Thermodynamic binding analysis of Notch transcription complexes from Drosophila melanogaster

Abstract

Notch is an intercellular signaling pathway that is highly conserved in metazoans and is essential for proper cellular specification during development and in the adult organism. Misregulated Notch signaling underlies or contributes to the pathogenesis of many human diseases, most notably cancer. Signaling through the Notch pathway ultimately results in changes in gene expression, which is regulated by the transcription factor CSL. Upon pathway activation, CSL forms a ternary complex with the intracellular domain of the Notch receptor (NICD) and the transcriptional coactivator Mastermind (MAM) that activates transcription from Notch target genes. While detailed in vitro studies have been conducted with mammalian and worm orthologous proteins, less is known regarding the molecular details of the Notch ternary complex in Drosophila. Here we thermodynamically characterize the assembly of the fly ternary complex using isothermal titration calorimetry. Our data reveal striking differences in the way the RAM (RBP-J associated molecule) and ANK (ankyrin) domains of NICD interact with CSL that is specific to the fly. Additional analysis using cross-species experiments suggest that these differences are primarily due to fly CSL, while experiments using point mutants show that the interface between fly CSL and ANK is likely similar to the mammalian and worm interface. Finally, we show that the binding of the fly RAM domain to CSL does not affect interactions of the corepressor Hairless with CSL. Taken together, our data suggest species-specific differences in ternary complex assembly that may be significant in understanding how CSL regulates transcription in different organisms.

Keywords: CSL; RBP-J; X-ray crystallography; isothermal titration calorimetry; notch signaling; protein−protein interactions; transcription.

Figure 1

Overview of CSL-mediated transcription regulation. A: Model of CSL functioning as a transcriptional switch. Left, pathway inactivity allows corepressors (CoR, magenta) to interact with CSL present on DNA in the regulatory regions of target genes, and thereby repress gene transcription. Right, when the pathway is active, the corepressor complex is exchanged for two coactivators, Notch intracellular domain (NICD, red and yellow) and Mastermind (Mam, gray) to activate transcription from Notch target genes. B: Ribbon diagram (left) and domain schematics (right) of the CSL-NICD-MAM ternary complex bound to DNA. Coloring is consistent in both images. CSL consists of three domains—NTD (cyan), BTD (green), and CTD (orange). A beta-strand that bridges all three domains of CSL is colored magenta. The NTD and BTD of CSL make contacts with the DNA (gray). The RAM domain (red) of NICD interacts solely with the BTD of CSL while the ANK domain (yellow) interacts with both the NTD and CTD of CSL. Mastermind (gray) binds as a long helix across a composite surface created by the ANK domain bound to the NTD and CTD of CSL. C: Model of ternary complex assembly. According to this model, the RAM domain (red) of NICD binds to the BTD of CSL (green) in a high affinity interaction. The ANK domain (yellow) of NICD interacts very weakly with CSL until the second coactivator, MAM (gray), is present, locking the complex into an active conformation.

Figure 2

Thermodynamic binding analysis of Notch proteins from Drosophila. Figure shows representative thermograms (raw heat signal and nonlinear least squares fit to the integrated data) for Su(H) binding Drosophila NICD. Each experiment was performed at 25°C, with 40 titrations of 7 μL injections spaced 120 s apart. The experimentally determined dissociation constant (K_d) is shown for each experiment. A: Su(H) binding dRAMANK. B: Su(H) binding dANK. C: Su(H) binding dRAM. D: CTD of Su(H) binding dANK.

Figure 3

Cross-species binding experiments (RBP-J + dNICD). Figure shows representative thermograms (raw heat signal and nonlinear least squares fit to the integrated data) for RBP-J binding Drosophila NICD. Each experiment was performed at 25°C, with 40 titrations of 7 μL injections spaced 120 s apart. The experimentally determined dissociation constant (K_d) is shown for each experiment. A: RBP-J binding dRAMANK. B: RBP-J binding dRAM. C: RBP-J does not bind dANK. D: CTD of RBP-J does not bind dANK. NBD, no binding detected.

Figure 4

Cross-species binding experiments (Su(H) + mNICD). Figure shows representative thermograms (raw heat signal and nonlinear least squares fit to the integrated data) for Su(H) binding mouse NICD. Each experiment was performed at 25°C, with forty titrations of 7 μL injections spaced 120 s apart. The experimentally determined dissociation constant (K_d) is shown for each experiment. A: Su(H) binding mRAMANK. B: Su(H) binding mRAM. C: Su(H) does not bind mANK. D: CTD of Su(H) does not bind mANK. NBD, no binding detected.

Figure 5

Characterizing the effect RAM has on Su(H)-Hairless interactions. Figure shows representative EMSAs in which ANK + MAM (A) or RAM + ANK + MAM (B) compete for binding to the preformed Su(H)-Hairless-DNA complex. The control lanes (1−5) for both EMSAs contain Su(H)-DNA, Su(H)-dRAMANK-DNA, Su(H)-dRAMANK-MAM-DNA, Su(H)-Hairless-DNA, and Su(H)-dANK-MAM-DNA, respectively. ANK was added in increasing amounts (Lanes 6−10) either with (B) or without (A) RAM. In both cases, ANK or RAM + ANK compete poorly for the Su(H)-Hairless complex.

Figure 6

Revised model of ternary complex assembly for Drosophila Notch proteins. In contrast to the mammalian and worm Notch proteins, our binding data suggest that the binding of Drosophila NICD to Su(H) is partitioned between its RAM and ANK domains, such that ANK has appreciable interactions with the CTD of Su(H) in the absence of MAM.