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. 2011 Dec 16;6(12):1321-6.
doi: 10.1021/cb200308e. Epub 2011 Nov 14.

Caught in the act: covalent cross-linking captures activator-coactivator interactions in vivo

Malathy Krishnamurthy, Amanda Dugan, Adaora Nwokoye, Yik-Hong Fung, Jody K Lancia, Chinmay Y Majmudar, Anna K Mapp

Caught in the act: covalent cross-linking captures activator-coactivator interactions in vivo

Malathy Krishnamurthy et al. ACS Chem Biol. .

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.

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Figures

Figure 1
Figure 1
(a) The transcriptional activation domain (TAD) of amphipathic activators can engage in high affinity interactions, such as those with masking proteins (mp), but the interactions between the TAD and coactivator complexes are more moderate in affinity and transient in nature (–21). (b) Amphipathic activators share little sequence homology but do share binding targets, at least in vitro. The photocrosslinking amino acid, Bpa, has been incorporated within the Gal4 TAD (positions of incorporation in red) with little impact on the function and binding profile of that TAD (22).
Figure 2
Figure 2
Incorporation of Bpa within the VP16 TAD. (a) Plasmids encoding the DNA binding domain (DBD) of LexA fused to either the N- or C-terminal VP16 TAD as well as a FLAG tag were constructed. The LexA DBD was utilized to exclusively examine transcriptional activation at the 2 unique LexA binding sites upstream of the LacZ reporter in S. cerevisiae. Positions at which Bpa mutagenesis was carried out are within regions of the VP16N or VP16C subdomains known to participate in coactivator binding (sites of incorporation highlighted in red). The loading control is an approximately 71 kDa, FLAG-detected yeast protein whose expression level does not vary with activator identity.(22) (b) Yeast cells bearing plasmids encoding the various LexA+VP16 constructs and the Bpa specific tRNA/synthetase pair expressed by pSNRtRNA-pBpaRS were grown in the presence or absence of 1 mM Bpa and analyzed by Western blot (c) LexA+VP16N L444Bpa and LexA+VP16C F475Bpa were assessed for their ability to upregulate transcription of an integrated LacZ reporter gene in S. cerevisiae as measured by liquid β-galactosidase assays. Each activity is the average of values from at least three independent experiments with the indicated error (SDOM). See Supporting Information Figure S7 for activity assays of the remaining mutants.
Figure 3
Figure 3
In vivo photocrosslinking captures the moderate affinity interaction between LexA+VP16 and the Mediator protein, Med15. a) VP16 has been shown to interact transiently with the coactivator Med15, as determined by measured kinetic rate constants. Equilibrium binding measurements place the affinity of the TAD for Med15 in the moderate category, with DNA-bound homodimers exhibiting the highest affinity (0.1 μM) and isolated TADs in the low to mid-micromolar range (16, 39). (b) Live yeast cells bearing plasmids expressing LexA+VP16N L444Bpa or LexA+VP16C F475Bpa fusion proteins, in addition to a plasmid expressing myc-Med15(1–416) were irradiated with UV light (365 nm) for 30 minutes. Subsequently, cell lysates were immunoprecipitated with αLexA and analyzed by Western blot (αmyc). For both constructs, a crosslink with Med15 is observed. Supporting Figure S1 shows expression of myc-Med15(1–416) and Supporting Figure S2 shows the full Western blot, complete with molecular weight references.
Figure 4
Figure 4
Analysis of VP16 crosslinking to the Swi/Snf coactivators, Snf2, Swi1 and Snf5. (a) The recruitment of the Swi/Snf chromatin remodeling complex by VP16 has been proposed to occur through interactions with the Snf2, Swi1 and Snf5 subunits although the direct binding partners in vivo have not been determined (17, 33, 34). (b) Live yeast cells expressing LexA+VP16C F475Bpa were irradiated with 365 nm light (30 minutes) and subsequently the cell lysates were immunoprecipitated with an antibody to Snf2 and resolved by Western blot (αFLAG), revealing a direct interaction between VP16C and endogenous Snf2. In line with previous biochemical experiments, when phenylalanine 479 in VP16C was mutated to either alanine or proline, crosslinking to Snf2 was abolished. (c,d) LexA+VP16C F475Bpa and LexA+VP16N L444Bpa were expressed in yeast strains lacking either Swi1 or Snf5 and the live yeast cells were irradiated with 365 nm light. Subsequently, cell lysates were immunoprecipitated (αLexA) and resolved by Western blot (αFLAG). In the individual blots for LexA+VP16N, the marks a and b denote crosslinked protein bands at the appropriate size for Swi1 and Snf5, respectively. In the individual blots for LexA+VP16C, the marks c, d, and e indicate bands at the appropriate size for Snf2, Swi1 and Snf5, respectively. (e) To test if Gal4 also contacts Snf2, crosslinking experiments were carried out with live yeast cells expressing LexA+Gal4 F867Bpa as in (b). The full Western blot of b) and e) can be found in Supplemental Figures S4 and S6, respectively.
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