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1.  Toremifene interacts with and destabilizes the Ebola virus glycoprotein 
Nature  2016;535(7610):169-172.
Ebola viruses (EBOVs) are responsible for repeated outbreaks of fatal infections, including the recent deadly epidemic in West Africa. There are currently no approved therapeutic drugs or vaccines for the disease. EBOV has a membrane envelope decorated by trimers of a glycoprotein (GP, cleaved by furin to form GP1 and GP2 subunits) which is solely responsible for host cell attachment, endosomal entry and membrane fusion1–7. GP is thus a primary target for the development of antiviral drugs. Here we report the first unliganded structure of EBOV GP, and complexes with an anticancer drug toremifene and the painkiller ibuprofen. The high-resolution apo structure gives a more complete and accurate picture of the molecule, and allows conformational changes introduced by antibody and receptor binding to be deciphered8–10. Unexpectedly both toremifene and ibuprofen bind in a cavity between the attachment (GP1) and fusion (GP2) subunits at the entrance to a large tunnel that links with equivalent tunnels from the other monomers of the trimer at the 3-fold axis. Protein-drug interactions, with both GP1 and GP2, are predominately hydrophobic. Residues lining the binding site are highly conserved amongst filoviruses except Marburg virus (MARV), suggesting that MARV may not bind these drugs. Thermal shift assays show up to a 14 °C decrease in protein melting temperature upon toremifene binding, while ibuprofen has only a marginal effect and is a less potent inhibitor. The results suggest that inhibitor binding destabilizes GP and triggers premature release of GP2, therefore preventing fusion between the viral and endosome membranes. Thus these complex structures reveal the mechanism of inhibition and may guide the development of more powerful anti-EBOV drugs.
doi:10.1038/nature18615
PMCID: PMC4947387  PMID: 27362232
2.  Hepatitis A virus and the origins of picornaviruses 
Nature  2014;517(7532):85-88.
Hepatitis A virus (HAV) remains enigmatic, despite some 1.4 million cases worldwide annually1. It differs radically from other picornaviruses, existing in an enveloped form2 and being unusually stable, both genetically and physically3, but has proved difficult to study. We report high-resolution X-ray structures for the mature virus and empty particles. The structures of the two particles are indistinguishable, apart from some disorder on the inside of the empty particle. The full virus contains the small viral protein VP4, while the empty particle harbors only the uncleaved precursor, VP0. The smooth particle surface is devoid of depressions which might correspond to receptor binding sites. Peptide scanning data extends the previously reported VP3 antigenic site4, while structure-based predictions5 suggest further epitopes. HAV contains no pocket factor, can withstand remarkably high temperature and low pH, with empty particles being even more robust than full particles. The virus probably uncoats via a novel mechanism, being built differently to other picornaviruses. It utilizes a VP2 ‘domain swap’ characteristic of insect picorna-like viruses6,7 and structure-based phylogenetic analysis places HAV between typical picornaviruses and the insect viruses. The enigmatic properties of HAV may reflect its position as a link between ‘modern’ picornaviruses and the more ‘primitive’ precursor insect viruses, for instance HAV retains the ability to move from cell-to-cell by transcytosis8,9.
doi:10.1038/nature13806
PMCID: PMC4773894  PMID: 25327248
3.  Crystal structure of the RNA-dependent RNA polymerase from influenza C virus 
Nature  2015;527(7576):114-117.
Negative-sense RNA viruses, such as influenza, encode large, multidomain RNA-dependent RNA polymerases that can both transcribe and replicate the viral RNA genome1. 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-snatching2. 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 states3,4. 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 FluPols3,4. 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.
doi:10.1038/nature15525
PMCID: PMC4783868  PMID: 26503046
4.  Structure of glycosylated NPC1 luminal domain C reveals insights into NPC2 and Ebola virus interactions 
Febs Letters  2016;590(5):605-612.
