Cryo-EM structure of a RAS/RAF recruitment complex.

RAF-family kinases are activated by recruitment to the plasma membrane by GTP-bound RAS, whereupon they initiate signaling through the MAP kinase cascade. Prior structural studies of KRAS with RAF have focused on the isolated RAS-binding and cysteine-rich domains of RAF (RBD and CRD, respectively), which interact directly with RAS. Here we describe cryo-EM structures of a KRAS bound to intact BRAF in an autoinhibited state with MEK1 and a 14-3-3 dimer. Analysis of this KRAS/BRAF/MEK1/14-3-3 complex reveals KRAS bound to the RAS-binding domain of BRAF, captured in two orientations. Core autoinhibitory interactions in the complex are unperturbed by binding of KRAS and in vitro activation studies confirm that KRAS binding is insufficient to activate BRAF, absent membrane recruitment. These structures illustrate the separability of binding and activation of BRAF by RAS and suggest stabilization of this pre-activation intermediate as an alternative therapeutic strategy to blocking binding of KRAS.

Isolation and characterization of single domain antibodies from banded houndshark (Triakis scyllium) targeting SARS-CoV-2 spike RBD protein.

The COVID-19 pandemic has significantly impacted human health for three years. To mitigate the spread of SARS-CoV-2, the development of neutralizing antibodies has been accelerated, including the exploration of alternative antibody formats such as single-domain antibodies. In this study, we identified variable new antigen receptors (VNARs) specific for the receptor binding domain (RBD) of SARS-CoV-2 by immunizing a banded houndshark (Triakis scyllium) with recombinant wild-type RBD. Notably, the CoV2NAR-1 clone showed high binding affinities in the nanomolar range to various RBDs and demonstrated neutralizing activity against SARS-CoV-2 pseudoviruses. These results highlight the potential of the banded houndshark as an animal model for the development of VNAR-based therapeutics or diagnostics against future pandemics.

Architecture of autoinhibited and active BRAF-MEK1-14-3-3 complexes.

RAF family kinases are RAS-activated switches that initiate signalling through the MAP kinase cascade to control cellular proliferation, differentiation and survival1-3. RAF activity is tightly regulated and inappropriate activation is a frequent cause of cancer4-6; however, the structural basis for RAF regulation is poorly understood at present. Here we use cryo-electron microscopy to determine autoinhibited and active-state structures of full-length BRAF in complexes with MEK1 and a 14-3-3 dimer. The reconstruction reveals an inactive BRAF-MEK1 complex restrained in a cradle formed by the 14-3-3 dimer, which binds the phosphorylated S365 and S729 sites that flank the BRAF kinase domain. The BRAF cysteine-rich domain occupies a central position that stabilizes this assembly, but the adjacent RAS-binding domain is poorly ordered and peripheral. The 14-3-3 cradle maintains autoinhibition by sequestering the membrane-binding cysteine-rich domain and blocking dimerization of the BRAF kinase domain. In the active state, these inhibitory interactions are released and a single 14-3-3 dimer rearranges to bridge the C-terminal pS729 binding sites of two BRAFs, which drives the formation of an active, back-to-back BRAF dimer. Our structural snapshots provide a foundation for understanding normal RAF regulation and its mutational disruption in cancer and developmental syndromes.

The C-terminal region of ATG101 bridges ULK1 and PtdIns3K complex in autophagy initiation.

The initiation of macroautophagy/autophagy is tightly regulated by the upstream ULK1 kinase complex, which affects many downstream factors including the PtdIns3K complex. The phosphorylation of the right position at the right time on downstream molecules is governed by proper complex formation. One component of the ULK1 complex, ATG101, known as an accessory protein, is a stabilizer of ATG13 in cells. The WF finger region of ATG101 plays an important role in the recruitment of WIPI1 (WD repeat domain, phosphoinositide interacting protein 1) and ZFYVE1 (zinc finger FYVE-type containing 1). Here, we report that the C-terminal region identified in the structure of the human ATG101-ATG13HORMA complex is responsible for the binding of the PtdIns3K complex. This region adopts a ß-strand conformation in free ATG101, but either an α-helix or random coil in our ATG101-ATG13HORMA complex, which protrudes from the core and interacts with other molecules. The C-terminal deletion of ATG101 shows a significant defect in the interaction with PtdIns3K components and subsequently impairs autophagosome formation. This result clearly presents an additional role of ATG101 for bridging the ULK1 and PtdIns3K complexes in the mammalian autophagy process. Abbreviations: ATG: autophagy related; BECN1: beclin 1; GFP: green fluorescent protein; HORMA: Hop1p/Rev7p/MAD2; HsATG13HORMA: HORMA domain of ATG13 from Homo sapiens; KO: knockout; MAD2: mitotic arrest deficient 2 like 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PIK3R4/VPS15: phosphoinositide-3-kinase regulatory subunit 4; PtdIns3K: phosphatidylinositol 3-kinase; RB1CC1/FIP200: RB1 inducible coiled-coil 1; SAXS: small-angle X-ray scattering; ScAtg13HORMA: HORMA domain of Atg13 from Sccharomyces cerevisiae; SEC-SAXS: size-exclusion chromatography with small-angle X-ray scattering; SpAtg13HORMA: HORMA domain of Atg13 from Schizosaccharomyces pombe; SQSTM1/p62: sequestosome 1; ULK1: unc51-like autophagy activating kinase 1; UVRAG: UV radiation resistance associated; WIPI1: WD repeat domain: phosphoinositide interacting 1; ZFYVE1/DFCP1: zinc finger FYVE-type containing 1.

