Using selenomethionyl derivatives to assign sequence in low-resolution structures of the AP2 clathrin adaptor.

Selenomethionine incorporation is a powerful technique for assigning sequence to regions of electron density at low resolution. Genetic introduction of methionine point mutations and the subsequent preparation and crystallization of selenomethionyl derivatives permits unambiguous sequence assignment by enabling the placement of the anomalous scatterers (Se atoms) thus introduced. Here, the use of this approach in the assignment of sequence in a part of the AP2 clathrin adaptor complex that is responsible for clathrin binding is described. AP2 plays a pivotal role in clathrin-mediated endocytosis, a tightly regulated process in which cell-surface transmembrane proteins are internalized from the plasma membrane by incorporation into lipid-enclosed transport vesicles. AP2 binds cargo destined for internalization and recruits clathrin, a large trimeric protein that helps to deform the membrane to produce the transport vesicle. By selenomethionine labelling of point mutants, it was shown that the clathrin-binding site is buried within a deep cleft of the AP2 complex. A membrane-stimulated conformational change in AP2 releases the clathrin-binding site from autoinhibition, thereby linking clathrin recruitment to membrane localization.We would like to thank the I02, I03 and I04-1 beamline staff at the Diamond Light Source (mx6641) and Chris Oubridge for advice and assistance with SeMet mapping of the hinge residues. D.J.O. and B.T.K. are supported by Wellcome Trust Principal Research Fellowship (090909/Z/09/Z). S.C.G. is supported by a Sir Henry Dale Fellowship from the Wellcome Trust and the Royal Society (098406/Z/12/Z). CIMR is supported by a Wellcome Trust Strategic Award (079895).This is the final version of the article. It first appeared from the International Union of Crystallography via http://dx.doi.org/10.1107/S205979831502158

Tegument Assembly and Secondary Envelopment of Alphaherpesviruses.

Alphaherpesviruses like herpes simplex virus are large DNA viruses characterized by their ability to establish lifelong latent infection in neurons. As for all herpesviruses, alphaherpesvirus virions contain a protein-rich layer called "tegument" that links the DNA-containing capsid to the glycoprotein-studded membrane envelope. Tegument proteins mediate a diverse range of functions during the virus lifecycle, including modulation of the host-cell environment immediately after entry, transport of virus capsids to the nucleus during infection, and wrapping of cytoplasmic capsids with membranes (secondary envelopment) during virion assembly. Eleven tegument proteins that are conserved across alphaherpesviruses have been implicated in the formation of the tegument layer or in secondary envelopment. Tegument is assembled via a dense network of interactions between tegument proteins, with the redundancy of these interactions making it challenging to determine the precise function of any specific tegument protein. However, recent studies have made great headway in defining the interactions between tegument proteins, conserved across alphaherpesviruses, which facilitate tegument assembly and secondary envelopment. We summarize these recent advances and review what remains to be learned about the molecular interactions required to assemble mature alphaherpesvirus virions following the release of capsids from infected cell nuclei.HSV-1 research in the laboratory of CMC is supported by the Leverhulme Trust (Grant RPG-2012-793) and the Biotechnology and Biological Sciences Research Council (Grant BB/M021424/1). SCG is a Sir Henry Dale Fellow jointly funded by the Wellcome Trust and the Royal Society (Grant Number 098406/Z/12/Z)This is the final version of the article. It first appeared from MDPI via http://dx.doi.org/10.3390/v709286

Non-native fold of the putative VPS39 zinc finger domain

Background: The multi-subunit homotypic fusion and vacuole protein sorting (HOPS) membrane-tethering complex is involved in regulating the fusion of late endosomes and autophagosomes with lysosomes in eukaryotes. The C-terminal regions of several HOPS components have been shown to be required for correct complex assembly, including the C-terminal really interesting new gene (RING) zinc finger domains of HOPS components VPS18 and VPS41. We sought to structurally characterise the putative C-terminal zinc finger domain of VPS39, which we hypothesised may be important for binding of VPS39 to cellular partners or to other HOPS components. Methods: We recombinantly expressed, purified and solved the crystal structure of the proposed zinc-binding region of VPS39. Results: In the structure, this region forms an anti-parallel β-hairpin that is incorporated into a homotetrameric eight-stranded β-barrel. However, the fold is stabilised by coordination of zinc ions by residues from the purification tag and an intramolecular disulphide bond between two predicted zinc ligands. Conclusions: We solved the structure of the VPS39 C-terminal domain adopting a non-native fold. Our work highlights the risk of non-native folds when purifying small zinc-containing domains with hexahistidine tags. However, the non-native structure we observe may have implications for rational protein design.This work was supported by a Sir Henry Dale Fellowship (098406), jointly funded by the Wellcome Trust and the Royal Society (to SCG). BGB is a Wellcome Trust PhD student. Remote synchrotron access was supported in part by the EU FP7 infrastructure grant BIOSTRUCT-X (Contract No. 283570

Molecular models should not be published without the corresponding atomic coordinates.