Niemann‐pick type C1 (NPC1) is an endo/lysosomal membrane protein involved in intracellular cholesterol trafficking, and its luminal domain C is an essential endosomal receptor for Ebola and Marburg viruses. We have determined the crystal structure of glycosylated NPC1 luminal domain C and find all seven possible sites are glycosylated. Mapping the disease mutations onto the glycosylated structure reveals a potential binding face for NPC2. Knowledge‐based docking of NPC1 onto Ebola viral glycoprotein and sequence analysis of filovirus susceptible and refractory species reveals four critical residues, H418, Q421, F502 and F504, some or all of which are likely responsible for the species‐specific susceptibility to the virus infection.
doi:10.1002/1873-3468.12089
PMCID: PMC4819692  PMID: 26846330
cholesterol transport; Ebola virus receptor; Ebola virus susceptibility; Niemann–Pick disease type C; NPC1; NPC2
5.  Crystal structures of penicillin‐binding protein 3 in complexes with azlocillin and cefoperazone in both acylated and deacylated forms 
Febs Letters  2016;590(2):288-297.
Penicillin‐binding protein 3 (PBP3) from Pseudomonas aeruginosa is the molecular target of β‐lactam‐based antibiotics. Structures of PBP3 in complexes with azlocillin and cefoperazone, which are in clinical use for the treatment of pseudomonad infections, have been determined to 2.0 Å resolution. Together with data from other complexes, these structures identify a common set of residues involved in the binding of β‐lactams to PBP3. Comparison of wild‐type and an active site mutant (S294A) showed that increased thermal stability of PBP3 following azlocillin binding was entirely due to covalent binding to S294, whereas cefoperazone binding produces some increase in stability without the covalent link. Consistent with this, a third crystal structure was determined in which the hydrolysis product of cefoperazone was noncovalently bound in the active site of PBP3. This is the first structure of a complex between a penicillin‐binding protein and cephalosporic acid and may be important in the design of new noncovalent PBP3 inhibitors.
doi:10.1002/1873-3468.12054
PMCID: PMC4764023  PMID: 26823174
azlocillin; cefoperazone; penicillin‐binding protein; Pseudomonas aeruginosa; thermal shift assay; β‐lactam antibiotics
6.  Structures of Coxsackievirus A16 Capsids with Native Antigenicity: Implications for Particle Expansion, Receptor Binding, and Immunogenicity 
Journal of Virology  2015;89(20):10500-10511.
ABSTRACT
Enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are the primary causes of the epidemics of hand-foot-and-mouth disease (HFMD) that affect more than a million children in China each year and lead to hundreds of deaths. Although there has been progress with vaccines for EV71, the development of a CVA16 vaccine has proved more challenging, and the EV71 vaccine does not give useful cross-protection, despite the capsid proteins of the two viruses sharing about 80% sequence identity. The structural details of the expanded forms of the capsids, which possess nonnative antigenicity, are now well understood, but high resolution information for the native antigenic form of CVA16 has been missing. Here, we remedy this with high resolution X-ray structures of both mature and natural empty CVA16 particles and also of empty recombinant viruslike particles of CVA16 produced in insect cells, a potential vaccine antigen. All three structures are unexpanded native particles and antigenically identical. The recombinant particles have recruited a lipid moiety to stabilize the native antigenic state that is different from the one used in a natural virus infection. As expected, the mature CVA16 virus is similar to EV71; however, structural and immunogenic comparisons highlight differences that may have implications for vaccine production.
IMPORTANCE Hand-foot-and-mouth disease is a serious public health threat to children in Asian-Pacific countries, resulting in millions of cases. EV71 and CVA16 are the two dominant causative agents of the disease that, while usually mild, can cause severe neurological complications, leading to hundreds of deaths. EV71 vaccines do not provide protection against CVA16. A CVA16 vaccine or bivalent EV71/CVA16 vaccine is therefore urgently needed. We report atomic structures for the mature CVA16 virus, a natural empty particle, and a recombinant CVA16 virus-like particle that does not contain the viral genome. All three particles have similar structures and identical antigenicity. The recombinant particles, produced in insect cells (a system suitable for making vaccine antigen), are stabilized by recruiting from the insect cells a small molecule that is different from that used by the virus in a normal infection. We present structural and immunogenic comparisons with EV71 to facilitate structure-based drug design and vaccine development.
doi:10.1128/JVI.01102-15
PMCID: PMC4580203  PMID: 26269176
7.  Localized reconstruction of subunits from electron cryomicroscopy images of macromolecular complexes 
Nature Communications  2015;6:8843.