A novel conformation of the LC3-interacting region motif revealed by the structure of a complex between LC3B and RavZ.

LC3-family member proteins play a critical role in autophagy, a cellular process responsible for the degradation of massive cellular components including intracellular pathogens. A variety of molecules involved in the autophagic pathway engage in specific interactions with a unique sequence motif referred to as the LIR (LC3-interacting region) motif. Although identification of conserved structural features of LIR motifs in complex with LC3-family members has established a canonical LIR motif, atypical conformations of LIR motifs have recently been revealed. Here, we determined the three-dimensional crystal structures of LC3B in complex with three different LIR motifs of RavZ from Legionella pneumophila, an intracellular pathogen that can manipulate the host autophagy system. The tandem LIR motifs located in the N-terminal region of RavZ adopt a novel ß-sheet conformation and thus provide specific ionic interactions with LC3B in addition to canonical hydrophobic plugged-in interactions. Consequently, these motifs possess higher binding affinity to LC3-family members than canonical LIR motifs, although the tandem repeats can only bind to one LC3 molecule. These findings broaden our understanding of the functional repertoire of LIR motifs in autophagy.

ACCORD: an assessment tool to determine the orientation of homodimeric coiled-coils.

The coiled-coil (CC) domain is a very important structural unit of proteins that plays critical roles in various biological functions. The major oligomeric state of CCs is a dimer, which can be either parallel or antiparallel. The orientation of each α-helix in a CC domain is critical for the molecular function of CC-containing proteins, but cannot be determined easily by sequence-based prediction. We developed a biochemical method for assessing differences between parallel and antiparallel CC homodimers and named it ACCORD (Assessment tool for homodimeric Coiled-Coil ORientation Decision). To validate this technique, we applied it to 15 different CC proteins with known structures, and the ACCORD results identified these proteins well, especially with long CCs. Furthermore, ACCORD was able to accurately determine the orientation of a CC domain of unknown directionality that was subsequently confirmed by X-ray crystallography and small angle X-ray scattering. Thus, ACCORD can be used as a tool to determine CC directionality to supplement the results of in silico prediction.

The 1:2 complex between RavZ and LC3 reveals a mechanism for deconjugation of LC3 on the phagophore membrane.

Hosts utilize macroautophagy/autophagy to clear invading bacteria; however, bacteria have also developed a specific mechanism to survive by manipulating the host cell autophagy mechanism. One pathogen, Legionella pneumophila, can hinder host cell autophagy by using the specific effector protein RavZ that cleaves phosphatidylethanolamine-conjugated LC3 on the phagophore membrane. However, the detailed molecular mechanisms associated with the function of RavZ have hitherto remained unclear. Here, we report on the biochemical characteristics of the RavZ-LC3 interaction, the solution structure of the 1:2 complex between RavZ and LC3, and crystal structures of RavZ showing different conformations of the active site loop without LC3. Based on our biochemical, structural, and cell-based analyses of RavZ and LC3, both distant flexible N- and C-terminal regions containing LC3-interacting region (LIR) motifs are important for substrate recognition. These results suggest a novel mechanism of RavZ action on the phagophore membrane and lay the groundwork for understanding how bacterial pathogens can survive autophagy.

Structure biology of selective autophagy receptors.

Autophagy is a process tightly regulated by various autophagy-related proteins. It is generally classified into non-selective and selective autophagy. Whereas non-selective autophagy is triggered when the cell is under starvation, selective autophagy is involved in eliminating dysfunctional organelles, misfolded and/or ubiquitylated proteins, and intracellular pathogens. These components are recognized by autophagy receptors and delivered to phagophores. Several selective autophagy receptors have been identified and characterized. They usually have some common domains, such as LC3-interacting- region (LIR) motif, a specific cargo interacting (ubiquitin-dependent or ubiquitin-independent) domain. Recently, structural data of these autophagy receptors has been described, which provides an insight of their function in the selective autophagic process. In this review, we summarize the most up-to-date findings about the structure-function of autophagy receptors that regulates selective autophagy.

