Article

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

ACS Chemical Biology (Impact Factor: 5.36). 12/2011; 6(12):1321-6. DOI: 10.1021/cb200308e
Source: PubMed

ABSTRACT 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.

0 Followers
 · 
158 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: Translocation of proteins from the cytosol across the mitochondrial inner membrane is driven by action of the matrix localized multi-subunit import motor, which is associated with the TIM23 translocon. The architecture of the import apparatus is not well understood. Here we report results of site-specific in vivo photocrosslinking along with genetic and coimmunoprecipitation analyses dissecting interactions between import motor subunits and the translocon. The translocon is composed of the two integral membrane proteins Tim23 and Tim17, each containing four membrane spanning segments. We found that Tim23 having a photoactivatable crosslinker in the matrix exposed loop between transmembrane domains 1 and 2 (loop 1) crosslinked to Tim44. Alterations in this loop destabilized interaction of Tim44 with the translocon. Analogously, Tim17 having a photoactivatable crosslinker in the matrix exposed loop between transmembrane segments 1 and 2 (loop 1) crosslinked to Pam17. Alterations in this loop caused destabilization of the interaction of Pam17 with the translocon. Substitution of individual photactivatable residues in Tim44 and Pam17 in regions we previously identified as important for translocon association resulted in crosslinking to Tim23 and Tim17, respectively. Our results are consistent with a model in which motor association is achieved via interaction of Tim23 with Tim44, which serves as a scaffold for association of other motor components, and of Tim17 with Pam17. As both Tim44 and Pam17 have been implicated as regulatory subunits of the motor, this positioning is conducive for responding to conformational changes in the translocon upon a translocating polypeptide entering the channel.
    Journal of Biological Chemistry 08/2014; 289(41). DOI:10.1074/jbc.M114.588152 · 4.60 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    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
  • [Show abstract] [Hide abstract]
    ABSTRACT: Vaccinia H1-related (VHR) phosphatase is a dual specificity phosphatase that is required for cell-cycle progression and plays a role in cell growth of certain cancers. Therefore, it represents a potential drug target. VHR is structurally and biochemically well characterized, yet its regulatory principles are still poorly understood. Understanding its regulation is important, not only to comprehend VHR's biological mechanisms and roles but also to determine its potential and druggability as a target in cancer. Here, we investigated the functional role of the unique "variable insert" region in VHR by selectively introducing the photo-crosslinkable amino acid para-benzoylphenylalanine (pBPA) using the amber suppression method. This approach led to the discovery of VHR dimerization, which was further confirmed using traditional chemical crosslinkers. F68 in VHR was discovered as a residue involved in the dimerization. We demonstrate that VHR can dimerize inside cells, and that VHR catalytic activity is reduced upon dimerization. Our results suggest that dimerization could occlude the active site of VHR, thereby blocking its accessibility to substrates. These findings indicate that the previously unknown transient self-association of VHR acts as a means for the negative regulation of its catalytic activity.
    ACS Chemical Biology 05/2014; 9(7). DOI:10.1021/cb500240n · 5.36 Impact Factor

Preview

Download
2 Downloads
Available from