The APC/C subunit Cdc16/Cut9 is a contiguous tetratricopeptide repeat superhelix with a homo-dimer interface similar to Cdc27

Abstract

The anaphase-promoting complex/cyclosome (APC/C), an E3 ubiquitin ligase responsible for controlling cell cycle transitions, is a multisubunit complex assembled from 13 different proteins. Numerous APC/C subunits incorporate multiple copies of the tetratricopeptide repeat (TPR). Here, we report the crystal structure of Schizosaccharomyces pombe Cut9 (Cdc16/Apc6) in complex with Hcn1 (Cdc26), showing that Cdc16/Cut9 is a contiguous TPR superhelix of 14 TPR units. A C-terminal block of TPR motifs interacts with Hcn1, whereas an N-terminal TPR block mediates Cdc16/Cut9 self-association through a homotypic interface. This dimer interface is structurally related to the N-terminal dimerization domain of Cdc27, demonstrating that both Cdc16/Cut9 and Cdc27 form homo-dimers through a conserved mechanism. The acetylated N-terminal Met residue of Hcn1 is enclosed within a chamber created from the Cut9 TPR superhelix. Thus, in complex with Cdc16/Cut9, the N-acetyl-Met residue of Hcn1, a putative degron for the Doa10 E3 ubiquitin ligase, is inaccessible for Doa10 recognition, protecting Hcn1/Cdc26 from ubiquitin-dependent degradation. This finding may provide a structural explanation for a mechanism to control the stoichiometry of proteins participating in multisubunit complexes.

Figure 1

Primary structure of Cdc16/Cut9/Apc6. (A) Multiple sequence alignment of Cdc16/Cut9/Apc6 orthologs; S. pombe, S. cerevisiae, Arabidopsis thaliana, Drosophila melongaster and H. sapiens. Red denotes invariant residues and yellow denotes conserved residues. Positions of observed TPR motifs and secondary structural elements are shown in light blue. Residues of the homotypic Cut9 dimer interface are indicated with red arrows. Mutated conserved ridge residues are indicated with blue arrows. Helices are labelled both by their structural TPR designations and by topology. Thus, the α1 helix of Cut9 has the TPR designation ‘1A' indicating the A helix of TPR1. A pair of inter-subunit salt bridges (Lys109–Asp306 and Arg135–Asp335) are invariant across all species. (B) Schematic of Cut9 showing 14 TPR motifs, dimerization module Cut9^Nterm and Cdc26/Hcn1-interacting domain, Cut9^Cterm (A) prepared using ALSCRIPT (Barton, 1993).

Figure 2

Cut9–Hcn1 is a hetero-tetramer. Cartoon representation of Cut9–Hcn1 is shown in orthogonal views. Two copies of Cut9 are shown in magenta and green, respectively, and Hcn1 are shown in yellow and cyan, respectively. indicates the two-fold symmetry axis. Cut9^Nterm and Cut9^Cterm domains are shown in each promoter, with lighter and darker shading, respectively. Figure is generated using PyMOL (http://www.pymol.org).

Figure 3

N-terminal dimerization domain of Cdc16/Cut9 and Cdc27 are structurally related. (A) Cartoon representation of Cut9^Nterm (B) Cdc27^Nterm after structural superimposition. indicates the two-fold symmetry axis. Superimpositions were performed using DALI (Holm et al, 2008), Z-score =14, r.m.s.d.=2.4 Å for 135 aligned residues. (C) Stereoview of a superimposition of Cut9^Nterm and Cdc27^Nterm.

Figure 4

Cdc16/Cut9^Nterm and Cdc27^Nterm share sequence similarity. (A) Structure-based alignment of dimerization domains of Cdc16/Cut9 and Cdc27 (TPR1–TPR4 and TPR5A of Cdc16/Cut9 and TPR1–TPR4 and α10 of Cdc27). Yellow denotes conserved residues. Positions of observed TPR motifs and secondary structural elements are shown in light blue. Blue arrows denote positions of structurally equivalent dimerization residues, with corresponding Encephalitozoon cuniculi Cdc27 and S. pombe Cut9 residue numbers indicated below. Interface cluster is indicated in parenthesis. Residues conserved between Cdc16/Cut9 and Cdc27 are not confined to TPR consensus positions: numbers below sequence correspond to consensus TPR positions: 8, 20 and 27 (in blue) are small non-residues, 4, 12, 17, 21, 28 and 30 (in red) are larger non-polar residues. (B) Stereoview of a superimposition of the Cdc16/Cut9 and Cdc27 homotypic dimer interface, showing two clusters of contacts defining the interface. Colour scheme as for Figure 3. For clarity, Leu274 and Tyr180 of Cut9 and Cdc27, respectively, are not included.

Figure 5

The N-terminal Met of Hcn1 is acetylated and enclosed within a chamber formed from the Cut9 TPR superhelix and homo-dimer interface. (A) 2Fo−Fc electron density map showing Hcn1. The N-terminus of Hcn1 is well ordered, and the N-acetyl-Met group is well defined. (B) N-terminal region of Hcn1 is shown in cyan with Cut9 shown as a surface representation, with secondary structure indicated.

Figure 6

MS/MS spectra corresponding to fragmentation of the N-terminally acetylated peptides. (A) Ac-¹MLRRNPTAIQITAEDVLAYDEE²².K (precursor ion 864.0981³⁺, mass error −0.82 p.p.m., ion score 92) from S. pombe Hcn1 digested with Glu-C. The fragmented mass series are consistent with N-acetylation (42 Da) of Met1, as the B2 mass of 287 Da corresponds to Met-Leu+(42 Da). (B) Ac-²AVNPELAPFTLSR¹⁴.G (precursor ion 728.8927²⁺, mass error −1.0 p.p.m., ion score 36.9) from endogeneous S. cerevisiae Cdc27 digested with trypsin. LC–MS/MS confidently identified acetylated N-terminal peptides, with high mass accuracy and comprehensive fragmentation data for both S. pombe Hcn1 and S. cerevisiae Cdc27.

Figure 7

A structurally conserved ridge defines one face of Cdc16/Cut9. (A) Two orthogonal views showing a surface representation of Cdc16/Cut9 colour coded according to structural conservation ramped from red to blue for low-to-high sequence conservation, based on sequences included in Figure 1. One Cut9–Hcn1 dimer of the tetramer is shown as a cartoon, with the surface of the symmetry-related molecule displayed. (B) Close-up view with mutated residues indicated. Conservation score is defined using ConSurf (Landau et al, 2005).