Crystal structure of Ski8p, a WD-repeat protein with dual roles in mRNA metabolism and meiotic recombination

Abstract

Ski8p is a WD-repeat protein with an essential role for the Ski complex assembly in an exosome-dependent 3'-to-5' mRNA decay. In addition, Ski8p is involved in meiotic recombination by interacting with Spo11p protein. We have determined the crystal structure of Ski8p from Saccharomyces cerevisiae at 2.2 A resolution. The structure reveals that Ski8p folds into a seven-bladed beta propeller. Mapping sequence conservation and hydrophobicities of amino acids on the molecular surface of Ski8p reveals a prominent site on the top surface of the beta propeller, which is most likely involved in mediating interactions of Ski8p with Ski3p and Spo11p. Mutagenesis combined with yeast two-hybrid and GST pull-down assays identified the top surface of the beta propeller as being required for Ski8p binding to Ski3p and Spo11p. The functional implications for Ski8p function in both mRNA decay and meiotic recombination are discussed.

Figure 1.

Overall structure of Ski8p. (A) Ribbon diagram of Ski8p showing the seven-bladed β propeller structure. Each blade consists of a four-stranded β-sheet (labeled A–D). The view is looking down the central axis of the propeller onto the “top” surface. The top surface is defined by the presence of the D–A loops connecting sequential blades. (B) View rotated 90° about the horizontal axis in A, looking at the propeller from the side. The top face and bottom face are marked according to the convention for WD-repeat protein structure. (C) Stereo view of a Cα trace of Ski8p with every tenth residue labeled and marked with a closed circle. Figures 1 ▶, 3 ▶, 4C ▶, and 5 ▶ were generated using MOLSCRIPT (Kraulis 1991).

Figure 2.

Sequence alignment of S. cerevisiae Ski8, S. macrospora Ski8, Schizosaccharmyces pombe Rec14, Homo sapiens Rec14, and Mus musculus Rec14. The secondary structures of S. cerevisiae Ski8 are shown. Invariant residues are white letters, similar residues are red, and others are black. Residues speculated for interactions with Ski3 and Spo11 are indicated by *.

Figure 3.

Comparison of Ski8p with Gβ and Tup1c. (A) Superposition of Ski8p, Gβ, and Tup1c. Ski8p is shown in red, Gβ in blue, and Tup1c in green. The view of Ski8p is as in Figure 1A ▶. (B) Same as A but with the view looking at the side of the propeller. (C) Superposition of all seven blades of Ski8p. The Cα backbone for each of the seven blades was aligned with respect to blade 1. Four strands of each blade are indicated by A, B, C, and D. Blade 1, green; blade 2, red; blade 3, yellow; blade 4, cyan; blade 5, blue; blade 6, maroon; blade 7, magenta. (D) Superposition of two typical blades, blade 1 (green) and blade 6 (maroon) containing the eponymous Trp–Asp motif. Four conserved residues, which are involved in the structural tetrad, are shown in stick models, and hydrogen bonds are indicated with dashed lines.

Figure 4.

Molecular surface views of Ski8p. (A) Surface representation of Ski8p showing the regions of high-to-low sequence conservation shared by the eukaryotic Ski8 proteins, corresponding to a color ramp from red to blue, respectively. Invariant residues are labeled. The view is as in Figure 1A ▶. (B) Molecular surface of Ski8p colored according to residue property, with hydrophobic residues green and other residues gray. The hydrophobic residues are labeled. The view is as in A. (C) The worm model showing the Cα backbones of Ski8p. Residues located either in hydrophobic patch or in conserved patch are shown in stick models. The view is as in A. A and B were produced using GRASP (Nicholls et al. 1991).

Figure 5.

Location of the protein–protein interactions site on the top face of the βpropeller. (A) Close-up view of the interface between the top face of the β propeller in Ski8p (yellow) and the loops of 2A–2B and 3C–3D from the symmetry related molecule (purple). Residues involved in the interface are shown in stick models. (B) Comparison of the protein–protein interactions on the top surface for three WD-repeat proteins. (Left) The top face of Ski8p (yellow) interacting with its symmetry related loop regions 3C–3D and 2A–2B (purple). (Middle) The top face of β-TrCP1 WD-40 domain (sky blue) with bound doubly phosphorylated β-catenin peptide (red). pdb code: 1p22. (Right) The top face of Gβ (light green) interacting with the helix of Gα (blue). pdb code: 1gp2. All residues involved in interaction on the top face of the β propeller are shown in CPK model.

Figure 6.

Mutational analysis of Ski8p mutants. (A) Effects of mutations at the top surface of the β propeller on the interactions of Ski8p with Ski3p and Spo11p, respectively. β-Galactosidase activity from various transformants estimated as described under Materials and Methods. The Ski8p-topmutant refers to a mutant Ski8p protein where seven residues (F20A, F89A, W125A, R237A, W293A, W311A, and F358A) were mutated to alanine. (Left) Ski8p versus Ski3p. (Right) Ski8p versus Spo11p. (B) Ski3p and Spo11p were translated in vitro in the presence of ³⁵S-methionine and examined for binding to the immobilized Ski8p variants. (C) Effects of mutations at the top surface of the β propeller on the interactions of Ski8 with Ski2p, Ski3p, respectively. Alanine substitutions for seven top residues of Ski8p (Ski8-topmutant) abolish the two-hybrid interaction between Ski3p and Ski8p, whereas such mutations have no effect on the interactions between Ski2p and Ski8p. Plus and minus signs indicate positive and negative interactions, respectively. F59A serves as negative control.