Electron cryomicroscopy can yield near-atomic resolution structures of highly ordered macromolecular complexes. Often however some subunits bind in a flexible manner, have different symmetry from the rest of the complex, or are present in sub-stoichiometric amounts, limiting the attainable resolution. Here we report a general method for the localized three-dimensional reconstruction of such subunits. After determining the particle orientations, local areas corresponding to the subunits can be extracted and treated as single particles. We demonstrate the method using three examples including a flexible assembly and complexes harbouring subunits with either partial occupancy or mismatched symmetry. Most notably, the method allows accurate fitting of the monomeric RNA-dependent RNA polymerase bound at the threefold axis of symmetry inside a viral capsid, revealing for the first time its exact orientation and interactions with the capsid proteins. Localized reconstruction is expected to provide novel biological insights in a range of challenging biological systems.
Electron cryomicroscopy can allow the elucidation of macromolecular structures; however, mismatches in symmetry between different components limit the attainable resolution. Here, the authors set out a computational method for extracting and retaining information from such components.
doi:10.1038/ncomms9843
PMCID: PMC4667630  PMID: 26534841
8.  Potent antiviral agents fail to elicit genetically-stable resistance mutations in either enterovirus 71 or Coxsackievirus A16 
Antiviral Research  2015;124:77-82.
Enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16) are the two major causative agents of hand, foot and mouth disease (HFMD), for which there are currently no licenced treatments. Here, the acquisition of resistance towards two novel capsid-binding compounds, NLD and ALD, was studied and compared to the analogous compound GPP3. During serial passage, EV71 rapidly became resistant to each compound and mutations at residues I113 and V123 in VP1 were identified. A mutation at residue 113 was also identified in CVA16 after passage with GPP3. The mutations were associated with reduced thermostability and were rapidly lost in the absence of inhibitors. In silico modelling suggested that the mutations prevented the compounds from binding the VP1 pocket in the capsid. Although both viruses developed resistance to these potent pocket-binding compounds, the acquired mutations were associated with large fitness costs and reverted to WT phenotype and sequence rapidly in the absence of inhibitors. The most effective inhibitor, NLD, had a very large selectivity index, showing interesting pharmacological properties as a novel anti-EV71 agent.
Highlights
•Resistance to each compound developed rapidly, mutations at residues I113 and V123 were identified.•Resistance mutations were associated with a large fitness cost.•The mutations resulted in reduced affinity for the pocket factor.•The most effective inhibitor, NLD, had a very large therapeutic window, and has potential as a novel anti-EV71 agent.
doi:10.1016/j.antiviral.2015.10.006
PMCID: PMC4678291  PMID: 26522770
Hand foot and mouth disease; Capsid binding; Drug resistance; Fitness; VP1 pocket; VP1, Viral Protein 1; IC50, Half-maximal inhibitory concentration; CC50, Half maximal cytotoxic concentration; TCID50, Half-maximal tissue culture infective dose; PV, poliovirus; CVB, Coxsackie virus B; WT, Wild Type; MTT, 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide; EV71, Enterovirus 71; CVA16, Coxsackie virus A16
9.  Structure of Ljungan virus provides insight into genome packaging of this picornavirus 
Nature Communications  2015;6:8316.
Picornaviruses are responsible for a range of human and animal diseases, but how their RNA genome is packaged remains poorly understood. A particularly poorly studied group within this family are those that lack the internal coat protein, VP4. Here we report the atomic structure of one such virus, Ljungan virus, the type member of the genus Parechovirus B, which has been linked to diabetes and myocarditis in humans. The 3.78-Å resolution cryo-electron microscopy structure shows remarkable features, including an extended VP1 C terminus, forming a major protuberance on the outer surface of the virus, and a basic motif at the N terminus of VP3, binding to which orders some 12% of the viral genome. This apparently charge-driven RNA attachment suggests that this branch of the picornaviruses uses a different mechanism of genome encapsidation, perhaps explored early in the evolution of picornaviruses.
The Ljungan virus is a picornavirus that lacks the internal coat protein VP4, and the packaging of its RNA genome is poorly understood. Here, the authors use cryo-electron microscopy to visualize this virus and suggest that it uses a different mechanism to other viruses for encapsidation of its genome.
doi:10.1038/ncomms9316
PMCID: PMC4633645  PMID: 26446437
10.  Structure Elucidation of Coxsackievirus A16 in Complex with GPP3 Informs a Systematic Review of Highly Potent Capsid Binders to Enteroviruses 
PLoS Pathogens  2015;11(10):e1005165.
