Structures of APC/C(Cdh1) with substrates identify Cdh1 and Apc10 as the D-box co-receptor

Abstract

The ubiquitylation of cell-cycle regulatory proteins by the large multimeric anaphase-promoting complex (APC/C) controls sister chromatid segregation and the exit from mitosis. Selection of APC/C targets is achieved through recognition of destruction motifs, predominantly the destruction (D)-box and KEN (Lys-Glu-Asn)-box. Although this process is known to involve a co-activator protein (either Cdc20 or Cdh1) together with core APC/C subunits, the structural basis for substrate recognition and ubiquitylation is not understood. Here we investigate budding yeast APC/C using single-particle electron microscopy and determine a cryo-electron microscopy map of APC/C in complex with the Cdh1 co-activator protein (APC/C(Cdh1)) bound to a D-box peptide at ∼10 Å resolution. We find that a combined catalytic and substrate-recognition module is located within the central cavity of the APC/C assembled from Cdh1, Apc10--a core APC/C subunit previously implicated in substrate recognition--and the cullin domain of Apc2. Cdh1 and Apc10, identified from difference maps, create a co-receptor for the D-box following repositioning of Cdh1 towards Apc10. Using NMR spectroscopy we demonstrate specific D-box-Apc10 interactions, consistent with a role for Apc10 in directly contributing towards D-box recognition by the APC/C(Cdh1) complex. Our results rationalize the contribution of both co-activator and core APC/C subunits to D-box recognition and provide a structural framework for understanding mechanisms of substrate recognition and catalysis by the APC/C.

Figure 1

Negative stain electron microscopy reconstructions of budding yeast APC/C show that substrate binding to APC/C^Cdh1 involves Cdh1 and Apc10. (a) APC/C^Cdh1, (b) APC/C, (c) APC/C^{ΔApc10·Cdh1}. Density assigned to Cdh1 and Apc10 is shown in magenta and blue, respectively. The resolution of the APC/C^Cdh1 binary complex is ~18-20 Å (Supplementary Fig. 10d). Negative stain EM reconstructions of (d) APC/C^Cdh1·Hsl1 complex, (e) APC/C^Cdh1·D-box, (f) APC/C^{Cdh1·KEN-box}. Lower panels in (d), (e) and (f) show details of the structural changes associated with Cdh1 and Apc10 in the presence of substrate compared with the superimposed binary APC/C^Cdh1 map represented in mesh. Hsl1 and D-box and KEN-box peptides were used at saturating concentrations to promote stoichiometric APC/C^Cdh1-substrate ternary complexes.

Figure 2

Cryo-electron microscopy reconstruction of budding yeast APC/C^Cdh1·D-box reveals the lattice-like architecture of the complex. Three views of the complex with (b) similar to views shown in figure 1. Resolution is ~10 Å (Supplementary Fig. 12c).

Figure 3

¹H-¹⁵N HSQC spectra of Apc10. Overlaid are spectra of the apo-protein and protein in the presence of stoichiometric excess of each of four peptides. The complete amide region (a) and for clarity expanded views of two boxed sub-regions (b, c) are shown. Spectra in the presence of either of the two D-box containing peptides show common changes with respect to the apo-protein spectrum, namely absence of the peaks seen in the apo-protein (black arrows) and new or shifted peaks not seen in the apo-spectrum (blue arrows). In contrast, spectra in the presence of either the Cdc13-derived peptide in which four residues of the D-box motif are mutated to alanine or a peptide containing a KEN-box motif are very similar to the apo-spectrum, retaining all of the peaks marked by black arrows. The spectrum with the mutant Cdc13 peptide does in some cases show low intensity peaks at the positions indicated by blue arrows (see expanded views b, c) indicating a very weak residual interaction. These spectra are consistent with a D-box dependent interaction with Apc10. (Peaks arising from natural abundance ¹⁵N amides in the unbound peptide that are protected from solvent exchange are indicated by an asterisk).

Figure 4

Cdh1, Apc10, Apc2 and Apc11 form a substrate recognition-catalytic module. (a) and (b). Two views of the cryo-EM APC/C^Cdh1·D-box complex. Protein density is represented by a mesh with fitted atomic coordinates of Cdh1 β-propeller (modelled), Apc10 (ref. 22), Apc2-Apc11 (modelled on Cul4a-Rbx1 of SCF) and Cdc27 (ref. 26). Only the N-terminal β-strand of Apc11 bound to the Apc2 CTD is modelled (orange). The two subunits of Cdc27 are shown in light and dark green. View in (a) shows the 2-fold symmetry axis of Cdc27. Density connecting Cdh1 to a TPR-super-helix of the Cdc27 dimer is indicated by an arrow. TPR motifs 8 to 10 of Cdc27, implicated in IR-tail recognition , are shown in lighter colourer. In (b) the final residue of Apc10 observed in the crystal structure (Ser 256), 25 residues N-terminal to the IR motif, is indicated by red spheres. (c) Details of the Cdh1 and Apc10 co-receptor for D-box. Both Cdh1 and Apc10 connect to Apc2. The N-terminus of Cdh1, including the C-box linking the WD40 domain to Apc2, is not modelled. Red arrow i denotes the conserved loop (residues His239 to Asp244) of Apc10 implicated in D-box recognition , red arrow ii denotes Lys162 and Arg163 of Apc10 responsible for APC/C affinity . Two models for a possible fit of D-box to the density interconnecting Cdh1 and Apc10 are shown in Supplementary Fig. 8. (d) Schematic of combined catalytic and substrate recognition module responsible for D-box binding and substrate ubiquitylation. D-box is represented as binding to an interface between Cdh1 and Apc10.