Structural basis for recognition of autophagic receptor NDP52 by the sugar receptor galectin-8.

Infectious bacteria are cleared from mammalian cells by host autophagy in combination with other upstream cellular components, such as the autophagic receptor NDP52 and sugar receptor galectin-8. However, the detailed molecular basis of the interaction between these two receptors remains to be elucidated. Here, we report the biochemical characterization of both NDP52 and galectin-8 as well as the crystal structure of galectin-8 complexed with an NDP52 peptide. The unexpected observation of nicotinamide adenine dinucleotide located at the carbohydrate-binding site expands our knowledge of the sugar-binding specificity of galectin-8. The NDP52-galectin-8 complex structure explains the key determinants for recognition on both receptors and defines a special orientation of N- and C-terminal carbohydrate recognition domains of galectin-8. Dimeric NDP52 forms a ternary complex with two monomeric galectin-8 molecules as well as two LC3C molecules. These results lay the groundwork for understanding how host cells target bacterial pathogens for autophagy.

Structures of the ribosome-inactivating protein from barley seeds reveal a unique activation mechanism.

Ribosome-inactivating protein (RIP), a defence protein found in various plants, possesses different chain architectures and activation mechanisms. The RIP from barley (bRIP) is a type I RIP and has sequence features that are divergent from those of type I and type II RIPs from dicotyledonous plants and even the type III RIP from maize. This study presents the first crystal structure of an RIP from a cereal crop, barley, in free, AMP-bound and adenine-bound states. For phasing, a codon-optimized synthetic brip1 gene was used and a vector was constructed to overexpress soluble bRIP fusion proteins; such expression has been verified in a number of cases. The overall structure of bRIP shows folding similar to that observed in other RIPs but also shows significant differences in specific regions, particularly in a switch region that undergoes a structural transition between a 3(10)-helix and a loop depending on the liganded state. The switch region is in a position equivalent to that of a proteolytically susceptible and putative ribosome-binding site in type III RIPs. Thus, the bRIP structure confirms the detailed enzymatic mechanism of this N-glycosidase and reveals a novel activation mechanism for type I RIPs from cereal crops.

Structure of the autophagic E2 enzyme Atg10.

Autophagy is a regulated degradation pathway that plays a critical role in all eukaryotic life cycles. One interesting feature of the core autophagic process, autophagosome formation, is similar to ubiquitination. One of two autophagic E2 enzymes, Atg10, interacts with Atg7 to receive Atg12, a ubiquitin-like molecule, and is also involved in the Atg12-Atg5 conjugation reaction. To date, no information on the interaction between Atg10 and Atg7 has been reported, although structural information is available pertaining to the individual components. Here, the crystal structure of Atg10 from Saccharomyces cerevisiae is described at 2.7 Å resolution. A significant improvement of the diffraction limit by heavy-atom derivatization was essential for structure determination. The core fold of yeast Atg10 is well conserved compared with those of Atg3 and other E2 enzymes. In contrast to other E2 enzymes, however, the autophagic E2 enzymes Atg3 and Atg10 possess insertion regions in the middle of the core fold and may be involved in protein function. The missing segment, which was termed the `FR-region', in Atg10 may be important for interaction with the E1 enzyme Atg7. This study provides a framework for understanding the E2 conjugation reaction in autophagy.

Insights into noncanonical E1 enzyme activation from the structure of autophagic E1 Atg7 with Atg8.

Autophagy is the degradation of cellular organelles via the lysosomal pathway. The autophagic ubiquitin-like (Ubl) molecule Atg8 is activated by the E1-like enzyme Atg7. As this noncanonical E1 enzyme's domain organization is unique among Ubl-activating E1 enzymes, the structural basis for its interactions with Atg8 and partner E2 enzymes remains obscure. Here we present the structure of the N-terminal domain of Atg7, revealing a unique protein fold and interactions with both autophagic E2 enzymes Atg3 and Atg10. The structure of the C-terminal domain of Atg7 in complex with Atg8 shows the mode of dimerization and mechanism of recognition of Atg8. Notably, the catalytic cysteine residue in Atg7 is positioned close to the C-terminal glycine of Atg8, its target for thioester formation, potentially eliminating the need for large conformational rearrangements characteristic of other E1s.