The replication of enterovirus 71 (EV71) and coxsackievirus A16 (CVA16), which are the major cause of hand, foot and mouth disease (HFMD) in children, can be inhibited by the capsid binder GPP3. Here, we present the crystal structure of CVA16 in complex with GPP3, which clarifies the role of the key residues involved in interactions with the inhibitor. Based on this model, in silico docking was performed to investigate the interactions with the two next-generation capsid binders NLD and ALD, which we show to be potent inhibitors of a panel of enteroviruses with potentially interesting pharmacological properties. A meta-analysis was performed using the available structural information to obtain a deeper insight into those structural features required for capsid binders to interact effectively and also those that confer broad-spectrum anti-enterovirus activity.
Author Summary
Hand, foot and mouth disease (HFMD), a common infectious disease of children documented worldwide is caused by enterovirus infection. Coxsackievirus A16 (CVA16) and enterovirus 71 (EV71) are the major causative agents of HFMD. Although EV71 vaccines have completed phase III clinical trials, there remains no vaccine against CVA16 or licensed drug against either CVA16 or EV71. In this study we show the crystal structure of CVA16 in complex with inhibitor GPP3, shedding light on the residues involved in the interaction with the virus capsid. Taking advantage of the crystal structure we employed a quantum mechanics polarized ligand docking (QMPLD) protocol to dock two recently designed capsid binders. These molecules have been already proven to be very powerful inhibitors of EV71 and here we show that they exhibit strong antiviral activity against a wide range of enterovirus.
Our research provides new insights into the design of inhibitors for the treatment of different pathogenic enterovirus infections.
doi:10.1371/journal.ppat.1005165
PMCID: PMC4613828  PMID: 26485389
11.  Diamond Light Source: status and perspectives 
Diamond Light Source, a third-generation synchrotron radiation (SR) facility in the UK, celebrated its 10th anniversary in 2012. A private limited company was set up in April 2002 to plan, construct and operate the new user-oriented SR facility, called in brief Diamond. It succeeded the Synchrotron Radiation Source in Daresbury, a second-generation synchrotron that opened in 1980 as the world's first dedicated X-ray-providing facility, closing finally in 2008, by which time Diamond's accelerators and first beamlines were operating and user experiments were under way. This theme issue of Philosophical Transactions of the Royal Society A gives some examples of the rich diversity of research done in the initial five years, with some glimpses of activity up to 2014. Speakers at the 10 year anniversary symposium were drawn from a small number of major thematic areas and each theme was elaborated by a few speakers whose contributions were placed into a broader context by a leading member of the UK academic community in the role of rapporteur. This introduction gives a summary of the design choices and strategic planning of Diamond as a coherent user facility, a snapshot of its present status and some consideration of future perspectives.
doi:10.1098/rsta.2013.0161
PMCID: PMC4308985  PMID: 25624517
Diamond Light Source; synchrotron radiation; X-ray physics
12.  Polyhedra structures and the evolution of the insect viruses 
Journal of Structural Biology  2015;192(1):88-99.
Polyhedra represent an ancient system used by a number of insect viruses to protect virions during long periods of environmental exposure. We present high resolution crystal structures of polyhedra for seven previously uncharacterised types of cypoviruses, four using ab initio selenomethionine phasing (two of these required over 100 selenomethionine crystals each). Approximately 80% of residues are structurally equivalent between all polyhedrins (pairwise rmsd ⩽1.5 Å), whilst pairwise sequence identities, based on structural alignment, are as little as 12%. These structures illustrate the effect of 400 million years of evolution on a system where the crystal lattice is the functionally conserved feature in the face of massive sequence variability. The conservation of crystal contacts is maintained across most of the molecular surface, except for a dispensable virus recognition domain. By spreading the contacts over so much of the protein surface the lattice remains robust in the face of many individual changes. Overall these unusual structural constraints seem to have skewed the molecule’s evolution so that surface residues are almost as conserved as the internal residues.
doi:10.1016/j.jsb.2015.08.009
PMCID: PMC4597613  PMID: 26291392
SeMet, selenomethionine; Protein microcrystals; Polyhedra; In vivo crystals; Cypovirus; Micro focus crystallography
13.  A RANKL mutant used as an inter-species vaccine for efficient immunotherapy of osteoporosis 
Scientific Reports  2015;5:14150.
