Transient and moderate affinity protein-protein interactions (PPIs) play a critical role in the regulation of essential cellular processes including protein folding, ubiquitylation, and transcription. A number of disease states are believed to be the result of aberrations within these protein networks; therefore, a longstanding therapeutic goal has been to design small molecules that can tunably modulate the constituent interactions (1–8). However, the discovery of small molecule modulators has been hindered by lack of structural and mechanistic information, in part due to the limitations of the approaches currently available for studying transient PPIs in their native environments. Techniques such as co-crystallization and co-purification in vitro and two-hybrid studies in vivo are best suited for probing stably associated proteins, but are less ideal for studying proteins that engage in modest-affinity and/or transient multi-protein binding interactions (9–12). Here we demonstrate the in vivo covalent capture of such binding partners of the prototypical activator VP16, focusing on the chromatin-modifying coactivator complex Swi/Snf. Through these in vivo photocrosslinking experiments we find that one region of VP16 contacts the Snf2 ATPase and a second relies upon the Snf5 scaffolding component for Swi/Snf binding, suggesting a cooperative recruitment mechanism for this complex at individual promoters. A similar in vivo analysis of the mechanistically related activator Gal4 reveals Snf2 to be a shared target, suggesting that the ATPase may be a viable target for small molecule intervention in the expanding roster of disease states that exhibit mis-regulated Swi/Snf (13–15). The success of using a genetically encoded photo-activatable amino acid for characterizing activator-coactivator complexes in vivo indicates that this strategy can be implemented more broadly for the capture and discovery of transient protein-protein interactions in their native contexts.
Transcriptional activators are signal responsive regulatory proteins that assemble the transcriptional machinery at the promoter of a gene through dynamic binding interactions with a variety of coactivator complexes, including chromatin-modifying, helicase, and scaffolding complexes (19, 23). Activators are modular in architecture and are minimally composed of a DNA binding domain (DBD) that localizes the activator to its cognate DNA binding site and a transcriptional activation domain (TAD) that mediates the majority of contacts with transcriptional complexes. Although the interactions between activators and suppressor proteins can be high affinity and specific in nature, activator-coactivator interactions are mediated through lower affinity, transient contacts (Figure 1a) (16–21). In vivo co-localization studies have defined the complexes that are recruited by activators during transcription, but they have not readily provided information on the direct coactivator targets within these complexes (24–26). For example, the well-characterized amphipathic activator VP16 has been shown to recruit the Swi/Snf chromatin-remodeling complex early in transcription initiation, as evidenced by both in vivo and in vitro co-localization studies (27–32). In vitro assays have identified several subunits within this complex as possible targets of VP16 but in vivo interaction studies have not distinguished which of the components are the relevant binding partner(s) (17, 33, 34). Thus there is a clear need for in vivo methodologies that can capture transient activator-coactivator interactions in their native environment.
In vivo crosslinking with the genetically incorporated, photo-labile amino acid p-benzoyl-L-phenylalanine (Bpa) has been demonstrated previously as a useful method for capturing direct, high affinity protein-protein interactions (22, 35–37). More specifically, Bpa placement within the TAD of the activator Gal4 did not impair function of the protein and photoactivation lead to covalent capture of its high affinity (low nanomolar KD) suppressor protein Gal80 (22, 38). However, while successful in the case of a very tight interaction, this method has not been employed in the case of moderate-affinity transient interactions such as those between activators and coactivators. In this study, we test the utility of in vivo Bpa crosslinking for capturing VP16-coactivator interactions and for resolving the identity of the Swi/Snf components targeted by this activator.