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Secreted class 3 semaphorins (Sema3s) form tripartite complexes with the plexin receptor and neuropilin coreceptor, which are both transmembrane proteins that together mediate semaphorin signal for neuronal axon guidance and other processes. Despite extensive investigations, the overall architecture of and the molecular interactions in the Sema3/plexin/neuropilin complex are incompletely understood. Here we present the cryo-EM structure of a near intact extracellular region complex of Sema3A, PlexinA4 and Neuropilin 1 (Nrp1) at 3.7 Å resolution. The structure shows a large symmetric 2:2:2 assembly in which each subunit makes multiple interactions with others. The two PlexinA4 molecules in the complex do not interact directly, but their membrane proximal regions are close to each other and poised to promote the formation of the intracellular active dimer for signaling. The structure reveals a previously unknown interface between the a2b1b2 module in Nrp1 and the Sema domain of Sema3A. This interaction places the a2b1b2 module at the top of the complex, far away from the plasma membrane where the transmembrane regions of Nrp1 and PlexinA4 embed. As a result, the region following the a2b1b2 module in Nrp1 must span a large distance to allow the connection to the transmembrane region, suggesting an essential role for the long non-conserved linkers and the MAM domain in neuropilin in the semaphorin/plexin/neuropilin complex., Secreted class 3 semaphorins (Sema3s) form tripartite complexes with a plexin receptor and neuropilin co-receptor to transduce signals for neuronal axon guidance and other processes. Here, the authors present the cryo-EM structure of the extracellular Sema3A/PlexinA4/Neuropilin1 complex that provides further insights into the interactions among semaphorin, plexin and neuropilin and reveals long flexible linkers in semaphorin and neuropilin that are important for complex formation.

The cyclic dinucleotide (CDN)-stimulator of interferon genes (STING) pathway plays an important role in the detection of viral and bacterial pathogens in animals. Previous studies have shown that the metazoan second messenger cyclic [G(2′,5′)pA(3′,5′)p] (2′,3′-cGAMP) generated by cyclic GMP-AMP synthase cGAS binds STING with high affinity compared with bacterial CDNs such as c-di-GMP, c-di-AMP, and 3′,3′-cGAMP. Despite recent progress indicating that the CDN-binding domain (CBD) of dimeric STING binds asymmetric 2′,3′-cGAMP preferentially over symmetric 3′,3′-CDNs, it remains an open question whether STING molecules, such as human STING, adopt a symmetric dimeric conformation to efficiently engage its asymmetric ligand. Here, structural studies of the CBD from porcine STING (STINGCBD) in complex with CDNs at 1.76–2.6 Å resolution revealed that porcine STINGCBD, unlike its human and mouse counterparts, can adopt an asymmetric ligand-binding pocket to accommodate the CDNs. We observed that the extensive interactions and shape complementarity between asymmetric 2′,3′-cGAMP and the ligand-binding pocket make it the most preferred ligand for porcine STING and that geometry constraints limit the binding between symmetric 3′,3′-CDN and porcine STING. The ligand-discrimination mechanism of porcine STING observed here expands our understanding of how the CDN–STING pathway is activated and of its role in antiviral defense.

RET is a receptor tyrosine kinase (RTK) that plays essential roles in development and has been implicated in several human diseases. Different from most of RTKs, RET requires not only its cognate ligands but also co-receptors for activation, the mechanisms of which remain unclear due to lack of high-resolution structures of the ligand/co-receptor/receptor complexes. Here, we report cryo-EM structures of the extracellular region ternary complexes of GDF15/GFRAL/RET, GDNF/GFRα1/RET, NRTN/GFRα2/RET and ARTN/GFRα3/RET. These structures reveal that all the four ligand/co-receptor pairs, while using different atomic interactions, induce a specific dimerization mode of RET that is poised to bring the two kinase domains into close proximity for cross-phosphorylation. The NRTN/GFRα2/RET dimeric complex further pack into a tetrameric assembly, which is shown by our cell-based assays to regulate the endocytosis of RET. Our analyses therefore reveal both the common mechanism and diversification in the activation of RET by different ligands.

Insulin signaling controls metabolic homeostasis. Here, we report the cryo-EM structure of full-length insulin receptor (IR) and insulin complex in the active state. This structure unexpectedly reveals that maximally four insulins can bind the ‘T’-shaped IR dimer at four distinct sites related by 2-fold symmetry. Insulins 1 and 1’ bind to sites 1 and 1’, formed by L1 of one IR protomer and α-CT and FnIII-1 of the other. Insulins 2 and 2’ bind to sites 2 and 2’ on FnIII-1 of each protomer. Mutagenesis and cellular assays show that both sites 1 and 2 are required for optimal insulin binding and IR activation. We further identify a homotypic FnIII-2–FnIII-2 interaction in mediating the dimerization of membrane proximal domains in the active IR dimer. Our results indicate that binding of multiple insulins at two distinct types of sites disrupts the autoinhibited apo-IR dimer and stabilizes the active dimer.