Anti-cytokine therapeutic antibodies have been demonstrated to be effective in the treatment of several auto-immune disorders. However, The problems in antibody manufacture and the immunogenicity caused by multiple doses of antibodies inspire people to use auto-cytokine as immunogen to induce anti-cytokine antibodies. Nevertheless, the tolerance for inducing immune response against self-antigen has hindered the wide application of the strategy. To overcome the tolerance, here we proposed a strategy using the inter-species cytokine as immunogen for active immunization (TISCAI) to induce anti-cytokine antibody. As a proof of concept, an inter-species cytokine RANKL was successfully used as immunogen to induce anti-RANKL immune response. Furthermore, to prevent undesirable side-effects, the human RANKL was mutated based on the crystal structure of the complex of human RANKL and its rodent counterpart receptor RANK. We found, the antibodies produced blocked the osteoclast development in vitro and osteoporosis in OVX rat models. The results demonstrated this strategy adopted is very useful for general anti-cytokine immunotherapy for different diseases settings.
doi:10.1038/srep14150
PMCID: PMC4585926  PMID: 26412210
14.  More powerful virus inhibitors from structure-based analysis of HEV71 capsid-binding molecules 
Enterovirus 71 (HEV71) epidemics amongst children and infants result mainly in mild symptoms, however, especially in the Asia-Pacific region, infection can be fatal. At present no therapies are available. We have used structural analysis of the complete virus to guide the design of HEV71 inhibitors. Analysis of complexes with four 3-(-4-pyridyl)-2-imidazolidinone derivatives with varying anti-HEV71 activities, pinpointed key structure-activity correlates. We then identified additional potentially beneficial substitutions, developed methods to reliably triage compounds by quantum mechanics-enhanced ligand docking, and synthesized two candidates. Structural analysis and in vitro assays confirmed the predicted binding modes and their ability to block viral infection. One ligand (IC50 = 25 pM) is an order of magnitude more potent than the best previously reported inhibitor, and is also more soluble. Our approach may be useful in the design of effective drugs for enterovirus infections.
doi:10.1038/nsmb.2769
PMCID: PMC4530014  PMID: 24509833
15.  Structure of CPV17 polyhedrin determined by the improved analysis of serial femtosecond crystallographic data 
Nature communications  2015;6:6435.
The X-ray free electron laser (XFEL) allows the analysis of small weakly diffracting protein crystals, but has required very many crystals to obtain good data. Here we use an XFEL to determine the room temperature atomic structure for the smallest known cytoplasmic polyhedrosis virus polyhedra, which we failed to solve at a synchrotron. These protein microcrystals, roughly a micron across, accrue within infected cells. We use a new physical model for XFEL diffraction, which better estimates the experimental signal, delivering a high-resolution XFEL structure (1.75 Å), using fewer crystals than previously required for this resolution. The crystal lattice and protein core are conserved compared to a polyhedrin with less than 10% sequence identity. We explain how the conserved biological phenotype, the crystal lattice, is maintained in the face of extreme environmental challenge and massive evolutionary divergence. Our improved methods should open up more challenging biological samples to XFEL analysis.
doi:10.1038/ncomms7435
PMCID: PMC4403592  PMID: 25751308
16.  Structure of CPV17 polyhedrin determined by the improved analysis of serial femtosecond crystallographic data 
Nature Communications  2015;6:6435.
The X-ray free-electron laser (XFEL) allows the analysis of small weakly diffracting protein crystals, but has required very many crystals to obtain good data. Here we use an XFEL to determine the room temperature atomic structure for the smallest cytoplasmic polyhedrosis virus polyhedra yet characterized, which we failed to solve at a synchrotron. These protein microcrystals, roughly a micron across, accrue within infected cells. We use a new physical model for XFEL diffraction, which better estimates the experimental signal, delivering a high-resolution XFEL structure (1.75 Å), using fewer crystals than previously required for this resolution. The crystal lattice and protein core are conserved compared with a polyhedrin with less than 10% sequence identity. We explain how the conserved biological phenotype, the crystal lattice, is maintained in the face of extreme environmental challenge and massive evolutionary divergence. Our improved methods should open up more challenging biological samples to XFEL analysis.
Serial femtosecond crystallography and the use of X-ray free-electron lasers (XFEL) promise to revolutionize structural biology. Here, the authors describe refinements that reduce the redundancy required to obtain quality XFEL data and report a 1.75-Å structure—not obtainable by synchrotron radiation—using less than 6,000 crystals.
doi:10.1038/ncomms7435
PMCID: PMC4403592  PMID: 25751308
17.  Tandem Fusion of Hepatitis B Core Antigen Allows Assembly of Virus-Like Particles in Bacteria and Plants with Enhanced Capacity to Accommodate Foreign Proteins 
PLoS ONE  2015;10(4):e0120751.
