Masaya Yamaguchi

Autoinhibition and phosphorylation-induced activation mechanisms of human cancer and autoimmune disease-related E3 protein Cbl-b

Cbl-b is a RING-type E3 ubiquitin ligase that functions as a negative regulator of T-cell activation and growth factor receptor and nonreceptor-type tyrosine kinase signaling. Cbl-b dysfunction is related to autoimmune diseases and cancers in humans. However, the molecular mechanism regulating its E3 activity is largely unknown. NMR and small-angle X-ray scattering analyses revealed that the unphosphorylated N-terminal region of Cbl-b forms a compact structure by an intramolecular interaction, which masks the interaction surface of the RING domain with an E2 ubiquitin-conjugating enzyme. Phosphorylation of Y363, located in the helix-linker region between the tyrosine kinase binding and the RING domains, disrupts the interdomain interaction to expose the E2 binding surface of the RING domain. Structural analysis revealed that the phosphorylated helix-RING region forms a compact structure in solution. Moreover, the phosphate group of pY363 is located in the vicinity of the interaction surface with UbcH5B to increase affinity by reducing their electrostatic repulsion. Thus, the phosphorylation of Y363 regulates the E3 activity of Cbl-b by two mechanisms: one is to remove the masking of the RING domain from the tyrosine kinase binding domain and the other is to form a surface to enhance binding affinity to E2.

Dual RING E3 Architectures Regulate Multiubiquitination and Ubiquitin Chain Elongation by APC/C.

Protein ubiquitination involves E1, E2, and E3 trienzyme cascades. E2 and RING E3 enzymes often collaborate to first prime a substrate with a single ubiquitin (UB) and then achieve different forms of polyubiquitination: multiubiquitination of several sites and elongation of linkage-specific UB chains. Here, cryo-EM and biochemistry show that the human E3 anaphase-promoting complex/cyclosome (APC/C) and its two partner E2s, UBE2C (aka UBCH10) and UBE2S, adopt specialized catalytic architectures for these two distinct forms of polyubiquitination. The APC/C RING constrains UBE2C proximal to a substrate and simultaneously binds a substrate-linked UB to drive processive multiubiquitination. Alternatively, during UB chain elongation, the RING does not bind UBE2S but rather lures an evolving substrate-linked UB to UBE2S positioned through a cullin interaction to generate a Lys11-linked chain. Our findings define mechanisms of APC/C regulation, and establish principles by which specialized E3-E2-substrate-UB architectures control different forms of polyubiquitination.

Cryo-EM of Mitotic Checkpoint Complex-Bound APC/C Reveals Reciprocal and Conformational Regulation of Ubiquitin Ligation

The mitotic checkpoint complex (MCC) coordinates proper chromosome biorientation on the spindle with ubiquitination activities of CDC20-activated anaphase-promoting complex/cyclosome (APC/C(CDC20)). APC/C(CDC20) and two E2s, UBE2C and UBE2S, catalyze ubiquitination through distinct architectures for linking ubiquitin (UB) to substrates and elongating polyUB chains, respectively. MCC, which contains a second molecule of CDC20, blocks APC/C(CDC20)-UBE2C-dependent ubiquitination of Securin and Cyclins, while differentially determining or inhibiting CDC20 ubiquitination to regulate spindle surveillance, checkpoint activation, and checkpointxa0termination. Here electron microscopy reveals conformational variation of APC/C(CDC20)-MCC underlying this multifaceted regulation. MCC binds APC/C-bound CDC20 to inhibit substrate access. However, rotation about the CDC20-MCC assembly andxa0conformational variability of APC/C modulate UBE2C-catalyzed ubiquitination of MCCs CDC20 molecule. Access of UBE2C is limiting for subsequentxa0polyubiquitination by UBE2S. We propose that conformational dynamics of APC/C(CDC20)-MCC modulate E2 activation and determine distinctive ubiquitination activities as part of a response mechanism ensuring accurate sister chromatid segregation.

Structural insights into Atg10-mediated formation of the autophagy-essential Atg12-Atg5 conjugate

The Atg12-Atg5 conjugate, which is formed by an ubiquitin-like conjugation system, is essential to autophagosome formation, a central event in autophagy. Despite its importance, the molecular mechanism of the Atg12-Atg5 conjugate formation has not been elucidated. Here, we report the solution and crystal structures of Atg10 and Atg5 homologs from Kluyveromyces marxianus (Km), a thermotolerant yeast. KmAtg10 comprises an E2-core fold with characteristic accessories, including two β strands, whereas KmAtg5 has two ubiquitin-like domains and a helical domain. The nuclear magnetic resonance experiments, mutational analyses, and crosslinking experiments showed that KmAtg10 directly recognizes KmAtg5, especially its C-terminal ubiquitin-like domain, by its characteristic two β strands. Kinetic analysis suggests that Tyr56 and Asn114 ofxa0KmAtg10 may place the side chain of KmAtg5 Lys145 into the optimal orientation for its conjugation reaction with Atg12. These structural features enable Atg10 to mediate the formation of the Atg12-Atg5 conjugate without a specific E3 enzyme.

