A map of human PRDM9 binding provides evidence for novel behaviors of PRDM9 and other zinc-finger proteins in meiosis.

PRDM9 binding localizes almost all meiotic recombination sites in humans and mice. However, most PRDM9-bound loci do not become recombination hotspots. To explore factors that affect binding and subsequent recombination outcomes, we mapped human PRDM9 binding sites in a transfected human cell line and measured PRDM9-induced histone modifications. These data reveal varied DNA-binding modalities of PRDM9. We also find that human PRDM9 frequently binds promoters, despite their low recombination rates, and it can activate expression of a small number of genes including CTCFL and VCX. Furthermore, we identify specific sequence motifs that predict consistent, localized meiotic recombination suppression around a subset of PRDM9 binding sites. These motifs strongly associate with KRAB-ZNF protein binding, TRIM28 recruitment, and specific histone modifications. Finally, we demonstrate that, in addition to binding DNA, PRDM9's zinc fingers also mediate its multimerization, and we show that a pair of highly diverged alleles preferentially form homo-multimers

Crystal structure and carbohydrate analysis of Nipah virus attachment glycoprotein:a template for antiviral and vaccine design

Two members of the paramyxovirus family, Nipah virus (NiV) and Hendra virus (HeV), are recent additions to a growing number of agents of emergent diseases which use bats as a natural host. Identification of ephrin-B2 and ephrin-B3 as cellular receptors for these viruses has enabled the development of immunotherapeutic reagents which prevent virus attachment and subsequent fusion. Here we present the structural analysis of the protein and carbohydrate components of the unbound viral attachment glycoprotein of NiV glycoprotein (NiV-G) at a 2.2-A resolution. Comparison with its ephrin-B2-bound form reveals that conformational changes within the envelope glycoprotein are required to achieve viral attachment. Structural differences are particularly pronounced in the 579-590 loop, a major component of the ephrin binding surface. In addition, the 236-245 loop is rather disordered in the unbound structure. We extend our structural characterization of NiV-G with mass spectrometric analysis of the carbohydrate moieties. We demonstrate that NiV-G is largely devoid of the oligomannose-type glycans that in viruses such as human immunodeficiency virus type 1 and Ebola virus influence viral tropism and the host immune response. Nevertheless, we find putative ligands for the endothelial cell lectin, LSECtin. Finally, by mapping structural conservation and glycosylation site positions from other members of the paramyxovirus family, we suggest the molecular surface involved in oligomerization. These results suggest possible pathways of virus-host interaction and strategies for the optimization of recombinant vaccines

Proteoglycan-Specific Molecular Switch for RPTPσ Clustering and Neuronal Extension

Heparan and chondroitin sulfate proteoglycans (HSPGs and CSPGs, respectively) regulate numerous cell surface signaling events, with typically opposite effects on cell function. CSPGs inhibit nerve regeneration through receptor protein tyrosine phosphatase sigma (RPTPσ). Here we report that RPTPσ acts bimodally in sensory neuron extension, mediating CSPG inhibition and HSPG growth promotion. Crystallographic analyses of a shared HSPG-CSPG binding site reveal a conformational plasticity that can accommodate diverse glycosaminoglycans with comparable affinities. Heparan sulfate and analogs induced RPTPσ ectodomain oligomerization in solution, which was inhibited by chondroitin sulfate. RPTPσ and HSPGs colocalize in puncta on sensory neurons in culture, whereas CSPGs occupy the extracellular matrix. These results lead to a model where proteoglycans can exert opposing effects on neuronal extension by competing to control the oligomerization of a common receptor

Initiation of T cell signaling by CD45 segregation at 'close contacts'.

It has been proposed that the local segregation of kinases and the tyrosine phosphatase CD45 underpins T cell antigen receptor (TCR) triggering, but how such segregation occurs and whether it can initiate signaling is unclear. Using structural and biophysical analysis, we show that the extracellular region of CD45 is rigid and extends beyond the distance spanned by TCR-ligand complexes, implying that sites of TCR-ligand engagement would sterically exclude CD45. We also show that the formation of 'close contacts', new structures characterized by spontaneous CD45 and kinase segregation at the submicron-scale, initiates signaling even when TCR ligands are absent. Our work reveals the structural basis for, and the potent signaling effects of, local CD45 and kinase segregation. TCR ligands have the potential to heighten signaling simply by holding receptors in close contacts.The authors thank R.A. Cornall, M.L. Dustin and P.A. van der Merwe for comments on the manuscript and S. Ikemizu for useful discussions about the structure. We also thank W. Lu and T. Walter for technical support with protein expression and crystallization, the staff at Diamond Light Source beamlines I02, I03 and I04-1 (proposal mx10627) and European Synchrotron Radiation Facility beamlines ID23EH1 and ID23EH2 for assistance at the synchrotrons, G. Sutton for assistance with MALS experiments, and M. Fritzsche for advice on the calcium analysis. This work was funded by the Wellcome Trust (098274/Z/12/Z to S.J.D.; 090532/Z/09/Z to R.J.C.G.; 090708/Z/09/Z to D.K.), the UK Medical Research Council (G0700232 to A.R.A.), the Royal Society (UF120277 to S.F.L.) and Cancer Research UK (C20724/A14414 to C.S.; C375/A10976 to E.Y.J.). The Oxford Division of Structural Biology is part of the Wellcome Trust Centre for Human Genetics, Wellcome Trust Core Award Grant Number 090532/Z/09/Z. We acknowledge financial support from Instruct, an ESFRI Landmark Project. The OPIC electron microscopy facility was funded by a Wellcome Trust JIF award (060208/Z/00/Z).This is the author accepted manuscript. The final version is available from Nature Publishing Group via https://doi.org/10.1038/ni.339