The core protein of the hepatitis B virus, HBcAg, assembles into highly immunogenic virus-like particles (HBc VLPs) when expressed in a variety of heterologous systems. Specifically, the major insertion region (MIR) on the HBcAg protein allows the insertion of foreign sequences, which are then exposed on the tips of surface spike structures on the outside of the assembled particle. Here, we present a novel strategy which aids the display of whole proteins on the surface of HBc particles. This strategy, named tandem core, is based on the production of the HBcAg dimer as a single polypeptide chain by tandem fusion of two HBcAg open reading frames. This allows the insertion of large heterologous sequences in only one of the two MIRs in each spike, without compromising VLP formation. We present the use of tandem core technology in both plant and bacterial expression systems. The results show that tandem core particles can be produced with unmodified MIRs, or with one MIR in each tandem dimer modified to contain the entire sequence of GFP or of a camelid nanobody. Both inserted proteins are correctly folded and the nanobody fused to the surface of the tandem core particle (which we name tandibody) retains the ability to bind to its cognate antigen. This technology paves the way for the display of natively folded proteins on the surface of HBc particles either through direct fusion or through non-covalent attachment via a nanobody.
doi:10.1371/journal.pone.0120751
PMCID: PMC4382129  PMID: 25830365
18.  The Human Otubain2-Ubiquitin Structure Provides Insights into the Cleavage Specificity of Poly-Ubiquitin-Linkages 
PLoS ONE  2015;10(1):e0115344.
Ovarian tumor domain containing proteases cleave ubiquitin (Ub) and ubiquitin-like polypeptides from proteins. Here we report the crystal structure of human otubain 2 (OTUB2) in complex with a ubiquitin-based covalent inhibitor, Ub-Br2. The ubiquitin binding mode is oriented differently to how viral otubains (vOTUs) bind ubiquitin/ISG15, and more similar to yeast and mammalian OTUs. In contrast to OTUB1 which has exclusive specificity towards Lys48 poly-ubiquitin chains, OTUB2 cleaves different poly-Ub linked chains. N-terminal tail swapping experiments between OTUB1 and OTUB2 revealed how the N-terminal structural motifs in OTUB1 contribute to modulating enzyme activity and Ub-chain selectivity, a trait not observed in OTUB2, supporting the notion that OTUB2 may affect a different spectrum of substrates in Ub-dependent pathways.
doi:10.1371/journal.pone.0115344
PMCID: PMC4295869  PMID: 25590432
19.  Expression, purification and crystallization of the ectodomain of the envelope glycoprotein E2 from Bovine viral diarrhoea virus  
The cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of the ectodomain of BVDV E2 are described.
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.
doi:10.1107/S1744309112049184
PMCID: PMC3539699  PMID: 23295482
Pestivirus; BVDV; envelope glycoprotein E2
20.  A national facility for biological cryo-electron microscopy 
This review provides a brief update on the use of cryo-electron microscopy for integrated structural biology, along with an overview of the plans for the UK national facility for electron microscopy being built at the Diamond synchrotron.
Three-dimensional electron microscopy is an enormously powerful tool for structural biologists. It is now able to provide an understanding of the molecular machinery of cells, disease processes and the actions of pathogenic organisms from atomic detail through to the cellular context. However, cutting-edge research in this field requires very substantial resources for equipment, infrastructure and expertise. Here, a brief overview is provided of the plans for a UK national three-dimensional electron-microscopy facility for integrated structural biology to enable internationally leading research on the machinery of life. State-of-the-art equipment operated with expert support will be provided, optimized for both atomic-level single-particle analysis of purified macromolecules and complexes and for tomography of cell sections. The access to and organization of the facility will be modelled on the highly successful macromolecular crystallography (MX) synchrotron beamlines, and will be embedded at the Diamond Light Source, facilitating the development of user-friendly workflows providing near-real-time experimental feedback.
doi:10.1107/S1399004714025280
PMCID: PMC4304693  PMID: 25615867
three-dimensional electron microscopy
21.  New methods for indexing multi-lattice diffraction data 
A new indexing method is presented which is capable of indexing multiple crystal lattices from narrow wedges of data. The efficacy of this method is demonstrated with both semi-synthetic multi-lattice data and real multi-lattice data recorded from microcrystals of ∼1 µm in size.