The NMR structure of the autophagy-related protein Atg8

Autophagy is the process through which the bulk degradation of cytoplasmic components by the lysosomal/vacuolar system occurs in response to starvation conditions (Nakatogawa et al. 2009). In autophagy, a double-membrane structure called an autophagosome sequesters a portion of the cytoplasm and fuses with the lysosome/ vacuole to deliver its contents into the organelle lumen. Recently, autophagy was found to have a crucial function in numerous biological processes including differentiation, antigen presentation and aging, and its dysfunction causes severe diseases such as neurodegeneration (Mizushima 2007). Atg8 is a ubiquitin like protein, and plays an essential role for autophagosome formation in Saccharomyces cerevisiae. Atg8 is unique in that it is conjugated to the lipid phosphatidylethanolamine (PE) by a ubiquitin-like system, called the Atg8 system. In the Atg8 system, nascent Atg8 is cleaved at its C-terminal arginine residue by Atg4, a cysteine protease (Kirisako et al. 2000), and the exposed C-terminal glycine is conjugated to PE by Atg7, an E1-like enzyme and Atg3, an E2-like enzyme. The Atg8-PE conjugate itself possesses membrane tethering and hemifusion ability and is essential in autophagosome formation, especially in a membrane expansion step (Nakatogawa et al. 2007). It was also known that Atg8 plays a critical role for target recognition in selective autophagy. For example, aminopeptidase I (Ape1) is selectively and constitutively transported into the vacuole through autophagic processes. During these processes, Ape1 is recognized by the receptor protein Atg19, which is further recognized by Atg8. These interactions may facilitate biogenesis of autophagosomal membranes around Ape1 and selective sequestration of the enzyme into the membranes (Nakatogawa et al. 2009). Structures of several Atg8 homologues have been determined (LC3; Sugawara et al. 2004, GABARAP; Bavro et al. 2002, Stangler et al. 2002, GATE-16; Paz et al. 2000, Trypanosoma brucei Atg8; Koopmann et al. 2009; reviewed by Noda et al. 2009). All of these homologues are comprised of two domains, an N-terminal a-helix domain and a C-terminal ubiquitin-like domain. However, S. cerevisiae Atg8 structure was only solved in the complex with the Atg19 peptide (Noda et al. 2008). Atg8 is suggested to take both an open and a closed conformations. The membrane biogenesis in autophagy is considered to be mediated through the open conformation that is oligomerized to initiate membrane fusion (Nakatogawa et al. 2007). This prompted us to study the structure of Atg8 at the peptide-free state. The assignment of Atg8 NMR signals was reported by Schwarten et al. (2009). We also determined the main chain signal assignment at both ligand-free and ligand-bound states (Noda et al. 2008). However, NMR signals of the N-terminal region and Arg47 of Atg8 at a ligand-free state were very weak or not detectable. Furthermore, Atg8 at the H. Kumeta M. Watanabe M. Yamaguchi K. Ogura W. Adachi Y. Fujioka N. N. Noda F. Inagaki (&) Laboratory of Structural Biology, Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita 12 Nishi 6, Kita-ku, Sapporo 060-0812, Japan e-mail: finagaki@pharm.hokudai.ac.jp

RING E3 mechanism for ubiquitin ligation to a disordered substrate visualized for human anaphase-promoting complex.

