Molecular mechanism of BMP signal control by Twisted gastrulation.

Twisted gastrulation (TWSG1) is an evolutionarily conserved secreted glycoprotein which controls signaling by Bone Morphogenetic Proteins (BMPs). TWSG1 binds BMPs and their antagonist Chordin to control BMP signaling during embryonic development, kidney regeneration and cancer. We report crystal structures of TWSG1 alone and in complex with a BMP ligand, Growth Differentiation Factor 5. TWSG1 is composed of two distinct, disulfide-rich domains. The TWSG1 N-terminal domain occupies the BMP type 1 receptor binding site on BMPs, whereas the C-terminal domain binds to a Chordin family member. We show that TWSG1 inhibits BMP function in cellular signaling assays and mouse colon organoids. This inhibitory function is abolished in a TWSG1 mutant that cannot bind BMPs. The same mutation in the Drosophila TWSG1 ortholog Tsg fails to mediate BMP gradient formation required for dorsal-ventral axis patterning of the early embryo. Our studies reveal the evolutionarily conserved mechanism of BMP signaling inhibition by TWSG1.

Crystal structure of LGR ligand α2/ß5 from Caenorhabditis elegans with implications for the evolution of glycoprotein hormones.

A family of leucine-rich-repeat-containing G-protein-coupled receptors (LGRs) mediate diverse physiological responses when complexed with their cognate ligands. LGRs are present in all metazoan animals. In humans, the LGR ligands include glycoprotein hormones (GPHs) chorionic gonadotropin (hCG), luteinizing hormone, follicle-stimulating hormone (hFSH), and thyroid-stimulating hormone (hTSH). These hormones are αß heterodimers of cystine-knot protein chains. LGRs and their ligand chains have coevolved. Ancestral hormone homologs, present in both bilaterian animals and chordates, are identified as α2ß5. We have used single-wavelength anomalous diffraction and molecular replacement to determine structures of the α2ß5 hormone from Caenorhabditis elegans (Ceα2ß5). Ceα2ß5 is unglycosylated, as are many other α2ß5 hormones. Both Hsα2ß5, the human homolog of Ceα2ß5, and hTSH activate the same receptor (hTSHR). Despite having little sequence similarity to vertebrate GPHs, apart from the cysteine patterns from core disulfide bridges, Ceα2ß5 is generally similar in structure to these counterparts; however, its α2 and ß5 subunits are more symmetric as compared with α and ß of hCG and hFSH. This quasisymmetry suggests a hypothetical homodimeric antecedent of the α2ß5 and αß heterodimers. Known structures together with AlphaFold models from the sequences for other LGR ligands provide representatives for the molecular evolution of LGR ligands from early metazoans through the present-day GPHs. The experimental Ceα2ß5 structure validates its AlphaFold model, and thus also that for Hsα2ß5; and interfacial characteristics in a model for the Hsα2ß5:hTSHR complex are similar to those found in an experimental hTSH:hTSHR structure.

Structural basis for proton coupled cystine transport by cystinosin.

Amino acid transporters play a key role controlling the flow of nutrients across the lysosomal membrane and regulating metabolism in the cell. Mutations in the gene encoding the transporter cystinosin result in cystinosis, an autosomal recessive metabolic disorder characterised by the accumulation of cystine crystals in the lysosome. Cystinosin is a member of the PQ-loop family of solute carrier (SLC) transporters and uses the proton gradient to drive cystine export into the cytoplasm. However, the molecular basis for cystinosin function remains elusive, hampering efforts to develop novel treatments for cystinosis and understand the mechanisms of ion driven transport in the PQ-loop family. To address these questions, we present the crystal structures of cystinosin from Arabidopsis thaliana in both apo and cystine bound states. Using a combination of in vitro and in vivo based assays, we establish a mechanism for cystine recognition and proton coupled transport. Mutational mapping and functional characterisation of human cystinosin further provide a framework for understanding the molecular impact of disease-causing mutations.

Hedgehog-Interacting Protein is a multimodal antagonist of Hedgehog signalling.