A new indexing method is presented which is capable of indexing multiple crystal lattices from narrow wedges of diffraction data. The method takes advantage of a simplification of Fourier transform-based methods that is applicable when the unit-cell dimensions are known a priori. The efficacy of this method is demonstrated with both semi-synthetic multi-lattice data and real multi-lattice data recorded from crystals of ∼1 µm in size, where it is shown that up to six lattices can be successfully indexed and subsequently integrated from a 1° wedge of data. Analysis is presented which shows that improvements in data-quality indicators can be obtained through accurate identification and rejection of overlapping reflections prior to scaling.
doi:10.1107/S1399004714017039
PMCID: PMC4188007  PMID: 25286849
indexing; multi-lattice data
22.  Unexpected structure for the N-terminal domain of hepatitis C virus envelope glycoprotein E1 
Nature Communications  2014;5:4874.
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.
Hepatitis C virus (HCV) gains entry into host cells via envelope glycoproteins E1 and E2. Here, El Omari et al. present the crystal structure of the N terminus of the E1 ectodomain of HCV and show that it adopts a different fold than predicted.
doi:10.1038/ncomms5874
PMCID: PMC4175578  PMID: 25224686
23.  Pushing the limits of sulfur SAD phasing: de novo structure solution of the N-terminal domain of the ectodomain of HCV E1 
The sulfur SAD phasing method was successfully used to determine the structure of the N-terminal domain of HCV E1 from low-resolution diffracting crystals by combining data from 32 crystals.
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 Å resolution, and after density modication and phase extension using a higher resolution native data set to 3.5 Å resolution model building was achievable.
doi:10.1107/S139900471401339X
PMCID: PMC4118829  PMID: 25084338
sulfur SAD; HCV; envelope glycoprotein E1
24.  Lysosome sorting of β-glucocerebrosidase by LIMP-2 is targeted by the mannose 6-phosphate receptor 
Nature Communications  2014;5:4321.
The integral membrane protein LIMP-2 has been a paradigm for mannose 6-phosphate receptor (MPR) independent lysosomal targeting, binding to β-glucocerebrosidase (β-GCase) and directing it to the lysosome, before dissociating in the late-endosomal/lysosomal compartments. Here we report structural results illuminating how LIMP-2 binds and releases β-GCase according to changes in pH, via a histidine trigger, and suggesting that LIMP-2 localizes the ceramide portion of the substrate adjacent to the β-GCase catalytic site. Remarkably, we find that LIMP-2 bears P-Man9GlcNAc2 covalently attached to residue N325, and that it binds MPR, via mannose 6-phosphate, with a similar affinity to that observed between LIMP-2 and β-GCase. The binding sites for β-GCase and the MPR are functionally separate, so that a stable ternary complex can be formed. By fluorescence lifetime imaging microscopy, we also demonstrate that LIMP-2 interacts with MPR in living cells. These results revise the accepted view of LIMP-2–β-GCase lysosomal targeting.
LIMP-2 is a membrane protein involved in β-glucocerebrosidase lysosomal targeting. Here Zhao et al. report structural and biochemical results showing how LIMP-2 interacts with β-glucocerebrosidase and the mannose 6-phosphate receptor, and propose a mechanism for LIMP-2-associated lysosomal targeting.
doi:10.1038/ncomms5321
PMCID: PMC4104448  PMID: 25027712
25.  Structure of the regulatory domain of the LysR family regulator NMB2055 (MetR-like protein) from Neisseria meningitidis  
Acta Crystallographica Section F  2012;68(Pt 7):730-737.
The crystal structure of the regulatory domain of NMB2055, a putative MetR regulator, was solved at 2.5 Å resolution.
The crystal structure of the regulatory domain of NMB2055, a putative MetR regulator from Neisseria meningitidis, is reported at 2.5 Å resolution. The structure revealed that there is a disulfide bond inside the predicted effector-binding pocket of the regulatory domain. Mutation of the cysteines (Cys103 and Cys106) that form the disulfide bond to serines resulted in significant changes to the structure of the effector pocket. Taken together with the high degree of conservation of these cysteine residues within MetR-related transcription factors, it is suggested that the Cys103 and Cys106 residues play an important role in the function of MetR regulators.
doi:10.1107/S1744309112010603
PMCID: PMC3388910  PMID: 22750853
MetR; Neisseria meningitidis; LysR-type regulator

Results 1-25 (105)