Significance The anaphase-promoting complex/cyclosome (APC) is a multisubunit RING E3 ubiquitin (Ub) ligase that regulates mitosis, meiosis, and numerous facets of neurobiology by targeting key regulatory proteins for Ub-mediated degradation. Despite great importance, it remains unclear how APC, or most of the other 600 RING E3s in humans, targets Ub to lysines in disordered substrates. Here, we report the structural and molecular basis for substrate ubiquitination by APC and its partner E2, UBCH10. UBCH10 is recruited to APC, activated for ubiquitination, and positioned for substrate targeting through multisite interactions with the APC cullin–RING core. We propose that many RING E3–E2 assemblies work similarly, with multisite interactions establishing specificity, harnessing ubiquitination machineries to accelerate searching for target lysines, and facilitating regulation. For many E3 ligases, a mobile RING (Really Interesting New Gene) domain stimulates ubiquitin (Ub) transfer from a thioester-linked E2∼Ub intermediate to a lysine on a remotely bound disordered substrate. One such E3 is the gigantic, multisubunit 1.2-MDa anaphase-promoting complex/cyclosome (APC), which controls cell division by ubiquitinating cell cycle regulators to drive their timely degradation. Intrinsically disordered substrates are typically recruited via their KEN-box, D-box, and/or other motifs binding to APC and a coactivator such as CDH1. On the opposite side of the APC, the dynamic catalytic core contains the cullin-like subunit APC2 and its RING partner APC11, which collaborates with the E2 UBCH10 (UBE2C) to ubiquitinate substrates. However, how dynamic RING–E2∼Ub catalytic modules such as APC11–UBCH10∼Ub collide with distally tethered disordered substrates remains poorly understood. We report structural mechanisms of UBCH10 recruitment to APCCDH1 and substrate ubiquitination. Unexpectedly, in addition to binding APC11’s RING, UBCH10 is corecruited via interactions with APC2, which we visualized in a trapped complex representing an APCCDH1–UBCH10∼Ub–substrate intermediate by cryo-electron microscopy, and in isolation by X-ray crystallography. To our knowledge, this is the first structural view of APC, or any cullin–RING E3, with E2 and substrate juxtaposed, and it reveals how tripartite cullin–RING–E2 interactions establish APC’s specificity for UBCH10 and harness a flexible catalytic module to drive ubiquitination of lysines within an accessible zone. We propose that multisite interactions reduce the degrees of freedom available to dynamic RING E3–E2∼Ub catalytic modules, condense the search radius for target lysines, increase the chance of active-site collision with conformationally fluctuating substrates, and enable regulation.

Mechanism of APC/CCDC20 activation by mitotic phosphorylation

Significance The ability of eukaryotic cells to pass their genomes properly from one cell generation to the next depends on the 1.2-MDa ubiquitin ligase complex APC/C (anaphase-promoting complex/cyclosome) and on the correct timing of its activation by the substrate adaptor CDC20 (cell division cycle 20). Although it has been known for two decades that mitotic APC/C phosphorylation is required for its activation by CDC20, the mechanistic basis of this process remained unknown, in part because the existence of numerous phospho-sites on APC/C made systematic mutagenesis approaches difficult. Here we have used the biGBac technique for the rapid assembly of multigene expression constructs to overcome this limitation and discovered that APC/C contains an autoinhibitory loop region that prevents CDC20 binding until it becomes phosphorylated in mitosis. Chromosome segregation and mitotic exit are initiated by the 1.2-MDa ubiquitin ligase APC/C (anaphase-promoting complex/cyclosome) and its coactivator CDC20 (cell division cycle 20). To avoid chromosome missegregation, APC/CCDC20 activation is tightly controlled. CDC20 only associates with APC/C in mitosis when APC/C has become phosphorylated and is further inhibited by a mitotic checkpoint complex until all chromosomes are bioriented on the spindle. APC/C contains 14 different types of subunits, most of which are phosphorylated in mitosis on multiple sites. However, it is unknown which of these phospho-sites enable APC/CCDC20 activation and by which mechanism. Here we have identified 68 evolutionarily conserved mitotic phospho-sites on human APC/C bound to CDC20 and have used the biGBac technique to generate 47 APC/C mutants in which either all 68 sites or subsets of them were replaced by nonphosphorylatable or phospho-mimicking residues. The characterization of these complexes in substrate ubiquitination and degradation assays indicates that phosphorylation of an N-terminal loop region in APC1 is sufficient for binding and activation of APC/C by CDC20. Deletion of the N-terminal APC1 loop enables APC/CCDC20 activation in the absence of mitotic phosphorylation or phospho-mimicking mutations. These results indicate that binding of CDC20 to APC/C is normally prevented by an autoinhibitory loop in APC1 and that its mitotic phosphorylation relieves this inhibition. The predicted location of the N-terminal APC1 loop implies that this loop controls interactions between the N-terminal domain of CDC20 and APC1 and APC8. These results reveal how APC/C phosphorylation enables CDC20 to bind and activate the APC/C in mitosis.

