RAM-induced allostery facilitates assembly of a notch pathway active transcription complex

Abstract

The Notch pathway is a conserved cell-to-cell signaling mechanism, in which extracellular signals are transduced into transcriptional outputs through the nuclear effector CSL. CSL is converted from a repressor to an activator through the formation of the CSL-NotchIC-Mastermind ternary complex. The RAM (RBP-J associated molecule) domain of NotchIC avidly interacts with CSL; however, its role in assembly of the CSL-NotchIC-Mastermind ternary complex is not understood. Here we provide a comprehensive thermodynamic, structural, and biochemical analysis of the RAM-CSL interaction for components from both mouse and worm. Our binding data show that RAM and CSL form a high affinity complex in the presence or absence of DNA. Our structural studies reveal a striking distal conformational change in CSL upon RAM binding, which creates a docking site for Mastermind to bind to the complex. Finally, we show that the addition of a RAM peptide in trans facilitates formation of the CSL-NotchIC-Mastermind ternary complex in vitro.

FIGURE 1.

Overview of transcriptional regulation mediated by CSL. Top, prior to pathway activation, CSL engages transcriptional corepressors (CoR), which recruit multiprotein repressor complexes that function to silence Notch target gene transcription through the action of histone deacetylases (HDAC) (32). Right, upon pathway activation, nuclear NotchIC binds to CSL through its RAM (RBP-jk associated molecule) (12) and ANK domain (18), which is thought to displace corepressors from CSL (33, 34). Bottom, the subsequent binding of Mastermind to CSL-NotchIC renders the ternary complex poised to activate transcription (35, 36). The DNA bound CSL-NotchIC-Mastermind complex recruits general transcription factors such as PCAF/GCN5 and CBP/p300 (37, 38), which contain histone acetylase (HAT) domains, to up-regulate transcription of Notch target genes. Left, transcription is terminated by the degradation of NotchIC, which is mediated by an E3 ubiquitin ligase that recognizes the phosphorylated C-terminal PEST domain, leading to disassembly of the activation complex (39).

FIGURE 2.

Overview of previously determined CSL structures. Ribbon diagrams for the coregulator-free worm CSL-DNA complex (A) and the worm CSL-NotchIC-Mastermind ternary complex (B) are shown (8, 9). CSL domains NTD, BTD, and CTD are colored cyan, green, and orange, respectively. A β-strand that bridges all three domains is colored magenta. For the ternary complex in B, the NotchIC RAM domain (RAM) and ANK are colored red and yellow, respectively; Mastermind (MM) is colored gray. The DNA is represented as a stick model. C, domain schematics for core CSL, NotchIC, and Mastermind are colored according to ribbon diagrams. D, structural alignment of CSL from the worm and human ternary complex structures (9, 10). Cα backbone representation of worm CSL (2FO1) in tricolor (cyan, green, and orange) overlaid onto human CSL (2F8X) colored gray. Alignment was performed over the NTD, highlighting the interdomain differences in BTD and CTD between the two CSL structures. Worm CSL in the context of the ternary complex structure undergoes large interdomain movements that are not observed in the human CSL structure. Despite these interdomain differences, a loop structure within NTD (henceforth referred to as the NTD loop) is in a similar open conformation in both CSL structures. In the coregulator-free worm CSL-DNA structure, the NTD loop is in a closed conformation. The structure-function explanation for this conformational change is evident, because opening of the NTD loop removes steric hindrances that would otherwise block the C-terminal helix of Mastermind binding to CSL, preventing ternary complex formation. Also shown are the site of RAM (red) binding to the BTD and the location of the NTD loop.

FIGURE 3.

Structures of worm CSL-RAM-DNA and mouse CSL-DNA complexes. Ribbon diagrams corresponding to worm CSL-RAM-DNA (A) and mouse CSL-DNA (B) structures reported here are shown. The domain coloring for CSL is the same as in Fig. 1. The RAM domain in A is represented as a stick model colored by atom. C, Cα overlays of BTD-RAM interactions determined here with BTD-RAM interaction from worm ternary complex structure, highlighting high degree of correspondence. The BTD is colored green, and RAM is colored yellow with hydrophobic tetrapeptide residues (VWMP) displayed as sticks. D, molecular surface representation of BTD-RAM interaction with BTD surface colored according to electrostatics: red, negative; blue, positive; gray, nonpolar. RAM is in a stick representation colored by atom. RAM binds in an extended conformation across a large hydrophobic surface and an electronegative patch on BTD. RAM also forms a β-stranded structure with a large loop on BTD that is also implicated in corepressor binding. All of the nonpolar and polar RAM-BTD interactions as well as the β-structure formed with the BTD loop are maintained in the worm CSL-RAM complexes determined here.

FIGURE 4.

