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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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Negative stain electron microscopy reconstructions of budding yeast APC/C show that substrate binding to APC/CCdh1 involves Cdh1 and Apc10. (a) APC/CCdh1, (b) APC/C, (c) APC/CΔApc10·Cdh1. Density assigned to Cdh1 and Apc10 is shown in magenta and blue, respectively. The resolution of the APC/CCdh1 binary complex is ~18-20 Å (Supplementary Fig. 10d). Negative stain EM reconstructions of (d) APC/CCdh1·Hsl1 complex, (e) APC/CCdh1·D-box, (f) APC/CCdh1·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/CCdh1 map represented in mesh. Hsl1 and D-box and KEN-box peptides were used at saturating concentrations to promote stoichiometric APC/CCdh1-substrate ternary complexes.
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Figure 1: Negative stain electron microscopy reconstructions of budding yeast APC/C show that substrate binding to APC/CCdh1 involves Cdh1 and Apc10. (a) APC/CCdh1, (b) APC/C, (c) APC/CΔApc10·Cdh1. Density assigned to Cdh1 and Apc10 is shown in magenta and blue, respectively. The resolution of the APC/CCdh1 binary complex is ~18-20 Å (Supplementary Fig. 10d). Negative stain EM reconstructions of (d) APC/CCdh1·Hsl1 complex, (e) APC/CCdh1·D-box, (f) APC/CCdh1·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/CCdh1 map represented in mesh. Hsl1 and D-box and KEN-box peptides were used at saturating concentrations to promote stoichiometric APC/CCdh1-substrate ternary complexes.

Mentions: The APC/C co-activator Cdh1 was identified in negative stain EM reconstructions as a prominent and discrete density feature present within the central cavity of APC/CCdh1 and absent from APC/C (Fig. 1a,b). Its disc-shaped density, characteristic of an exposed WD40 β-propeller domain, is connected to the APC/C via an edge-on interface. Overall, with the exception of the Cdh1 density, APC/C and APC/CCdh1 are similar, and the large conformational changes that accompany co-activator binding to vertebrate APC/C 19,21 are not evident. An ellipsoid-shaped density feature, resembling the β-sandwich of Apc10 13,22, situated adjacent to, but not in contact with Cdh1, is more prominent in the presence of Cdh1 (Fig. 1a,b). Its close proximity to Cdh1 was intriguing in view of the role of Apc10 in contributing towards substrate recognition 6, and the D-box-dependent processivity of the ubiquitylation reaction 5,7. To unequivocally identify Apc10, we generated APC/CΔApc10 in complex with Cdh1 (APC/CΔApc10-Cdh1). The resultant APC/CΔApc10-Cdh1 map showed complete loss of this ellipsoid density (Fig. 1c), confirming its identity as Apc10. Deletion of Apc10 also resulted in a depletion of Cdh1 density around the circumference of the β-propeller most distant from its contact to APC/C (Fig. 1c). Since deletion of Apc10 does not affect the APC/C subunit composition 6 or abrogate Cdh1 binding (Supplementary Fig. 2), the partial loss of Cdh1 density is indicative of an increased flexibility of Cdh1’s WD40 domain. This finding and the reduced density for Apc10 in APC/C imply conformational interdependence of Apc10 and Cdh1.


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)

Negative stain electron microscopy reconstructions of budding yeast APC/C show that substrate binding to APC/CCdh1 involves Cdh1 and Apc10. (a) APC/CCdh1, (b) APC/C, (c) APC/CΔApc10·Cdh1. Density assigned to Cdh1 and Apc10 is shown in magenta and blue, respectively. The resolution of the APC/CCdh1 binary complex is ~18-20 Å (Supplementary Fig. 10d). Negative stain EM reconstructions of (d) APC/CCdh1·Hsl1 complex, (e) APC/CCdh1·D-box, (f) APC/CCdh1·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/CCdh1 map represented in mesh. Hsl1 and D-box and KEN-box peptides were used at saturating concentrations to promote stoichiometric APC/CCdh1-substrate ternary complexes.
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Related In: Results  -  Collection

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Figure 1: Negative stain electron microscopy reconstructions of budding yeast APC/C show that substrate binding to APC/CCdh1 involves Cdh1 and Apc10. (a) APC/CCdh1, (b) APC/C, (c) APC/CΔApc10·Cdh1. Density assigned to Cdh1 and Apc10 is shown in magenta and blue, respectively. The resolution of the APC/CCdh1 binary complex is ~18-20 Å (Supplementary Fig. 10d). Negative stain EM reconstructions of (d) APC/CCdh1·Hsl1 complex, (e) APC/CCdh1·D-box, (f) APC/CCdh1·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/CCdh1 map represented in mesh. Hsl1 and D-box and KEN-box peptides were used at saturating concentrations to promote stoichiometric APC/CCdh1-substrate ternary complexes.
Mentions: The APC/C co-activator Cdh1 was identified in negative stain EM reconstructions as a prominent and discrete density feature present within the central cavity of APC/CCdh1 and absent from APC/C (Fig. 1a,b). Its disc-shaped density, characteristic of an exposed WD40 β-propeller domain, is connected to the APC/C via an edge-on interface. Overall, with the exception of the Cdh1 density, APC/C and APC/CCdh1 are similar, and the large conformational changes that accompany co-activator binding to vertebrate APC/C 19,21 are not evident. An ellipsoid-shaped density feature, resembling the β-sandwich of Apc10 13,22, situated adjacent to, but not in contact with Cdh1, is more prominent in the presence of Cdh1 (Fig. 1a,b). Its close proximity to Cdh1 was intriguing in view of the role of Apc10 in contributing towards substrate recognition 6, and the D-box-dependent processivity of the ubiquitylation reaction 5,7. To unequivocally identify Apc10, we generated APC/CΔApc10 in complex with Cdh1 (APC/CΔApc10-Cdh1). The resultant APC/CΔApc10-Cdh1 map showed complete loss of this ellipsoid density (Fig. 1c), confirming its identity as Apc10. Deletion of Apc10 also resulted in a depletion of Cdh1 density around the circumference of the β-propeller most distant from its contact to APC/C (Fig. 1c). Since deletion of Apc10 does not affect the APC/C subunit composition 6 or abrogate Cdh1 binding (Supplementary Fig. 2), the partial loss of Cdh1 density is indicative of an increased flexibility of Cdh1’s WD40 domain. This finding and the reduced density for Apc10 in APC/C imply conformational interdependence of Apc10 and Cdh1.

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