In PNAS, Romero et al. (1) present models of how glucocerebrosidase (GCase) interacts with saposin C (SAPC) and membranes. Unfortunately, the authors do not publish representative atomic coordinates or molecular dynamics trajectories for their models, denying researchers the opportunity to scrutinize the data Romero et al. (1) use to draw their functional conclusions. Access to these data is an important issue for structural biologists (2), and the open release of experimentally determined structural data has been the accepted practice for many years (3). Indeed, Romero et al. rely on several such publically available structures to carry out their study.J.E.D. is supported by a Royal Society University Research Fellowship (UF100371). S.C.G. is supported by a Sir Henry Dale Fellowship co-funded by the Royal Society and Wellcome Trust (098406/Z/12/B)

The lipid transfer protein Saposin B does not directly bind CD1d for lipid antigen loading.

Background: Lipid antigens are presented on the surface of cells by the CD1 family of glycoproteins, which have structural and functional similarity to MHC class I molecules. The hydrophobic lipid antigens are embedded in membranes and inaccessible to the lumenal lipid-binding domain of CD1 molecules. Therefore, CD1 molecules require lipid transfer proteins for lipid loading and editing. CD1d is loaded with lipids in late endocytic compartments, and lipid transfer proteins of the saposin family have been shown to play a crucial role in this process. However, the mechanism by which saposins facilitate lipid binding to CD1 molecules is not known and is thought to involve transient interactions between protein components to ensure CD1-lipid complexes can be efficiently trafficked to the plasma membrane for antigen presentation. Of the four saposin proteins, the importance of Saposin B (SapB) for loading of CD1d is the most well-characterised. However, a direct interaction between CD1d and SapB has yet to be described. Methods: In order to determine how SapB might load lipids onto CD1d, we used purified, recombinant CD1d and SapB and carried out a series of highly sensitive binding assays to monitor direct interactions. We performed equilibrium binding analysis, chemical cross-linking and co-crystallisation experiments, under a range of different conditions. Results: We could not demonstrate a direct interaction between SapB and CD1d using any of these binding assays. Conclusions: This work strongly indicates that the role of SapB in lipid loading does not involve direct binding to CD1d. We discuss the implication of this for our understanding of lipid loading of CD1d and propose several factors that may influence this process

Integrated stratigraphy of the Kimmeridge Clay Formation (Upper Jurassic) based on exposures and boreholes in south Dorset, UK

For the purposes of a high-resolution multi-disciplinary study of the Upper Jurassic Kimmeridge Clay Formation, two boreholes were drilled at Swanworth Quarry and one at Metherhills, south Dorset, UK. Together, the cores represent the first complete section through the entire formation close to the type section. We present graphic logs that record the stratigraphy of the cores, and outline the complementary geophysical and analytical data sets (gamma ray, magnetic susceptibility, total organic carbon, carbonate, [delta]13Corg). Of particular note are the new borehole data from the lowermost part of the formation which does not crop out in the type area. Detailed logs are available for download from the Kimmeridge Drilling Project web-site at http://kimmeridge.earth.ox.ac.uk/. Of further interest is a mid-eudoxus Zone positive shift in the [delta]13Corg record, a feature that is also registered in Tethyan carbonate successions, suggesting that it is a regional event and may therefore be useful for correlation. The lithostratigraphy of the cores has been precisely correlated with the nearby cliff section, which has also been examined and re-described. Magnetic-susceptibility and spectral gamma-ray measurements were made at a regular spacing through the succession, and facilitate core-to-exposure correlation. The strata of the exposure and core have been subdivided into four main mudrock lithological types: (a) medium-dark–dark-grey marl; (b) medium-dark–dark grey–greenish black shale; (c) dark-grey–olive-black laminated shale; (d) greyish-black–brownish-black mudstone. The sections also contain subordinate amounts of siltstone, limestone and dolostone. Comparison of the type section with the cores reveals slight lithological variation and notable thickness differences between the coeval strata. The proximity of the boreholes and different parts of the type section to the Purbeck–Isle of Wight Disturbance is proposed as a likely control on the thickness changes

Assessing wellbeing at school entry using the strengths and difficulties questionnaire: professional perspectives

