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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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Cryo-electron microscopy reconstruction of budding yeast APC/CCdh1·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).
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Figure 2: Cryo-electron microscopy reconstruction of budding yeast APC/CCdh1·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).

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)

Cryo-electron microscopy reconstruction of budding yeast APC/CCdh1·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).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3037847&req=5

Figure 2: Cryo-electron microscopy reconstruction of budding yeast APC/CCdh1·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).
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.

Show MeSH