Hiroyuki Kumeta

Structural basis of target recognition by Atg8/LC3 during selective autophagy

Autophagy is a non‐selective bulk degradation process in which isolation membranes enclose a portion of cytoplasm to form double‐membrane vesicles, called autophagosomes, and deliver their inner constituents to the lytic compartments. Recent studies have also shed light on another mode of autophagy that selectively degrades various targets. Yeast Atg8 and its mammalian homologue LC3 are ubiquitin‐like modifiers that are localized on isolation membranes and play crucial roles in the formation of autophagosomes. These proteins are also involved in selective incorporation of specific cargo molecules into autophagosomes, in which Atg8 and LC3 interact with Atg19 and p62, receptor proteins for vacuolar enzymes and disease‐related protein aggregates, respectively. Using X‐ray crystallography and NMR, we herein report the structural basis for Atg8–Atg19 and LC3–p62 interactions. Remarkably, Atg8 and LC3 were shown to interact with Atg19 and p62, respectively, in a quite similar manner: they recognized the side‐chains of Trp and Leu in a four‐amino acid motif, WXXL, in Atg19 and p62 using hydrophobic pockets conserved among Atg8 homologues. Together with mutational analyses, our results show the fundamental mechanism that allows Atg8 homologues, in association with WXXL‐containing proteins, to capture specific cargo molecules, thereby endowing isolation membranes and/or their assembly machineries with target selectivity.

The structure of Atg4B-LC3 complex reveals the mechanism of LC3 processing and delipidation during autophagy.

Atg8 is conjugated to phosphatidylethanolamine (PE) by ubiquitin‐like conjugation reactions. Atg8 has at least two functions in autophagy: membrane biogenesis and target recognition. Regulation of PE conjugation and deconjugation of Atg8 is crucial for these functions in which Atg4 has a critical function by both processing Atg8 precursors and deconjugating Atg8–PE. Here, we report the crystal structures of catalytically inert human Atg4B (HsAtg4B) in complex with processed and unprocessed forms of LC3, a mammalian orthologue of yeast Atg8. On LC3 binding, the regulatory loop and the N‐terminal tail of HsAtg4B undergo large conformational changes. The regulatory loop masking the entrance of the active site of free HsAtg4B is lifted by LC3 Phe119, so that a groove is formed along which the LC3 tail enters the active site. At the same time, the N‐terminal tail masking the exit of the active site of HsAtg4B in the free form is detached from the enzyme core and a large flat surface is exposed, which might enable the enzyme to access the membrane‐bound LC3–PE.

Solution Structures of Cytosolic RNA Sensor MDA5 and LGP2 C-terminal Domains: IDENTIFICATION OF THE RNA RECOGNITION LOOP IN RIG-I-LIKE RECEPTORS

The RIG-I like receptor (RLR) comprises three homologues: RIG-I (retinoic acid-inducible gene I), MDA5 (melanoma differentiation-associated gene 5), and LGP2 (laboratory of genetics and physiology 2). Each RLR senses different viral infections by recognizing replicating viral RNA in the cytoplasm. The RLR contains a conserved C-terminal domain (CTD), which is responsible for the binding specificity to the viral RNAs, including double-stranded RNA (dsRNA) and 5′-triphosphated single-stranded RNA (5′ppp-ssRNA). Here, the solution structures of the MDA5 and LGP2 CTD domains were solved by NMR and compared with those of RIG-I CTD. The CTD domains each have a similar fold and a similar basic surface but there is the distinct structural feature of a RNA binding loop; The LGP2 and RIG-I CTD domains have a large basic surface, one bank of which is formed by the RNA binding loop. MDA5 also has a large basic surface that is extensively flat due to open conformation of the RNA binding loop. The NMR chemical shift perturbation study showed that dsRNA and 5′ppp-ssRNA are bound to the basic surface of LGP2 CTD, whereas dsRNA is bound to the basic surface of MDA5 CTD but much more weakly, indicating that the conformation of the RNA binding loop is responsible for the sensitivity to dsRNA and 5′ppp-ssRNA. Mutation study of the basic surface and the RNA binding loop supports the conclusion from the structure studies. Thus, the CTD is responsible for the binding affinity to the viral RNAs.

Structural basis for the transforming activity of human cancer-related signaling adaptor protein CRK

CRKI (SH2-SH3) and CRKII (SH2-SH3-SH3) are splicing isoforms of the oncoprotein CRK that regulate transcription and cytoskeletal reorganization for cell growth and motility by linking tyrosine kinases to small G proteins. CRKI shows substantial transforming activity, whereas the activity of CRKII is low, and phosphorylated CRKII has no biological activity whatsoever. The molecular mechanisms underlying the distinct biological activities of the CRK proteins remain elusive. We determined the solution structures of CRKI, CRKII and phosphorylated CRKII by NMR and identified the molecular mechanism that gives rise to their activities. Results from mutational analysis using rodent 3Y1 fibroblasts were consistent with those from the structural studies. Together, these data suggest that the linker region modulates the binding of CRKII to its targets, thus regulating cell growth and motility.