<p>Background: Emotional and behavioural disorders in early childhood are related to poorer academic attainment and school engagement, and difficulties already evident at the point of starting school can affect a child’s later social and academic development. Successful transfer from pre-school settings to primary education is helped by communication between pre-school staff and primary school teachers. Typically, in Scotland, pre-school establishments prepare individual profiles of children before they start school around the age of five years, highlighting their strengths and development needs, for transfer to primary schools. There is, however, no consistent approach to the identification of potential social, emotional and behavioural problems. In 2010, in one local authority area in Scotland, the Strengths and Difficulties Questionnaire (SDQ) was introduced for children about to start school as a routine, structured, component of the transition process to help teachers plan support arrangements for classes and individual children. The SDQ assesses emotional, conduct, hyperactivity/ inattention and peer-relationship problems as well as pro-social behaviour. In order to be an effective means of communicating social and emotional functioning, the use of instruments such as the SDQ needs to be practicable. Finding out the views of pre-school education staff with experience of assessing children using the SDQ was, therefore, essential to establish its future utility.</p> <p>Aim: The purpose of this study was to explore the views of pre-school education staff about assessing social and emotional wellbeing of children at school entry using the SDQ. The objectives were to examine the opinions of pre-school workers about completing the SDQ and to elicit their thoughts on the value of doing this and their perceptions of the usefulness of the information collected.</p> <p>Method: Pre-school establishments were approached using a purposive sampling strategy in order to achieve a mix of local authority (n=14) and ‘partnership’ establishments (n=8) as well as different socio-economic areas. Semi-structured interviews (n=25) were conducted with pre-school head teachers (n=14) and child development officers (n=11) in order to explore the process of completing the SDQ along with perceptions of its value. The interviews were transcribed verbatim and analysed thematically.</p> <p>Results: In general, staff in pre-school establishments viewed the use of the SDQ positively. It was seen as a chance to highlight the social and emotional development of children rather than just their academic or educational ability. Most felt that the SDQ had not identified anything they did not already know about a child. A minority, nevertheless, suggested that a previously unrecognised potential difficulty was brought to light, most commonly emotional problems. Completing the SDQ was felt to be relatively straightforward even though the staff felt under pressure from competing priorities. Concerns were, however, raised about the potential of labelling a child at an early stage of formal education.</p> <p>Conclusion: The findings from this small scale study suggest that, from the point of view of pre-school education staff, it is feasible to assess children systematically for social and behavioural problems as part of the routine transition process at school entry.</p&gt

Application of error-prone PCR to functionally probe the morbillivirus Haemagglutinin protein.

The enveloped morbilliviruses utilise conserved proteinaceous receptors to enter host cells: SLAMF1 or Nectin-4. Receptor binding is initiated by the viral attachment protein Haemagglutinin (H), with the viral Fusion protein (F) driving membrane fusion. Crystal structures of the prototypic morbillivirus measles virus H with either SLAMF1 or Nectin-4 are available and have served as the basis for improved understanding of this interaction. However, whether these interactions remain conserved throughout the morbillivirus genus requires further characterisation. Using a random mutagenesis approach, based on error-prone PCR, we targeted the putative receptor binding site for SLAMF1 interaction on peste des petits ruminants virus (PPRV) H, identifying mutations that inhibited virus-induced cell-cell fusion. These data, combined with structural modelling of the PPRV H and ovine SLAMF1 interaction, indicate this region is functionally conserved across all morbilliviruses. Error-prone PCR provides a powerful tool for functionally characterising functional domains within viral proteins

Molecular basis of cullin-3 (Cul3) ubiquitin ligase subversion by vaccinia virus protein A55.

BTB-Kelch proteins are substrate-specific adaptors for cullin-3 (Cul3) RING-box-based E3 ubiquitin ligases, mediating protein ubiquitylation for subsequent proteasomal degradation. Vaccinia virus encodes three BTB-Kelch proteins: A55, C2, and F3. Viruses lacking A55 or C2 have altered cytopathic effects in cultured cells and altered pathology in vivo Previous studies have shown that the ectromelia virus orthologue of A55 interacts with Cul3 in cells. We report that the N-terminal BTB-BACK (BB) domain of A55 binds directly to the Cul3 N-terminal domain (Cul3-NTD), forming a 2:2 complex in solution. We solved the structure of an A55BB/Cul3-NTD complex from anisotropic crystals diffracting to 2.3/3.7 Å resolution in the best/worst direction, revealing that the overall interaction and binding interface closely resemble the structures of cellular BTB/Cul3-NTD complexes, despite low sequence identity between A55 and cellular BTB domains. Surprisingly, despite this structural similarity, the affinity of Cul3-NTD for A55BB was stronger than for cellular BTB proteins. Glutamate substitution of the A55 residue Ile-48, adjacent to the canonical φX(D/E) Cul3-binding motif, reduced affinity of A55BB for Cul3-NTD by at least 2 orders of magnitude. Moreover, Ile-48 and the φX(D/E) motif are conserved in A55 orthologues from other poxviruses, but not in the vaccinia virus proteins C2 or F3. The high-affinity interaction between A55BB and Cul3-NTD suggests that, in addition to directing the Cul3-RING E3 ligase complex to degrade cellular/viral target proteins that are normally unaffected, A55 may also sequester Cul3 from cellular adaptor proteins, thereby protecting substrates of these cellular adaptors from ubiquitylation and degradation.Wellcome Trust, Royal Societ