Comparison of CSL structures. The figure shows Cα overlays for mouse and worm CSL structures determined here against previously determined worm and human CSL structures, highlighting differences in domain dispositions and conformation of the NTD loop. CSL domains are colored as in Fig. 1. For comparison, a structurally equivalent Cα atom is denoted as a sphere in the NTD loops. For overlays performed in B and C, the alignment was done over the entire CSL molecule; for overlays in A and D, the alignment was done only over the NTD of CSL, because of the substantial interdomain movements of BTD and CTD about the NTD. A, overlay of the coregulator-free form of worm CSL (apo) in tricolor with worm CSL from the ternary complex (NIC+MM) colored gray, with RMSD greater than 2.4 Å for all Cα atoms; note the open and closed conformations of the NTD loop. B, overlay of coregulator-free mouse CSL (apo) in tricolor with human CSL from the ternary complex (NIC+MM) colored gray, with RMSD of ∼0.8 Å for corresponding Cα atoms; note the open and closed conformations of the NTD loop similar to worm structures in A. C, overlay of coregulator-free worm CSL (apo) in tricolor with worm CSL-RAM colored gray, with RMSD of less than 0.8 Å for corresponding Cα atoms; note the open and closed conformations of the NTD loop. D, overlay of worm CSL-RAM in tricolor with worm CSL from ternary complex (NIC+MM) colored gray, with RMSD greater than 2.4 Å for all Cα atoms; note the similar open conformations of the NTD loop.

FIGURE 5.

Analysis of NTD loop conformations. The figure shows detailed structural comparisons of the NTD loop from CSL proteins in the coregulator-free, complexed with RAM, and ternary complex forms represented in cross-eyed stereo pairs, and emphasizes the steric clash of Mastermind with the closed conformation of the NTD loop. The NTD of CSL and the C-terminal helix of Mastermind are depicted as ribbon diagrams and colored cyan and black, respectively. A, comparison of NTD loop conformations from the coregulator-free structure of mouse CSL (cyan loop) and human CSL from the ternary complex (gray loop). Residues Ile¹³¹-Gln¹³⁹ and Ile⁹¹-Gln⁹⁹ in the NTD loop for mouse and human CSL, respectively, are represented as sticks and colored by atom type. The side chains of Gln¹³⁶ and Leu⁵⁹ from mouse CSL and human Mastermind, respectively, are colored red to represent the putative steric clash between the NTD loop in the closed conformation with Mastermind. For B-D, worm CSL residues I292-Q300, which correspond to the NTD loop, are drawn as sticks and colored by atom. The side chains of Arg²⁹⁹ and Leu⁹⁹ from worm CSL and Mastermind, respectively, are colored red to indicate potential steric clashes. B, comparison of NTD loop conformations for coregulator-free structure of worm CSL (cyan loop) with worm CSL from the ternary complex (gray loop). C, comparison of NTD loop conformations for coregulator-free structure of worm CSL (cyan loop) with worm CSL from RAM complex (magenta loop). D, comparison of NTD loop conformations for worm CSL-RAM complex (magenta loop) with worm CSL from the ternary complex (gray loop).

FIGURE 6.

EMSA analysis of ternary complex assembly. The figure shows the contribution of RAM peptide to formation of the CSL-NotchIC-Mastermind ternary complex. Components of each binding reaction are denoted above each gel. EMSAs showing additional controls are included in the supplemental data (supplemental Fig. S4). A, assembly of worm ternary complex. The concentration of CSL is 1 μm, and the concentrations of all other components (RAMANK, RAM, ANK, Mastermind, and DNA) are 10 μm. For lanes 7-9, increasing concentrations of ANK 10, 20, and 30 μm, respectively, were utilized in the binding reaction. The worm ternary complex does not form without RAM (lanes 7-9); however, the addition of an exogenous RAM peptide allows for the ternary complex to form (lane 4). B, assembly of mouse ternary complex. The concentration of DNA is 1.5 μm; the concentrations of CSL, RAMANK, and ANK are 1.0 μm, except for lanes 7-9, in which the concentrations of ANK are 0, 2.5, and 5.0 μm, respectively. The concentration of Mastermind and RAM are 10 μm. The mouse ternary complex forms with or without RAM (lanes 4, 6, 8, and 9). C, efficiency of mouse ternary complex formation with and without exogenous RAM peptide. For all lanes, the concentration of CSL, Mastermind, RAM, and DNA are 1, 10, 10, and 1.5 μm, respectively. Increasing concentrations of ANK (0.025, 0.1, 0.5, and 2.0 μm) are included in lanes 2-5 and 7-10. The addition of the RAM peptide (lanes 2-5) increases the efficiency of ternary complex formation, as compared with ternary complex formation without RAM peptide (lanes 7-10). D, quantitation of mouse ternary complex formation. The data points were generated from integration of band intensities in C from three independent experiments. The bar graph shows the percentages of ternary complex formation (y axis) as a function of ANK concentration (x axis). A control peptide, consisting of a scrambled RAM sequence, had no effect on ternary complex formation (data not shown).

FIGURE 7.

Revised model of ternary complex assembly. The figure diagrams the sequence of events leading to formation of transcriptionally active ternary complex. CSL is drawn bound to DNA with all three functional domains, NTD (N), BTD (B), and CTD (C), which are colored cyan, green, and orange, respectively. The RAM and ANK domains of NotchIC are colored red and yellow, respectively. Mastermind (Mm) is colored gray. A, upon pathway activation, RAM binding to CSL both targets NotchIC to CSL and triggers an allosteric change in the NTD of CSL, which is denoted by a red asterisk. B, two possibilities exist: Mastermind (Mm) interacts with the complex to direct ANK binding to CSL (top) or ANK interacts with the CTD of CSL (bottom), creating the complete Mastermind docking site. The second scenario is more likely, because of the tethering of ANK to CSL through RAM, which would dramatically increase the local concentration of ANK (30). C, either case leads to formation of CSL-NotchIC-Mastermind ternary complexes, occupying sites at Notch target genes.