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The ULK complex initiates the autophagosome formation, and has recently been implicated in selective autophagy by interacting with autophagy receptors through its FIP200 subunit. However, the structural mechanism underlying the interactions of autophagy receptors with FIP200 and the relevant regulatory mechanism remain elusive. Here, we discover that the interactions of FIP200 Claw domain with autophagy receptors CCPG1 and Optineurin can be regulated by the phosphorylation in their respective FIP200-binding regions. We determine the crystal structures of FIP200 Claw in complex with the phosphorylated CCPG1 and Optineurin, and elucidate the detailed molecular mechanism governing the interactions of FIP200 Claw with CCPG1 and Optineurin as well as their potential regulations by kinase-mediated phosphorylation. In addition, we define the consensus FIP200 Claw-binding motif, and find other autophagy receptors that contain this motif within their conventional LC3-interacting regions. In all, our findings uncover a general and phosphoregulatable binding mode shared by many autophagy receptors to interact with FIP200 Claw for autophagosome biogenesis, and are valuable for further understanding the molecular mechanism of selective autophagy. Cooperation between the ULK complex and autophagy receptors mediates targeting cargoes to autophagosomes. Here, the authors show that interactions of ULK subunit FIP200 with autophagy receptors CCPG1 and Optineurin can be regulated by phosphorylation, suggesting a general binding mode shared by autophagy receptors.

Optineurin is an important autophagy receptor involved in several selective autophagy processes, during which its function is regulated by TBK1. Mutations of optineurin and TBK1 are both associated with neurodegenerative diseases. However, the mechanistic basis underlying the specific interaction between optineurin and TBK1 is still elusive. Here we determine the crystal structures of optineurin/TBK1 complex and the related NAP1/TBK1 complex, uncovering the detailed molecular mechanism governing the optineurin and TBK1 interaction, and revealing a general binding mode between TBK1 and its associated adaptor proteins. In addition, we demonstrate that the glaucoma-associated optineurin E50K mutation not only enhances the interaction between optineurin and TBK1 but also alters the oligomeric state of optineurin, and the ALS-related TBK1 E696K mutation specifically disrupts the optineurin/TBK1 complex formation but has little effect on the NAP1/TBK1 complex. Thus, our study provides mechanistic insights into those currently known disease-causing optineurin and TBK1 mutations found in patients. Mutations in optineurin that cause defects in the interaction with TBK1 are associated with neurodegenerative diseases. Here, the authors report the structure of this complex, and outline a general binding mode for these proteins.

Macroautophagy maintains cellular and organismal homeostasis, and entails de novo synthesis of double-membrane autophagosome. The effective formation of autophagosome requires the recruitment of the ATG12~ATG5-ATG16L1 complex to the pre-autophagosomal structure by relevant ATG16L1-binding autophagic factors including FIP200. However, the molecular mechanism governing the specific interaction of ATG16L1 with FIP200 remains elusive. Here, we uncover that ATG16L1 contains a FIP200-interacting region (FIR), which not only can directly bind FIP200 Claw domain, but also can serve as an atypical ATG8-interacting motif to selectively recognize mammalian ATG8 family proteins (ATG8s). We determine the high-resolution crystal structures of ATG16L1 FIR in complex with FIP200 Claw and GABARAPL1, respectively, and elucidate the molecular mechanism underlying the interactions of ATG16L1 with FIP200 and ATG8s. To distinguish the precise contribution of FIP200 from ATG8s for binding to ATG16L1 FIR in autophagy, we develop a ATG16L1 mutant that can exclusively interact with ATG8s but not FIP200. Finally, using relevant cell-based functional assays, we demonstrate that the interaction of ATG16L1 with FIP200 is indispensable for the effective autophagic flux. In conclusion, our findings provide mechanistic insights into the interactions of ATG16L1 with FIP200 and ATG8s, and are valuable for further understanding the function of ATG16L1 in autophagy. ATG16L1 is a key autophagic protein. Here, the authors elucidate the molecular basis of ATG16L1 interactions with FIP200 and ATG8 family proteins, showing that the interaction of ATG16L1 with FIP200 is indispensable for the effective autophagic flux.

