Structural basis of the heterodimerization of the MST and RASSF SARAH domains in the Hippo signalling pathway

Abstract

Despite recent progress in research on the Hippo signalling pathway, the structural information available in this area is extremely limited. Intriguingly, the homodimeric and heterodimeric interactions of mammalian sterile 20-like (MST) kinases through the so-called `SARAH' (SAV/RASSF/HPO) domains play a critical role in cellular homeostasis, dictating the fate of the cell regarding cell proliferation or apoptosis. To understand the mechanism of the heterodimerization of SARAH domains, the three-dimensional structures of an MST1-RASSF5 SARAH heterodimer and an MST2 SARAH homodimer were determined by X-ray crystallography and were analysed together with that previously determined for the MST1 SARAH homodimer. While the structure of the MST2 homodimer resembled that of the MST1 homodimer, the MST1-RASSF5 heterodimer showed distinct structural features. Firstly, the six N-terminal residues (Asp432-Lys437), which correspond to the short N-terminal 3₁₀-helix h1 kinked from the h2 helix in the MST1 homodimer, were disordered. Furthermore, the MST1 SARAH domain in the MST1-RASSF5 complex showed a longer helical structure (Ser438-Lys480) than that in the MST1 homodimer (Val441-Lys480). Moreover, extensive polar and nonpolar contacts in the MST1-RASSF5 SARAH domain were identified which strengthen the interactions in the heterodimer in comparison to the interactions in the homodimer. Denaturation experiments performed using urea also indicated that the MST-RASSF heterodimers are substantially more stable than the MST homodimers. These findings provide structural insights into the role of the MST1-RASSF5 SARAH domain in apoptosis signalling.

Keywords: Hippo signalling pathway; MST; RASSF; SARAH domains.

Figure 1

Sequence comparison of the SARAH domains of hMST1, hMST2, hWW45, hRASSF5, hRASSF1, dHPO, dSAV and dRASSF. Colours represent the degree of homology. Red blocks denote regions of sequence identity across all homologues. Partially conserved residues are shown in orange to blue accordingly. For all domain subfamilies, members from human (h) and Drosophila (d) are shown.

Figure 2

Overall structures of the SARAH dimers. Structures of the MST1–RASSF5 SARAH heterodimer (a, d), the MST2 SARAH homodimer (b, e) and the MST1 SARAH homodimer (c, f) are shown as ribbon diagrams and surface representations, respectively. For ease of comparison, the orientations shown in all panels are identical. For the MST1–RASSF5 SARAH heterodimer (a, d), the blue ribbon represents the backbone structure of the MST1 SARAH domain and the pink ribbon represents that of the RASSF5 SARAH domain. (g) Comparison of the monomer structures of the MST1 SARAH domain in the homodimer (light green) and in the heterodimer (blue). Note that the short h1 helix of the MST1 SARAH domain is missing and the length of the MST1 h2 helix is extended in the MST1–RASSF5 heterodimer.

Figure 3

Comparison of the dimeric interactions of SARAH domains based on computational alanine scanning. (a, b, c) Ribbon representations depicting the side chains of residues having dimeric interactions derived from the computational alanine scanning of SARAH dimeric interfaces are shown for the MST1–RASSF5 SARAH heterodimer (a), the MST2 SARAH homodimer (b) and the MST1 SARAH homodimer (c). Residues with ΔΔG _bind > 1.0 kcal mol⁻¹ in computational alanine scanning are represented as stick models. Among the residues, Trp369, Ile374 and Glu387 of RASSF5 and Phe437 and Leu440 of MST2 are not seen in the figure and are not labelled for clarity. Residues that have polar interactions in the dimeric interface are shown in green. Red balls represent the water molecules mediating the hydrogen bonds between the two protomers. For the MST1–RASSF5 SARAH heterodimer (a), the light blue ribbon represents the backbone structure of the MST1 SARAH domain and the light pink ribbon represents that of the RASSF5 SARAH domain. (d, e) Ribbon representations with the side chains of the hydrophobic core formed by the N-terminal helix–turn–helix region are shown for the MST1 SARAH homodimer (d) and the MST1–RASFF5 SARAH heterodimer (e). Residues with ΔΔG _bind > 1.0 kcal mol⁻¹ in computational alanine scanning are shown as yellow sticks. Red sticks represent the side chains that interact with the aromatic residues in the hydrophobic core. Line models represent residues involved in the inter-protomer hydrogen bonding with smaller values of ΔΔG _bind than 1.0 kcal mol⁻¹.

Figure 4

Comparison of the urea-induced denaturation curves of SARAH domains. The secondary-structural changes in the SARAH domains upon increases in the urea concentration were observed by monitoring the molar ellipticity [θ] at 222 nm in far-UV circular dichroism (CD) for the MST1–RASSF5 SARAH heterodimer (a), the MST1 SARAH homodimer (b), the MST2–RASSF5 SARAH heterodimer (c), the MST2 SARAH homodimer (d) and a double-mutant MST1–RASSF5 (E388A and K398A of RASSF5) SARAH heterodimer (e). Dotted lines indicate the transition concentration (C _m) of urea for the denaturation of α-helical structures.

Figure 5

Schematic model showing how the MST1 kinase domain gains motional freedom by heterodimerization with RASSF5 in the membrane environment. The MST1 SARAH promoter undergoes structural change when it binds to the RASSF5 SARAH domain to form a heterodimer. By extension of the h2 helix and unfolding of the h1 helix, the MST1 SARAH domain in the heterodimer can provide motional freedom to the MST1 catalytic domain. The grey ribbon represents the MST1 SARAH domain in the homodimer and the blue ribbon denotes the MST1 SARAH domain in the heterodimer with RASSF5. The magenta ribbon represents the RASSF5 SARAH domain in the heterodimer with MST1. The blue horizontal bar represents a cellular membrane where the prenylated Ras protein anchors.