Hedgehog (HH) morphogen signalling, crucial for cell growth and tissue patterning in animals, is initiated by the binding of dually lipidated HH ligands to cell surface receptors. Hedgehog-Interacting Protein (HHIP), the only reported secreted inhibitor of Sonic Hedgehog (SHH) signalling, binds directly to SHH with high nanomolar affinity, sequestering SHH. Here, we report the structure of the HHIP N-terminal domain (HHIP-N) in complex with a glycosaminoglycan (GAG). HHIP-N displays a unique bipartite fold with a GAG-binding domain alongside a Cysteine Rich Domain (CRD). We show that HHIP-N is required to convey full HHIP inhibitory function, likely by interacting with the cholesterol moiety covalently linked to HH ligands, thereby preventing this SHH-attached cholesterol from binding to the HH receptor Patched (PTCH1). We also present the structure of the HHIP C-terminal domain in complex with the GAG heparin. Heparin can bind to both HHIP-N and HHIP-C, thereby inducing clustering at the cell surface and generating a high-avidity platform for SHH sequestration and inhibition. Our data suggest a multimodal mechanism, in which HHIP can bind two specific sites on the SHH morphogen, alongside multiple GAG interactions, to inhibit SHH signalling.

Multiple liquid crystalline geometries of highly compacted nucleic acid in a dsRNA virus.

Characterizing the genome of mature virions is pivotal to understanding the highly dynamic processes of virus assembly and infection. Owing to the different cellular fates of DNA and RNA, the life cycles of double-stranded (ds)DNA and dsRNA viruses are dissimilar. In terms of nucleic acid packing, dsDNA viruses, which lack genome segmentation and intra-capsid transcriptional machinery, predominantly display single-spooled genome organizations1-8. Because the release of dsRNA into the cytoplasm triggers host defence mechanisms9, dsRNA viruses retain their genomes within a core particle that contains the enzymes required for RNA replication and transcription10-12. The genomes of dsRNA viruses vary greatly in the degree of segmentation. In members of the Reoviridae family, genomes consist of 10-12 segments and exhibit a non-spooled arrangement mediated by RNA-dependent RNA polymerases11-14. However, whether this arrangement is a general feature of dsRNA viruses remains unknown. Here, using cryo-electron microscopy to resolve the dsRNA genome structure of the tri-segmented bacteriophage ɸ6 of the Cystoviridae family, we show that dsRNA viruses can adopt a dsDNA-like single-spooled genome organization. We find that in this group of viruses, RNA-dependent RNA polymerases do not direct genome ordering, and the dsRNA can adopt multiple conformations. We build a model that encompasses 90% of the genome, and use this to quantify variation in the packing density and to characterize the different liquid crystalline geometries that are exhibited by the tightly compacted nucleic acid. Our results demonstrate that the canonical model for the packing of dsDNA can be extended to dsRNA viruses.

The structure of a prokaryotic viral envelope protein expands the landscape of membrane fusion proteins.

Lipid membrane fusion is an essential function in many biological processes. Detailed mechanisms of membrane fusion and the protein structures involved have been mainly studied in eukaryotic systems, whereas very little is known about membrane fusion in prokaryotes. Haloarchaeal pleomorphic viruses (HRPVs) have a membrane envelope decorated with spikes that are presumed to be responsible for host attachment and membrane fusion. Here we determine atomic structures of the ectodomains of the 57-kDa spike protein VP5 from two related HRPVs revealing a previously unreported V-shaped fold. By Volta phase plate cryo-electron tomography we show that VP5 is monomeric on the viral surface, and we establish the orientation of the molecules with respect to the viral membrane. We also show that the viral membrane fuses with the host cytoplasmic membrane in a process mediated by VP5. This sheds light on protein structures involved in prokaryotic membrane fusion.

Structure-Based Classification Defines the Discrete Conformational Classes Adopted by the Arenaviral GP1.

