Conformational locking upon cooperative assembly of notch transcription complexes

Abstract

The Notch intracellular domain (NICD) forms a transcriptional activation complex with the DNA-binding factor CSL and a transcriptional co-activator of the Mastermind family (MAML). The "RAM" region of NICD recruits Notch to CSL, facilitating the binding of MAML at the interface between the ankyrin (ANK) repeat domain of NICD and CSL. Here, we report the X-ray structure of a human MAML1/RAM/ANK/CSL/DNA complex, and probe changes in component dynamics upon stepwise assembly of a MAML1/NICD/CSL complex using HX-MS. Association of CSL with NICD exerts remarkably little effect on the exchange kinetics of the ANK domain, whereas MAML1 binding greatly retards the exchange kinetics of ANK repeats 2-3. These exchange patterns identify critical features contributing to the cooperative assembly of Notch transcription complexes (NTCs), highlight the importance of MAML recruitment in rigidifying the ANK domain and stabilizing its interface with CSL, and rationalize the requirement for MAML1 in driving cooperative dimerization of NTCs on paired-site DNA.

Figure 1. Components of nuclear Notch transcription complexes

(A) Top: domain organization of intracellular Notch1 (NICD), denoting the RAM region, ANK repeats, a transcriptional activation domain (TAD), and PEST sequence implicated in receptor degradation. Middle: domain organization of CSL, denoting the N-terminal domain (NTD), beta-trefoil domain (BTD), and C-temrinal domain (CTD). Bottom: organization of MAML1, identifying the N-terminal polypeptide required for assembly of NTCs (red), a region implicated in binding p300 (purple), and the C-terminal portion implicated in recruitment of Cyclin C:cdk8 complexes (white). Domains of the proteins utilized in these studies are colored blue (Notch1), yellow (CSL), and red (MAML1). (B) Schematic of the core components of the human NTC, illustrating the Notch ANK and RAM regions (blue), the MAML1 helix (red), and the three domains of CSL (yellow) on DNA (black cylinder).

Figure 2. supported by Figure S1 and Table S1. X-ray crystal structure of a human NTC core containing MAML1, RAM, ANK, CSL, and cognate DNA

(A) The human NTC structure showing the ANK repeats in blue, the CSL protein in yellow, the MAML1 polypeptide in red, DNA in orange, and the RAM polypeptide in cyan sticks. (B) Comparison of the human RAM-CSL interface (right) with that seen in the X-ray structure of the worm NTC (pink, left); RAM peptides: sticks; BTD: transparent surface over cartoon representation. An alignment of the RAM residues observed in the two structures is shown below the structures. Residues in red occupy analogous positions in the two complexes.

Figure 3. supported by Figures S2 and S3, and Tables S2 and S3. Global Hydrogen Exchange Profiles

Time course of exchange, comparing deuteration of RAMANK (A), CSL (B), and MAML1 (C) as isolated proteins, and in RAMANK-CSL (A, B) and RAMANK-CSL-MAML1 (A–C) complexes. The relative difference in deuteration upon formation of RAMANK-CSL and RAMANK-CSL-MAML1 complexes, compared to isolated RAMANK (A) and CSL (B) proteins, is also plotted in the right-hand panels. (D) Percent exchange at the 10-second time point for various combinations of MAML1 with RAMANK and CSL. Error bars represent standard deviations from three or more replicates. Error bars for the left-hand panels in (A–C) are frequently smaller than the peaks themselves.

Figure 4. supported by Figures S2 and S3A. Local Exchange Profile of MAML1

(A) Cartoon representation of the MAML-1 polypeptide (red), with contacting residues from CSL (left) and ANK (center) illustrated in orange and purple mesh, respectively. The right panel shows illustrates the contact interface in the context of the full NTC. (B) Time course of exchange, comparing deuterium uptake of the MAML1 polypeptide in isolation with that observed in the CSL-RAMANK-MAML1 complex. The mass difference between MAML1 peptides (uncomplexed minus complexed) is mapped onto a cartoon representation of its conformation in the full complex, colored on a siding scale from maroon (>6 Da) to dark blue (no difference). The black bar to the left denotes the sequence of the MAML1 polypeptide, with residue numbers and lines indicating the boundaries of specific peptides mapped onto the structure.

Figure 5. supported by Figures S2, S3B, S4, and S5. Local Exchange Profile of RAMANK

(A) Cartoon representation of the ANK domain of Notch1 (blue), with contacting residues from CSL and MAML1 illustrated in green and purple mesh, respectively. (B) Interface of the RAM peptide (sticks and green mesh) with the BTD of CSL (yellow cartoon trace). (C–K) Semilogarithmic plots showing the time course of exchange, comparing deuteration of peptides from RAMANK alone (red squares), RAMANK in complex with CSL (green circles), and RAMANK in complex with CSL and MAML1 (inverted blue triangles). The region of RAMANK represented by each peptide in panels C–K is indicated by the blue shading in the cartoon at the upper right corner of the plot. Error bars represent the range derived from analysis of duplicate measurements.

Figure 6. supported by Figures S2, S3C, S5, and S6. Local Exchange Profile of CSL

(A) Cartoon representation of CSL (yellow), with contacting residues from RAMANK and MAML1 illustrated in green and brown mesh, respectively. (B) Cartoon representation of the BTD interacting with the RAM peptide (sticks). BTD residues contacting RAM are illustrated in green mesh. (C–E) Semilogarithmic plots of exchange as a function of time for BTD peptides in the interface with RAM (C), NTD peptides in contact with MAML1 (D), and CTD peptides in the interface with ANK and MAML1 (E). Plots compare extent of deuteration for CSL alone (red squares), CSL when complexed with RAMANK (green circles), and CSL in complex with RAMANK and MAML1 (inverted blue triangles). Error bars represent the range derived from analysis of duplicate measurements.