The crystal structure of a partial mouse Notch-1 ankyrin domain: repeats 4 through 7 preserve an ankyrin fold

Abstract

Folding and stability of proteins containing ankyrin repeats (ARs) is of great interest because they mediate numerous protein-protein interactions involved in a wide range of regulatory cellular processes. Notch, an ankyrin domain containing protein, signals by converting a transcriptional repression complex into an activation complex. The Notch ANK domain is essential for Notch function and contains seven ARs. Here, we present the 2.2 A crystal structure of ARs 4-7 from mouse Notch 1 (m1ANK). These C-terminal repeats were resistant to degradation during crystallization, and their secondary and tertiary structures are maintained in the absence of repeats 1-3. The crystallized fragment adopts a typical ankyrin fold including the poorly conserved seventh AR, as seen in the Drosophila Notch ANK domain (dANK). The structural preservation and stability of the C-terminal repeats shed a new light onto the mechanism of hetero-oligomeric assembly during Notch-mediated transcriptional activation.

Figure 1.

(A) Simplified schematic diagram of the Notch-mediated transcriptional “switch.” NICD is composed of a membrane proximal RAM domain (yellow), seven ARs (red), and C-terminal PEST, OPA domains (navy). Binding of NICD to CSL displaces transcriptional repressors (SMRT, HDAC, CIR) and leads to recruitment of transcriptional activators MAM and HAT. (B) A 2.2 Å crystal structure of mouse Notch1 ARs 3β–7. There are two molecules in the asymmetric unit of the crystal. Repeats 3β–7 of molecule A are colored red, yellow, green, brown, and cyan, respectively. Repeats of molecule B are colored gray. (C) Structural overlay of partial ANK domain of mouse Notch-1 with ARs is color coded as in B and the dANK domain of the Drosophila Notch receptor is shown in a gray transparent worm representation.

Figure 2.

SDS PAGE analysis of RAMANK during purification and crystallization. (A) Four-step chromatography purification of the RAMANK protein: lane 1, Talon column; lane 2, Q Sepharose; lane 3, gel filtration; lane 4, TEV cleavage; lane 5, second Talon column. (B) Samples taken from crystallization drop on the second day of crystallization (lane 1), and after 7 d in the crystallization drop (lane 2). Dissolved crystals contained only a 14.4 kDa band (lane 3).

Figure 3.

Sequence conservation among four Notch homologs in the mouse. (A) Structure-based sequence alignment of ARs 2–7 of mouse Notch1, Notch2, Notch3, and Notch4. Conserved, semiconserved, and nonconserved residues are colored green, yellow, and white, respectively. Secondary structure elements corresponding to inner and outer helices of each AR are colored red and blue, respectively. The figure was generated using the program ALSCRIPT (Barton 1993). (B) Sequence conservation mapped onto the molecular surface of the mouse Notch-1 ANK domain. Repeats 2 and 3 were modeled from the crystal structure of the dANK domain (Zweifel et al. 2003). The color scheme is identical to A. Orientation of panel 1 is illustrated by a ribbon diagram of the individual AR where the tips of the β-hairpin loops and inner helices are looking at the reader. Panel 2 is a 180° rotation along the Y axis, representing the molecular surface of the outer helices of ARs. The figure was generated using the program GRASP (Nicholls et al. 1991).