The emergence of Old and New World arenaviruses from rodent reservoirs persistently threatens human health. The GP1 subunit of the envelope-displayed arenaviral glycoprotein spike complex (GPC) mediates host cell recognition and is an important determinant of cross-species transmission. Previous structural analyses of Old World arenaviral GP1 glycoproteins, alone and in complex with a cognate GP2 subunit, have revealed that GP1 adopts two distinct conformational states distinguished by differences in the orientations of helical regions of the molecule. Here, through comparative study of the GP1 glycoprotein architectures of Old World Loei River virus and New World Whitewater Arroyo virus, we show that these rearrangements are restricted to Old World arenaviruses and are not induced solely by the pH change that is associated with virus endosomal trafficking. Our structure-based phylogenetic analysis of arenaviral GP1s provides a blueprint for understanding the discrete structural classes adopted by these therapeutically important targets.IMPORTANCE The genetically and geographically diverse group of viruses within the family Arenaviridae includes a number of zoonotic pathogens capable of causing fatal hemorrhagic fever. The multisubunit GPC glycoprotein spike complex displayed on the arenavirus envelope is a key determinant of species tropism and a primary target of the host humoral immune response. Here, we show that the receptor-binding GP1 subcomponent of the GPC spike from Old World but not New World arenaviruses adopts a distinct, pH-independent conformation in the absence of the cognate GP2. Our analysis provides a structure-based approach to understanding the discrete conformational classes sampled by these therapeutically important targets, informing strategies to develop arenaviral glycoprotein immunogens that resemble GPC as presented on the mature virion surface.

Structural basis for antibacterial peptide self-immunity by the bacterial ABC transporter McjD.

Certain pathogenic bacteria produce and release toxic peptides to ensure either nutrient availability or evasion from the immune system. These peptides are also toxic to the producing bacteria that utilize dedicated ABC transporters to provide self-immunity. The ABC transporter McjD exports the antibacterial peptide MccJ25 in Escherichia coli Our previously determined McjD structure provided some mechanistic insights into antibacterial peptide efflux. In this study, we have determined its structure in a novel conformation, apo inward-occluded and a new nucleotide-bound state, high-energy outward-occluded intermediate state, with a defined ligand binding cavity. Predictive cysteine cross-linking in E. coli membranes and PELDOR measurements along the transport cycle indicate that McjD does not undergo major conformational changes as previously proposed for multi-drug ABC exporters. Combined with transport assays and molecular dynamics simulations, we propose a novel mechanism for toxic peptide ABC exporters that only requires the transient opening of the cavity for release of the peptide. We propose that shielding of the cavity ensures that the transporter is available to export the newly synthesized peptides, preventing toxic-level build-up.

Crystal structure of the RNA-dependent RNA polymerase from influenza C virus.

Negative-sense RNA viruses, such as influenza, encode large, multidomain RNA-dependent RNA polymerases that can both transcribe and replicate the viral RNA genome. In influenza virus, the polymerase (FluPol) is composed of three polypeptides: PB1, PB2 and PA/P3. PB1 houses the polymerase active site, whereas PB2 and PA/P3 contain, respectively, cap-binding and endonuclease domains required for transcription initiation by cap-snatching. Replication occurs through de novo initiation and involves a complementary RNA intermediate. Currently available structures of the influenza A and B virus polymerases include promoter RNA (the 5' and 3' termini of viral genome segments), showing FluPol in transcription pre-initiation states. Here we report the structure of apo-FluPol from an influenza C virus, solved by X-ray crystallography to 3.9 Å, revealing a new 'closed' conformation. The apo-FluPol forms a compact particle with PB1 at its centre, capped on one face by PB2 and clamped between the two globular domains of P3. Notably, this structure is radically different from those of promoter-bound FluPols. The endonuclease domain of P3 and the domains within the carboxy-terminal two-thirds of PB2 are completely rearranged. The cap-binding site is occluded by PB2, resulting in a conformation that is incompatible with transcription initiation. Thus, our structure captures FluPol in a closed, transcription pre-activation state. This reveals the conformation of newly made apo-FluPol in an infected cell, but may also apply to FluPol in the context of a non-transcribing ribonucleoprotein complex. Comparison of the apo-FluPol structure with those of promoter-bound FluPols allows us to propose a mechanism for FluPol activation. Our study demonstrates the remarkable flexibility of influenza virus RNA polymerase, and aids our understanding of the mechanisms controlling transcription and genome replication.

Unexpected structure for the N-terminal domain of hepatitis C virus envelope glycoprotein E1.

