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

da Fonseca PC, Kong EH, Zhang Z, Schreiber A, Williams MA, Morris EP, Barford D - Nature (2010)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Section of Structural Biology, Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, London SW3 6JB, UK.

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.

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Cdh1, Apc10, Apc2 and Apc11 form a substrate recognition-catalytic module. (a) and (b). Two views of the cryo-EM APC/CCdh1·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 23, 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 7, red arrow ii denotes Lys162 and Arg163 of Apc10 responsible for APC/C affinity 7. 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.
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Figure 4: Cdh1, Apc10, Apc2 and Apc11 form a substrate recognition-catalytic module. (a) and (b). Two views of the cryo-EM APC/CCdh1·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 23, 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 7, red arrow ii denotes Lys162 and Arg163 of Apc10 responsible for APC/C affinity 7. 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.

Mentions: To explore the structure of APC/CCdh1·D-box in more detail, we collected cryo-EM images of the complex and determined its structure at ~10 Å resolution. The cryo-EM map reproduces the overall features of the APC/CCdh1·D-box map generated from negatively stained particles, but with greatly enhanced detail and resolution (Fig. 2, Supplementary Fig. 6,7). Similar to the APC/CCdh1·D-box ternary complex obtained from negative stain EM, the cryo-EM reconstruction shows density connecting Cdh1 and Apc10 (Figs. 2,4). Docking the crystal structure of Apc10 (refs 13,22) and the modelled Cdh1 WD40 domain into their respective densities, indicates additional unassigned density linking Cdh1 to Apc10 (Fig. 4a,c). Strikingly, the best fit of Apc10 into the cryo-EM map positions a highly conserved loop, required for D-box recognition 7, adjacent to the density linking Apc10 with Cdh1. In contrast, residues on Apc10’s opposite surface that contribute to APC/C interactions 7, are oriented towards Apc2 (Fig. 4c).


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

da Fonseca PC, Kong EH, Zhang Z, Schreiber A, Williams MA, Morris EP, Barford D - Nature (2010)

Cdh1, Apc10, Apc2 and Apc11 form a substrate recognition-catalytic module. (a) and (b). Two views of the cryo-EM APC/CCdh1·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 23, 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 7, red arrow ii denotes Lys162 and Arg163 of Apc10 responsible for APC/C affinity 7. 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.
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Figure 4: Cdh1, Apc10, Apc2 and Apc11 form a substrate recognition-catalytic module. (a) and (b). Two views of the cryo-EM APC/CCdh1·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 23, 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 7, red arrow ii denotes Lys162 and Arg163 of Apc10 responsible for APC/C affinity 7. 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.
Mentions: To explore the structure of APC/CCdh1·D-box in more detail, we collected cryo-EM images of the complex and determined its structure at ~10 Å resolution. The cryo-EM map reproduces the overall features of the APC/CCdh1·D-box map generated from negatively stained particles, but with greatly enhanced detail and resolution (Fig. 2, Supplementary Fig. 6,7). Similar to the APC/CCdh1·D-box ternary complex obtained from negative stain EM, the cryo-EM reconstruction shows density connecting Cdh1 and Apc10 (Figs. 2,4). Docking the crystal structure of Apc10 (refs 13,22) and the modelled Cdh1 WD40 domain into their respective densities, indicates additional unassigned density linking Cdh1 to Apc10 (Fig. 4a,c). Strikingly, the best fit of Apc10 into the cryo-EM map positions a highly conserved loop, required for D-box recognition 7, adjacent to the density linking Apc10 with Cdh1. In contrast, residues on Apc10’s opposite surface that contribute to APC/C interactions 7, are oriented towards Apc2 (Fig. 4c).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Section of Structural Biology, Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, London SW3 6JB, UK.

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.

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