsdC to IsdE. The resulting structural model was validated with a chimeric version of the homologous transporter IsdA. Overall, our results show that the ultra-weak interactions between Isd transporters are governed by bona fide protein structural motifs.
    Journal of Biological Chemistry 03/2012; 287(20):16477-87. DOI:10.1074/jbc.M112.346700 · 4.57 Impact Factor
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