Hepatitis C virus (HCV) infection remains a major health problem worldwide. HCV entry into host cells and membrane fusion are achieved by two envelope glycoproteins, E1 and E2. We report here the 3.5-Å resolution crystal structure of the N-terminal domain of the HCV E1 ectodomain, which reveals a complex network of covalently linked intertwined homodimers that do not harbour the expected truncated class II fusion protein fold.

Pushing the limits of sulfur SAD phasing: de novo structure solution of the N-terminal domain of the ectodomain of HCV E1.

Single-wavelength anomalous dispersion of S atoms (S-SAD) is an elegant phasing method to determine crystal structures that does not require heavy-atom incorporation or selenomethionine derivatization. Nevertheless, this technique has been limited by the paucity of the signal at the usual X-ray wavelengths, requiring very accurate measurement of the anomalous differences. Here, the data collection and structure solution of the N-terminal domain of the ectodomain of HCV E1 from crystals that diffracted very weakly is reported. By combining the data from 32 crystals, it was possible to solve the sulfur substructure and calculate initial maps at 7 Šresolution, and after density modication and phase extension using a higher resolution native data set to 3.5 Šresolution model building was achievable.

Tracking in atomic detail the functional specializations in viral RecA helicases that occur during evolution.

Many complex viruses package their genomes into empty protein shells and bacteriophages of the Cystoviridae family provide some of the simplest models for this. The cystoviral hexameric NTPase, P4, uses chemical energy to translocate single-stranded RNA genomic precursors into the procapsid. We previously dissected the mechanism of RNA translocation for one such phage, 12, and have now investigated three further highly divergent, cystoviral P4 NTPases (from 6, 8 and 13). High-resolution crystal structures of the set of P4s allow a structure-based phylogenetic analysis, which reveals that these proteins form a distinct subfamily of the RecA-type ATPases. Although the proteins share a common catalytic core, they have different specificities and control mechanisms, which we map onto divergent N- and C-terminal domains. Thus, the RNA loading and tight coupling of NTPase activity with RNA translocation in 8 P4 is due to a remarkable C-terminal structure, which wraps right around the outside of the molecule to insert into the central hole where RNA binds to coupled L1 and L2 loops, whereas in 12 P4, a C-terminal residue, serine 282, forms a specific hydrogen bond to the N7 of purines ring to confer purine specificity for the 12 enzyme.

Plate tectonics of virus shell assembly and reorganization in phage φ8, a distant relative of mammalian reoviruses.

The hallmark of a virus is its capsid, which harbors the viral genome and is formed from protein subunits, which assemble following precise geometric rules. dsRNA viruses use an unusual protein multiplicity (120 copies) to form their closed capsids. We have determined the atomic structure of the capsid protein (P1) from the dsRNA cystovirus Φ8. In the crystal P1 forms pentamers, very similar in shape to facets of empty procapsids, suggesting an unexpected assembly pathway that proceeds via a pentameric intermediate. Unlike the elongated proteins used by dsRNA mammalian reoviruses, P1 has a compact trapezoid-like shape and a distinct arrangement in the shell, with two near-identical conformers in nonequivalent structural environments. Nevertheless, structural similarity with the analogous protein from the mammalian viruses suggests a common ancestor. The unusual shape of the molecule may facilitate dramatic capsid expansion during phage maturation, allowing P1 to switch interaction interfaces to provide capsid plasticity.

Structural basis for LMO2-driven recruitment of the SCL:E47bHLH heterodimer to hematopoietic-specific transcriptional targets.

Cell fate is governed by combinatorial actions of transcriptional regulators assembling into multiprotein complexes. However, the molecular details of how these complexes form are poorly understood. One such complex, which contains the basic-helix-loop-helix heterodimer SCL:E47 and bridging proteins LMO2:LDB1, critically regulates hematopoiesis and induces T cell leukemia. Here, we report the crystal structure of (SCL:E47)bHLH:LMO2:LDB1LID bound to DNA, providing a molecular account of the network of interactions assembling this complex. This reveals an unexpected role for LMO2. Upon binding to SCL, LMO2 induces new hydrogen bonds in SCL:E47, thereby strengthening heterodimer formation. This imposes a rotation movement onto E47 that weakens the heterodimer:DNA interaction, shifting the main DNA-binding activity onto additional protein partners. Along with biochemical analyses, this illustrates, at an atomic level, how hematopoietic-specific SCL sequesters ubiquitous E47 and associated cofactors and supports SCL's reported DNA-binding-independent functions. Importantly, this work will drive the design of small molecules inhibiting leukemogenic processes.