Noncanonical recognition and UBL loading of distinct E2s by autophagy-essential Atg7

Autophagy requires ubiquitin-like Atg8 and Atg12 conjugation systems, where Atg7 has a critical role as the sole E1 enzyme. Although Atg7 recognizes two distinct E2s, Atg3 and Atg10, it is not understood how Atg7 correctly loads these E2s with their cognate ubiquitin-like proteins, Atg8 and Atg12. Here, we report the crystal structures of the N-terminal domain of Atg7 bound to Atg10 or Atg3 of thermotolerant yeast and plant homologs. The observed Atg7-Atg10 and Atg7-Atg3 interactions, which resemble each other but are quite distinct from the canonical E1-E2 interaction, makes Atg7 suitable for transferring Atg12 to Atg10 and Atg8 to Atg3 by a trans mechanism. Notably, in vitro experiments showed that Atg7 loads Atg3 and Atg10 with Atg8 and Atg12 in a nonspecific manner, which suggests that cognate conjugate formation in vivo is not an intrinsic quality of Atg7.

Structure of an APC3-APC16 Complex: Insights into Assembly of the Anaphase-Promoting Complex/Cyclosome.

The anaphase-promoting complex/cyclosome (APC/C) is a massive E3 ligase that controls mitosis by catalyzing ubiquitination of key cell cycle regulatory proteins. The APC/C assembly contains two subcomplexes: the Platform centers around a cullin-RING-like E3 ligase catalytic core; the Arc Lamp is a hub that mediates transient association with regulators and ubiquitination substrates. The Arc Lamp contains the small subunits APC16, CDC26, and APC13, and tetratricopeptide repeat (TPR) proteins (APC7, APC3, APC6, and APC8) that homodimerize and stack with quasi-2-fold symmetry. Within the APC/C complex, APC3 serves as center for regulation. APC3s TPR motifs recruit substrate-binding coactivators, CDC20 and CDH1, via their C-terminal conserved Ile-Arg (IR) tail sequences. Human APC3 also binds APC16 and APC7 and contains a >200-residue loop that is heavily phosphorylated during mitosis, although the basis for APC3 interactions and whether loop phosphorylation is required for ubiquitination are unclear. Here, we map the basis for human APC3 assembly with APC16 and APC7, report crystal structures of APC3Δloop alone and in complex with the C-terminal domain of APC16, and test roles of APC3s loop and IR tail binding surfaces in APC/C-catalyzed ubiquitination. The structures show how one APC16 binds asymmetrically to the symmetric APC3 dimer and, together with biochemistry and prior data, explain how APC16 recruits APC7 to APC3, show how APC3s C-terminal domain is rearranged in the full APC/C assembly, and visualize residues in the IR tail binding cleft important for coactivator-dependent ubiquitination. Overall, the results provide insights into assembly, regulation, and interactions of TPR proteins and the APC/C.

The Thermotolerant Yeast Kluyveromyces marxianus Is a Useful Organism for Structural and Biochemical Studies of Autophagy

Background: Autophagosome formation is mediated by multiple autophagy-related (Atg) proteins. Results: Essential Atg proteins of K. marxianus, which have superior thermostability and solubility, are identified. Conclusion: K. marxianus can be used as a novel organism to study autophagy. Significance: K. marxianus proteins are broadly applicable as tools for in vitro studies, not only in autophagy field but also in other fields. Autophagy is a conserved degradation process in which autophagosomes are generated by cooperative actions of multiple autophagy-related (Atg) proteins. Previous studies using the model yeast Saccharomyces cerevisiae have provided various insights into the molecular basis of autophagy; however, because of the modest stability of several Atg proteins, structural and biochemical studies have been limited to a subset of Atg proteins, preventing us from understanding how multiple Atg proteins function cooperatively in autophagosome formation. With the goal of expanding the scope of autophagy research, we sought to identify a novel organism with stable Atg proteins that would be advantageous for in vitro analyses. Thus, we focused on a newly isolated thermotolerant yeast strain, Kluyveromyces marxianus DMKU3-1042, to utilize as a novel system elucidating autophagy. We developed experimental methods to monitor autophagy in K. marxianus cells, identified the complete set of K. marxianus Atg homologs, and confirmed that each Atg homolog is engaged in autophagosome formation. Biochemical and bioinformatic analyses revealed that recombinant K. marxianus Atg proteins have superior thermostability and solubility as compared with S. cerevisiae Atg proteins, probably due to the shorter primary sequences of KmAtg proteins. Furthermore, bioinformatic analyses showed that more than half of K. marxianus open reading frames are relatively short in length. These features make K. marxianus proteins broadly applicable as tools for structural and biochemical studies, not only in the autophagy field but also in other fields.