Structural basis of Atg8 activation by a homodimeric E1, Atg7.

E1 enzymes activate ubiquitin-like proteins and transfer them to cognate E2 enzymes. Atg7, a noncanonical E1, activates two ubiquitin-like proteins, Atg8 and Atg12, and plays a crucial role in autophagy. Here, we report crystal structures of full-length Atg7 and its C-terminal domain bound to Atg8 and MgATP, as well as a solution structure of Atg8 bound to the extreme C-terminal domain (ECTD) of Atg7. The unique N-terminal domain (NTD) of Atg7 is responsible for Atg3 (E2) binding, whereas its C-terminal domain is comprised of a homodimeric adenylation domain (AD) and ECTD. The structural and biochemical data demonstrate that Atg8 is initially recognized by the C-terminal tail of ECTD and is then transferred to an AD, where the Atg8 C terminus is attacked by the catalytic cysteine to form a thioester bond. Atg8 is then transferred via a trans mechanism to the Atg3 bound to the NTD of the opposite protomer within a dimer.

The Crystal Structure of Atg3, an Autophagy-related Ubiquitin Carrier Protein (E2) Enzyme that Mediates Atg8 Lipidation

Atg3 is an E2-like enzyme that catalyzes the conjugation of Atg8 and phosphatidylethanolamine (PE). The Atg8-PE conjugate is essential for autophagy, which is the bulk degradation process of cytoplasmic components by the vacuolar/lysosomal system. We report here the crystal structure of Saccharomyces cerevisiae Atg3 at 2.5-Å resolution. Atg3 has an α/β-fold, and its core region is topologically similar to canonical E2 enzymes. Atg3 has two regions inserted in the core region, one of which consists of ∼80 residues and has a random coil structure in solution and another with a long α-helical structure that protrudes from the core region as far as 30Å. In vivo and in vitro analyses suggested that the former region is responsible for binding Atg7, an E1-like enzyme, and that the latter is responsible for binding Atg8. A sulfate ion was bound near the catalytic cysteine of Atg3, suggesting a possible binding site for the phosphate moiety of PE. The structure of Atg3 provides a molecular basis for understanding the unique lipidation reaction that Atg3 carries out.

Attachment of an NMR-invisible solubility enhancement tag using a sortase-mediated protein ligation method

Sample solubility is essential for structural studies of proteins by solution NMR. Attachment of a solubility enhancement tag, such as GB1, MBP and thioredoxin, to a target protein has been used for this purpose. However, signal overlap of the tag with the target protein often made the spectral analysis difficult. Here we report a sortase-mediated protein ligation method to eliminate NMR signals arising from the tag by preparing the isotopically labeled target protein attached with the non-labeled GB1 tag at the C-terminus.

Autophagy-related Protein 8 (Atg8) Family Interacting Motif in Atg3 Mediates the Atg3-Atg8 Interaction and Is Crucial for the Cytoplasm-to-Vacuole Targeting Pathway

The autophagy-related protein 8 (Atg8) conjugation system is essential for the formation of double-membrane vesicles called autophagosomes during autophagy, a bulk degradation process conserved among most eukaryotes. It is also important in yeast for recognizing target vacuolar enzymes through the receptor protein Atg19 during the cytoplasm-to-vacuole targeting (Cvt) pathway, a selective type of autophagy. Atg3 is an E2-like enzyme that conjugates Atg8 with phosphatidylethanolamine. Here, we show that Atg3 directly interacts with Atg8 through the WEDL sequence, which is distinct from canonical interaction between E2 and ubiquitin-like modifiers. Moreover, NMR experiments suggest that the mode of interaction between Atg8 and Atg3 is quite similar to that between Atg8/LC3 and the Atg8 family interacting motif (AIM) conserved in autophagic receptors, such as Atg19 and p62. Thus, the WEDL sequence in Atg3 is a canonical AIM. In vitro analyses showed that Atg3 AIM is crucial for the transfer of Atg8 from the Atg8∼Atg3 thioester intermediate to phosphatidylethanolamine but not for the formation of the intermediate. Intriguingly, in vivo experiments showed that it is necessary for the Cvt pathway but not for starvation-induced autophagy. Atg3 AIM attenuated the inhibitory effect of Atg19 on Atg8 lipidation in vitro, suggesting that Atg3 AIM may be important for the lipidation of Atg19-bound Atg8 during the Cvt pathway.

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.

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