Expression, purification and crystallization of the ectodomain of the envelope glycoprotein E2 from Bovine viral diarrhoea virus.

Bovine viral diarrhoea virus (BVDV) is an economically important animal pathogen which is closely related to Hepatitis C virus. Of the structural proteins, the envelope glycoprotein E2 of BVDV is the major antigen which induces neutralizing antibodies; thus, BVDV E2 is considered as an ideal target for use in subunit vaccines. Here, the expression, purification of wild-type and mutant forms of the ectodomain of BVDV E2 and subsequent crystallization and data collection of two crystal forms grown at low and neutral pH are reported. Native and multiple-wavelength anomalous dispersion (MAD) data sets have been collected and structure determination is in progress.

Structure of a pestivirus envelope glycoprotein E2 clarifies its role in cell entry.

Enveloped viruses have developed various adroit mechanisms to invade their host cells. This process requires one or more viral envelope glycoprotein to achieve cell attachment and membrane fusion. Members of the Flaviviridae such as flaviviruses possess only one envelope glycoprotein, E, whereas pestiviruses and hepacivirus encode two glycoproteins, E1 and E2. Although E2 is involved in cell attachment, it has been unclear which protein is responsible for membrane fusion. We report the crystal structures of the homodimeric glycoprotein E2 from the pestivirus bovine viral diarrhea virus 1 (BVDV1) at both neutral and low pH. Unexpectedly, BVDV1 E2 does not have a class II fusion protein fold, and at low pH the N-terminal domain is disordered, similarly to the intermediate postfusion state of E2 from sindbis virus, an alphavirus. Our results suggest that the pestivirus and possibly the hepacivirus fusion machinery are unlike any previously observed.

Structure of the leukemia oncogene LMO2: implications for the assembly of a hematopoietic transcription factor complex.

The LIM only protein 2 (LMO2) is a key regulator of hematopoietic stem cell development whose ectopic expression in T cells leads to the onset of acute lymphoblastic leukemia. Through its LIM domains, LMO2 is thought to function as the scaffold for a DNA-binding transcription regulator complex, including the basic helix-loop-helix proteins SCL/TAL1 and E47, the zinc finger protein GATA-1, and LIM-domain interacting protein LDB1. To understand the role of LMO2 in the formation of this complex and ultimately to dissect its function in normal and aberrant hematopoiesis, we solved the crystal structure of LMO2 in complex with the LID domain of LDB1 at 2.4 Å resolution. We observe a largely unstructured LMO2 kept in register by the LID binding both LIM domains. Comparison of independently determined crystal structures of LMO2 reveals large movements around a conserved hinge between the LIM domains. We demonstrate that such conformational flexibility is necessary for binding of LMO2 to its partner protein SCL/TAL1 in vitro and for the function of this complex in vivo. These results, together with molecular docking and analysis of evolutionarily conserved residues, yield the first structural model of the DNA-binding complex containing LMO2, LDB1, SCL/TAL1, and GATA-1.

Purification, crystallization and preliminary X-ray analysis of a fusion of the LIM domains of LMO2 and the LID domain of Ldb1.