The unconventional myosin VIIa (MYO7A) is one of the five proteins that form a network of complexes involved in formation of stereocilia. Defects in these proteins cause syndromic deaf-blindness in humans [Usher syndrome I (USH1)]. Many disease-causing mutations occur in myosin tail homology 4-protein 4.1, ezrin, radixin, moesin (MyTH4-FERM) domains in the myosin tail that binds to another USH1 protein, Sans. We report the crystal structure of MYO7A MyTH4-FERM domains in complex with the central domain (CEN) of Sans at 2.8 angstrom resolution. The MyTH4 and FERM domains form an integral structural and functional supramodule binding to two highly conserved segments (CEN1 and 2) of Sans. The MyTH4-FERM/CEN complex structure provides mechanistic explanations for known deafness-causing mutations in MYO7A MyTH4-FERM. The structure will also facilitate mechanistic and functional studies of MyTH4-FERM domains in other myosins.

Ubiquitination plays vital roles in modulating pathogen-host cell interactions. RNF213, a E3 ligase, can catalyze the ubiquitination of lipopolysaccharide (LPS) and is crucial for antibacterial immunity in mammals. Shigella flexneri , an LPS-containing pathogenic bacterium, has developed mechanisms to evade host antibacterial defenses during infection. However, the precise strategies by which S. flexneri circumvents RNF213-mediated antibacterial immunity remain poorly understood. Here, through comprehensive biochemical, structural and cellular analyses, we reveal that the E3 effector IpaH1.4 of S. flexneri can directly target human RNF213 via a specific interaction between the IpaH1.4 LRR domain and the RING domain of RNF213, and mediate the ubiquitination and proteasomal degradation of RNF213 in cells. Furthermore, we determine the cryo-EM structure of human RNF213 and the crystal structure of the IpaH1.4 LRR/RNF213 RING complex, elucidating the molecular mechanism underlying the specific recognition of RNF213 by IpaH1.4. Finally, our cell based functional assays demonstrate that the targeting of host RNF213 by IpaH1.4 promotes S. flexneri proliferation within infected cells. In summary, our work uncovers an unprecedented strategy employed by S. flexneri to subvert the key host immune factor RNF213, thereby facilitating bacterial proliferation during invasion. RNF213 is a key player in fighting against various invasive pathogens in mammals. Here, the authors show that pathogenic Shigella flexneri can use its effector IpaH1.4 to directly target and subvert RNF213 to evade host antibacterial immunity.

The p97-UFD1L-NPLOC4 ATPase unfolds numerous proteins for proteasomal degradation, but whether it suffices to pull proteins out of lipid bilayer remains unclear. Here, we identify a conserved ubiquitin-binding helix (UBH) in many UBX-containing p97 adapters, including FAF2, across yeast, plants, and metazoans. The UBH-UBX substantially facilitates the engagement of ubiquitinated substrates with p97-UFD1L-NPLOC4, and enhances p97 motor’s working ATPase and unfolding activities by approximately twofold. Using purified p97-UFD1L-NPLOC4-FAF2 UBH-UBX , we reconstitute membrane protein extraction from the ER and mitochondria, establishing p97-UFD1L-NPLOC4-FAF2 (p97-UNF) as a power-enhanced unfoldase. Deleting UBH or disrupting UBH-ubiquitin interaction impairs substrate targeting, reduces p97-UNF’s working ATPase and unfolding activities, and abolishes membrane protein extraction and degradation. We propose that UBH-UBX module amplifies p97’s mechanical output power, enabling the removal of challenging substrates from large assemblies and ensuring rapid responses to protein misfolding or regulatory signals in diverse physiological processes. p97-UFD1L-NPLOC4 unfolds proteins for proteasomal degradation, but whether it suffices to extract proteins from lipid bilayers is unclear. We show that the UBH-UBX module in FAF2 and its homologs double p97’s ATPase and unfolding activity, and drives membrane protein extraction.

Myosin VI plays crucial roles in diverse cellular processes. In autophagy, Myosin VI can facilitate the maturation of autophagosomes through interactions with Tom1 and the autophagy receptors, Optineurin, NDP52 and TAX1BP1. Here, we report the high-resolution crystal structure of the C-terminal cargo-binding domain (CBD) of Myosin VI in complex with Tom1, which elucidates the mechanistic basis underpinning the specific interaction between Myosin VI and Tom1, and uncovers that the C-terminal CBD of Myosin VI adopts a unique cargo recognition mode to interact with Tom1 for tethering. Furthermore, we show that Myosin VI can serve as a bridging adaptor to simultaneously interact with Tom1 and autophagy receptors through two distinct interfaces. In all, our findings provide mechanistic insights into the interactions of Myosin VI with Tom1 and relevant autophagy receptors, and are valuable for further understanding the functions of these proteins in autophagy and the cargo recognition modes of Myosin VI. Myosin VI can facilitate the maturation of autophagosomes in autophagy through interactions with Tom1 and autophagy receptors. Here authors report the structure of the cargobinding domain of Myosin VI in complex with Tom1, which provides insights into Myosin IV’s cargo recognition modes.