Histidine biosynthesis, which is absent in animals, was shown to be highly conserved among gram-negative bacteria, thus making it an attractive target for antibiotic design. There are many fusion forms of enzymes in the histidine biosynthetic pathway and people still have limited knowledge about their domain organizations and catalytic mechanisms, due to the lack of structural information. Here we report the first crystal structure of Shigella flexneri bi-functional enzyme HisIE (SfHisIE) that functions in the 2nd and 3rd steps in the histidine biosynthetic pathway. This structure shows that HisIE exists as dimers with two loops (fusion loop) connecting the individual dimer of HisE and HisI in its N-terminus and C-terminus respectively. Our mutagenesis study shows mutations in this fusion loop are lethal for bacteria indicating the advantage of gene fusion in Histidine biosynthesis. Structural analysis revealed several highly conserved residues in the putative ligand binding grooves of HisE and HisI, showing an evolutionarily conserved catalytic mechanism shared among gram negative-bacteria.

The influenza virulence factor NS1 protein interacts with the cellular NS1-BP protein to promote splicing and nuclear export of the viral M mRNAs. The viral M1 mRNA encodes the M1 matrix protein and is alternatively spliced into the M2 mRNA, which is translated into the M2 ion channel. These proteins have key functions in viral trafficking and budding. To uncover the NS1-BP structural and functional activities in splicing and nuclear export, we performed proteomics analysis of nuclear NS1-BP binding partners and showed its interaction with constituents of the splicing and mRNA export machineries. NS1-BP BTB domains form dimers in the crystal. Full-length NS1-BP is a dimer in solution and forms at least a dimer in cells. Mutations suggest that dimerization is important for splicing. The central BACK domain of NS1-BP interacts directly with splicing factors such as hnRNP K and PTBP1 and with the viral NS1 protein. The BACK domain is also the site for interactions with mRNA export factor Aly/REF and is required for viral M mRNA nuclear export. The crystal structure of the C-terminal Kelch domain shows that it forms a β-propeller fold, which is required for the splicing function of NS1-BP. This domain interacts with the polymerase II C-terminal domain and SART1, which are involved in recruitment of splicing factors and spliceosome assembly, respectively. NS1-BP functions are not only critical for processing a subset of viral mRNAs but also impact levels and nuclear export of a subset of cellular mRNAs encoding factors involved in metastasis and immunity.

FAM46C, a non-canonical poly(A) polymerase, is frequently mutated in multiple myeloma. Loss-of-function of FAM46C promotes cell survival of multiple myeloma, suggesting a tumor-suppressive role. FAM46C is also essential for fastening sperm head and flagellum, indispensable for male fertility. The molecular mechanisms of these functions of FAM46C remain elusive. We report the crystal structure of FAM46C to provide the basis for its poly(A) polymerase activity and rationalize mutations associated with multiple myeloma. In addition, we found that FAM46C interacts directly with the serine/threonine kinase Plk4, the master regulator of centrosome duplication. We present the structure of FAM46C in complex with the Cryptic Polo-Box 1–2 domains of Plk4. Our structure-based mutational analyses show that the interaction with Plk4 recruits FAM46C to centrosomes. Our data suggest that Plk4-mediated localization of FAM46C enables its regulation of centrosome structure and functions, which may underlie the roles for FAM46C in cell proliferation and sperm development.

Holliday junction (HJ) is a hallmark intermediate in DNA recombination and must be processed by dissolution (for double HJ) or resolution to ensure genome stability. Although HJ resolvases have been identified in all domains of life, there is a long-standing effort to search in prokaryotes and eukarya for proteins promoting HJ migration. Here, we report the structural and functional characterization of a novel ATPase, Sulfolobus islandicus PilT N-terminal-domain-containing ATPase (SisPINA), encoded by the gene adjacent to the resolvase Hjc coding gene. PINA is conserved in archaea and vital for S. islandicus viability. Purified SisPINA forms hexameric rings in the crystalline state and in solution, similar to the HJ migration helicase RuvB in Gram-negative bacteria. Structural analysis suggests that ATP binding and hydrolysis cause conformational changes in SisPINA to drive branch migration. Further studies reveal that SisPINA interacts with SisHjc and coordinates HJ migration and cleavage.