LMO2 (LIM domain only 2), also known as rhombotin-2, is a transcriptional regulator that is essential for normal haematopoietic development. In malignant haematopoiesis, its ectopic expression in T cells is involved in the pathogenesis of leukaemia. LMO2 contains four zinc-finger domains and binds to the ubiquitous nuclear adaptor protein Ldb1 via the LIM-interaction domain (LID). Together, they act as scaffolding proteins and bridge important haematopoietic transcription factors such as SCL/Tal1, E2A and GATA-1. Solving the structure of the LMO2:Ldb1-LID complex would therefore be a first step towards understanding how haematopoietic specific protein complexes form and would also provide an attractive target for drug development in anticancer therapy, especially for T-cell leukaemia. Here, the expression, purification, crystallization and data collection of a fusion protein consisting of the two LIM domains of LMO2 linked to the LID domain of Ldb1 via a flexible linker is reported. The crystals belonged to space group C2, with unit-cell parameters a = 179.9, b = 51.5, c = 114.7 Å, ß = 90.1°, and contained five molecules in the asymmetric unit. Multiple-wavelength anomalous dispersion (MAD) data have been collected at the zinc X-ray absorption edge to a resolution of 2.8 Šand the data were used to solve the structure of the LMO2:Ldb1-LID complex. Refinement and analysis of the electron-density map is in progress.

Structural basis for non-competitive product inhibition in human thymidine phosphorylase: implications for drug design.

HTP (human thymidine phosphorylase), also known as PD-ECGF (platelet-derived endothelial cell growth factor) or gliostatin, has an important role in nucleoside metabolism. HTP is implicated in angiogenesis and apoptosis and therefore is a prime target for drug design, including antitumour therapies. An HTP structure in a closed conformation complexed with an inhibitor has previously been solved. Earlier kinetic studies revealed an ordered release of thymine followed by ribose phosphate and product inhibition by both ligands. We have determined the structure of HTP from crystals grown in the presence of thymidine, which, surprisingly, resulted in bound thymine with HTP in a closed dead-end complex. Thus thymine appears to be able to reassociate with HTP after its initial ordered release before ribose phosphate and induces the closed conformation, hence explaining the mechanism of non-competitive product inhibition. In the active site in one of the four HTP molecules within the crystal asymmetric unit, additional electron density is present. This density has not been previously seen in any pyrimidine nucleoside phosphorylase and it defines a subsite that may be exploitable in drug design. Finally, because our crystals did not require proteolysed HTP to grow, the structure reveals a loop (residues 406-415), disordered in the previous HTP structure. This loop extends across the active-site cleft and appears to stabilize the dimer interface and the closed conformation by hydrogen-bonding. The present study will assist in the design of HTP inhibitors that could lead to drugs for anti-angiogenesis as well as for the potentiation of other nucleoside drugs.

Mutations distal to the substrate site can affect varicella zoster virus thymidine kinase activity: implications for drug design.

Varicella zoster virus encodes a thymidine kinase responsible for the activation of antiherpetic nucleoside prodrugs such as acyclovir. In addition, herpes virus thymidine kinases are being explored in gene/chemotherapy strategies aimed at developing novel antitumor therapies. To investigate and improve compound selectivity, we report here structure-based site-directed mutagenesis studies of varicella zoster virus thymidine kinase (VZVTK). Earlier reports showed that mutating residues at the core of the VZVTK active site invariably destroyed activity; hence, we targeted more distal residues. Based on the VZVTK crystal structure, we constructed six mutants (E59S, R84V, H97Y/A, and Y21H/E) and tested substrate activity and competitive inhibition for several compound series. All VZVTK mutants tested retained significant phosphorylation activity with dThd as substrate, apart from Y21E (350-fold diminution in the k(cat)/K(m)). Some mutations give slightly improved affinities: bicyclic nucleoside analogs (BCNAs) with a p-alkyl-substituted phenyl group seem to require aromatic ring stacking interactions with residue 97 for optimal inhibitory effect. Mutation Y21E decreased the IC(50) value for the BCNA 3-(2'-deoxy-beta-D-ribofuranosyl)-6-octyl-2,3-dihydrofuro[2,3-d]pyrimidin-2-one (Cf1368) 4-fold, whereas mutation Y21H increased the IC(50) value by more than 15-fold. These results suggest that residue 21 is important for BCNA selectivity and might explain why HSV1TK is unable to bind BCNAs. Other mutants, such as the E59S and R84V thymidine kinases, which in wild-type VZVTK stabilize the dimer interface, give opposite results regarding the level of sensitivity to BCNAs. The work described here shows that distal mutations that affect the VZVTK active-site may help in the design of more selective substrates for gene suicide therapy or as anti-varicella zoster virus drugs.