Oxepinone rings represent one of structurally unusual motifs of natural products and the biosynthesis of oxepinones is not fully understood. 1,5- Seco -vibralactone ( 3 ) features an oxepinone motif and is a stable metabolite isolated from mycelial cultures of the mushroom Boreostereum vibrans . Cyclization of 3 forms vibralactone ( 1 ) whose β-lactone-fused bicyclic core originates from 4-hydroxybenzoate, yet it remains elusive how 4-hydroxybenzoate is converted to 3 especially for the oxepinone ring construction in the biosynthesis of 1 . In this work, using activity-guided fractionation together with proteomic analyses, we identify an NADPH/FAD-dependent monooxygenase VibO as the key enzyme performing a crucial ring-expansive oxygenation on the phenol ring to generate the oxepin-2-one structure of 3 . The crystal structure of VibO reveals that it forms a dimeric phenol hydroxylase-like architecture featured with a unique substrate-binding pocket adjacent to the bound FAD. Computational modeling and solution studies provide insight into the likely VibO active site geometry, and suggest possible involvement of a flavin-C4a-OO(H) intermediate. Vibralactone is a strong lipase inhibitor and a bicyclic β-lactone containing an oxepinone ring, whose biosynthetic construction was unknown. Here the authors identify an oxepinone-building flavin monooxygenase VibO that is involved in the biosynthesis of vibralactone, and determine its X-ray crystal structure.

Syntaxin17, a key autophagosomal N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) protein, can associate with ATG8 family proteins SNAP29 and VAMP8 to facilitate the membrane fusion process between the double-membraned autophagosome and single-membraned lysosome in mammalian macroautophagy. However, the inherent properties of Syntaxin17 and the mechanistic basis underlying the interactions of Syntaxin17 with its binding proteins remain largely unknown. Here, using biochemical, NMR, and structural approaches, we systemically characterized Syntaxin17 as well as its interactions with ATG8 family proteins, SNAP29 and VAMP8. We discovered that Syntaxin17 alone adopts an autoinhibited conformation mediated by a direct interaction between its Habc domain and the Qa- SNARE motif. In addition, we revealed that the Qa-SNARE region of Syntaxin17 contains one LC3-interacting region (LIR) motif, which preferentially binds to GABARAP subfamily members. Importantly, the GABARAP binding of Syntaxin17 can release its autoinhibited state. The determined crystal structure of the Syntaxin17 LIR–GABARAP complex not only provides mechanistic insights into the interaction between Syntaxin17 and GABARAP but also reveals an unconventional LIR motif with a C-terminally extended 310 helix for selectively binding to ATG8 family proteins. Finally, we also elucidated structural arrangements of the autophagic Syntaxin17–SNAP29–VAMP8 SNARE core complex, and uncovered its conserved biochemical and structural characteristics common to all other SNAREs. In all, our findings reveal three distinct states of Syntaxin17, and provide mechanistic insights into the Syntaxin17-mediated autophagosome–lysosome fusion process.

The prolyl hydroxylation of hypoxia-inducible factor 1α (HIF-1α) mediated by the EGLN–pVHL pathway represents a classic signalling mechanism that mediates cellular adaptation under hypoxia. Here we identify RIPK1, a known regulator of cell death mediated by tumour necrosis factor receptor 1 (TNFR1), as a target of EGLN1–pVHL. Prolyl hydroxylation of RIPK1 mediated by EGLN1 promotes the binding of RIPK1 with pVHL to suppress its activation under normoxic conditions. Prolonged hypoxia promotes the activation of RIPK1 kinase by modulating its proline hydroxylation, independent of the TNFα–TNFR1 pathway. As such, inhibiting proline hydroxylation of RIPK1 promotes RIPK1 activation to trigger cell death and inflammation. Hepatocyte-specific Vhl deficiency promoted RIPK1-dependent apoptosis to mediate liver pathology. Our findings illustrate a key role of the EGLN–pVHL pathway in suppressing RIPK1 activation under normoxic conditions to promote cell survival and a model by which hypoxia promotes RIPK1 activation through modulating its proline hydroxylation to mediate cell death and inflammation in human diseases, independent of TNFR1. Zhang, Xu, Liu, Wang et al. identify an inhibitory mechanism for RIPK1 kinase through EGLN1/pVHL-mediated proline hydroxylation, which is disrupted upon prolonged hypoxia that activates RIPK1 activity to promote cell death and inflammation.