Structures of APC/C(Cdh1) with substrates identify Cdh1 and Apc10 as the D-box co-receptor.
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.
Section of Structural Biology, Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, London SW3 6JB, UK.
Paula C A
PC
da Fonseca
Eric H
EH
Kong
Ziguo
Z
Zhang
Anne
A
Schreiber
Mark A
MA
Williams
Edward P
EP
Morris
David
D
Barford
D020816:Amino Acid Motifs; D064173:Anaphase-Promoting Complex-Cyclosome; D064197:Apc10 Subunit, Anaphase-Promoting Complex-Cyclosome; D064190:Apc2 Subunit, Anaphase-Promoting Complex-Cyclosome; D055162:Biocatalysis; D064200:Cdh1 Proteins; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D020285:Cryoelectron Microscopy; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000737:chemistry*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D013379:Substrate Specificity; D043743:Ubiquitin-Protein Ligase Complexes / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D054875:Ubiquitination
D064197:Apc10 Subunit, Anaphase-Promoting Complex-Cyclosome; C577583:CDH1 protein, S cerevisiae; D064200:Cdh1 Proteins; D018797:Cell Cycle Proteins; C577456:DOC1 protein, S cerevisiae; D010455:Peptides; D029701:Saccharomyces cerevisiae Proteins; D043743:Ubiquitin-Protein Ligase Complexes; C577346:APC2 protein, S cerevisiae; D064173:Anaphase-Promoting Complex-Cyclosome; D064190:Apc2 Subunit, Anaphase-Promoting Complex-Cyclosome