The GIPC family adaptor proteins mediate endocytosis by tethering cargo proteins to the myosin VI motor. The structural mechanisms for the GIPC/cargo and GIPC/myosin VI interactions remained unclear. PlexinD1, a transmembrane receptor that regulates neuronal and cardiovascular development, is a cargo of GIPCs. GIPC-mediated endocytic trafficking regulates PlexinD1 signaling. Here, we unravel the mechanisms of the interactions among PlexinD1, GIPCs and myosin VI by a series of crystal structures of these proteins in apo or bound states. GIPC1 forms a domain-swapped dimer in an autoinhibited conformation that hinders binding of both PlexinD1 and myosin VI. PlexinD1 binding to GIPC1 releases the autoinhibition, promoting its interaction with myosin VI. GIPCs and myosin VI interact through two distinct interfaces and form an open-ended alternating array. Our data support that this alternating array underlies the oligomerization of the GIPC/Myosin VI complexes in solution and cells. DOI: http://dx.doi.org/10.7554/eLife.27322.001

Summary Cyclic dinucleotides are a newly expanded class of second messengers that contribute to the regulation of multiple different pathways in bacterial, eukaryotic, and archaeal cells. The recently identified Vibrio cholerae dinucleotide cyclase (DncV, the gene product of VC0179 ) can generate three different cyclic dinucleotides and preferentially synthesize a hybrid cyclic-GMP-AMP. Here, we report the crystal structural and functional studies of DncV. We unexpectedly observed a 5-methyltetrahydrofolate diglutamate (5MTHFGLU2) molecule bound in a surface pocket opposite the nucleotide substrate-binding groove of DncV. Subsequent mutagenesis and functional studies showed that the enzymatic activity of DncV is regulated by folate-like molecules, suggesting the existence of a signaling pathway that links folate-like metabolism cofactors to the regulation of cyclic dinucleotide second messenger synthesis. Sequence analysis showed that the residues involved in 5MTHFGLU2 binding are highly conserved in DncV orthologs, implying the presence of this regulation mechanism in a wide variety of bacteria.

The opportunistic pathogen Pseudomonas aeruginosa uses the type VI secretion system (T6SS) to deliver the muramidase Tse3 into the periplasm of rival bacteria to degrade their peptidoglycan (PG). Concomitantly, P. aeruginosa uses the periplasm-localized immunity protein Tsi3 to prevent potential self-intoxication caused by Tse3, and thus gains an edge over rival bacteria in fierce niche competition. Here, we report the crystal structures of Tse3 and the Tse3-Tsi3 complex. Tse3 contains an annexin repeat-like fold at the N-terminus and a G-type lysozyme fold at the C-terminus. One loop in the N-terminal domain (Loop 12) and one helix (α9) from the C-terminal domain together anchor Tse3 and the Tse3-Tsi3 complex to membrane in a calcium-dependent manner in vitro, and this membrane-binding ability is essential for Tse3's activity. In the C-terminal domain, a Y-shaped groove present on the surface likely serves as the PG binding site. Two calcium-binding motifs are also observed in the groove and these are necessary for Tse3 activity. In the Tse3-Tsi3 structure, three loops of Tsi3 insert into the substrate-binding groove of Tse3, and three calcium ions present at the interface of the complex are indispensable for the formation of the Tse3-Tsi3 complex.

Terpenoids are a large and highly diverse group of natural products, with the most chemically diverse pool of structures. Terpene synthase is the key enzyme in the process of terpenoid synthesis. In this paper, the first diterpene synthase (CYC) of bacterial origin was successfully crystallized. Native and SeMet-derivative crystals diffracted to 1.75 and 2.6 Å resolution, respectively. The native crystal belonged to space group P212121, with unit-cell parameters a = 59.10, b = 101.73, c = 108.93 Å, and contained two molecules per asymmetric unit. The SeMet-derivative crystal belonged to space group P21, with unit-cell parameters a = 58.64, b = 109.47, c = 58.73 Å, β = 119.35°, and had two molecules per asymmetric unit.

Pseudomonas aeruginosa uses the type VI secretion system (T6SS) to inject effector proteins into rival cells in niche competition. Tse3, one of the effectors of T6SS, is delivered into the periplasm of recipient cells. Tse3 functions as a muramidase that degrades the β-1,4-linkage between N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) in peptidoglycan, thus leading to lysis of the recipient cells and providing a competitive advantage to the donor cells. Here, the preliminary crystallographic study of Tse3 is reported. A crystal of Tse3 diffracted to 1.5 Å resolution. It belonged to space group C121, with unit-cell parameters a = 166.99, b = 70.13, c = 41.94 Å, α = 90.00, β = 90.52, γ = 90.00° and one molecule per asymmetric unit.

Ribosome-recycling factor (RRF) plays an essential role in the fourth step of protein synthesis in prokaryotes. RRF combined with elongation factor G (EF-G) disassembles the post-termination ribosome complex and recycles the protein synthesis machine for the next round of translation. A reductive-methylation-modified RRF fromThermoanaerobacter tengcongensis(TteRRF) has been crystallized using the vapour-diffusion method. The crystal grew in a condition consisting of 0.1 Mcitric acid pH 3.5, 3.0 MNaCl and 50 mg ml−1methylated protein solution at 289 K. A complete data set was collected from a crystal to 2.80 Å resolution using synchrotron radiation at 100 K. The crystal belonged to space groupP6122/P6522 with unit-cell parametersa=b= 103.26,c= 89.17 Å. The asymmetric unit was estimated to contain one molecule ofTteRRF.