A Dual Binding Mode for RhoGTPases in Plexin Signalling

A novel binding site for RhoGTPases on the intracellular region of plexins induces a trimeric ligand—receptor arrangement that appears crucial for plexin function

Defining the molecular basis of interaction between R3 receptor-type protein tyrosine phosphatases and VE-cadherin

Receptor-type protein tyrosine phosphatases (RPTPs) of the R3 subgroup play key roles in the immune, vascular and nervous systems. They are characterised by a large ectodomain comprising multiple FNIII-like repeats, a transmembrane domain, and a single intracellular phosphatase domain. The functional role of the extracellular region has not been clearly defined and potential roles in ligand interaction, di-merization, and regulation of cell-cell contacts have been reported. Here bimolecular fluorescence complementation (BiFC) in live cells was used to examine the molecular basis for the interaction of VE-PTP with VE-cadherin, two proteins involved in endothelial cell contact and maintenance of vascu-lar integrity. The potential of other R3-PTPs to interact with VE-cadherin was also explored using this method. Quantitative BiFC analysis, using a VE-PTP construct expressing only the ectodomain and transmembrane domain, revealed a specific interaction with VE-cadherin, when compared with con-trols. Controls were sialophorin, an unrelated membrane protein with a large ectodomain, and a mem-brane anchored C-terminal Venus-YFP fragment, lacking both ectodomain and transmembrane do-mains. Truncation of the first 16 FNIII-like repeats from the ectodomain of VE-PTP indicated that re-moval of this region is not sufficient to disrupt the interaction with VE-cadherin, although it occurs predominantly in an intracellular location. A construct with a deletion of only the 17th domain of VE-PTP was, in contrast to previous studies, still able to interact with VE-cadherin, although this also was predominantly intracellular. Other members of the R3-PTP family (DEP-1, GLEPP1 and SAP-1) also exhibited the potential to interact with VE-cadherin. The direct interaction of DEP-1 with VE-cadherin is likely to be of physiological relevance since both proteins are expressed in endothelial cells. Together the data presented in the study suggest a role for both the ectodomain and transmembrane domain of R3-PTPs in interaction with VE-cadherin

The Glycan Shield of HIV Is Predominantly Oligomannose Independently of Production System or Viral Clade

The N-linked oligomannose glycans of HIV gp120 are a target for both microbicide and vaccine design. The extent of cross-clade conservation of HIV oligomannose glycans is therefore a critical consideration for the development of HIV prophylaxes. We measured the oligomannose content of virion-associated gp120 from primary virus from PBMCs for a range of viral isolates and showed cross-clade elevation (62–79%) of these glycans relative to recombinant, monomeric gp120 (∼30%). We also confirmed that pseudoviral production systems can give rise to notably elevated gp120 oligomannose levels (∼98%), compared to gp120 derived from a single-plasmid viral system using the HIVLAI backbone (56%). This study highlights differences in glycosylation between virion-associated and recombinant gp120

Calcitonin receptor N-glycosylation enhances peptide hormone affinity by controlling receptor dynamics

The class B G protein-coupled receptor (GPCR) calcitonin receptor (CTR) is a drug target for osteoporosis and diabetes. N-glycosylation of asparagine 130 in its extracellular domain (ECD) enhances calcitonin hormone affinity with the proximal GlcNAc residue mediating this effect through an unknown mechanism. Here, we present two crystal structures of salmon calcitonin-bound, GlcNAc-bearing CTR ECD at 1.78 and 2.85 Å resolutions and analyze the mechanism of the glycan effect. The N130 GlcNAc does not contact the hormone. Surprisingly, the structures are nearly identical to a structure of hormone-bound, N-glycan-free ECD, which suggested that the GlcNAc might affect CTR dynamics not observed in the static crystallographic snapshots. Hydrogen-deuterium exchange mass spectrometry and molecular dynamics simulations revealed that glycosylation stabilized a β-sheet adjacent to the N130 GlcNAc and the N-terminal α-helix near the peptide-binding site, while increasing flexibility of the peptide-binding site turret loop. These changes due to N-glycosylation increased the ligand on-rate and decreased its off rate. The glycan effect extended to RAMP-CTR amylin receptor complexes and was also conserved in the related CGRP receptor. These results reveal that N-glycosylation can modulate GPCR function by altering receptor dynamics

Factors influencing success of clinical genome sequencing across a broad spectrum of disorders

To assess factors influencing the success of whole-genome sequencing for mainstream clinical diagnosis, we sequenced 217 individuals from 156 independent cases or families across a broad spectrum of disorders in whom previous screening had identified no pathogenic variants. We quantified the number of candidate variants identified using different strategies for variant calling, filtering, annotation and prioritization. We found that jointly calling variants across samples, filtering against both local and external databases, deploying multiple annotation tools and using familial transmission above biological plausibility contributed to accuracy. Overall, we identified disease-causing variants in 21% of cases, with the proportion increasing to 34% (23/68) for mendelian disorders and 57% (8/14) in family trios. We also discovered 32 potentially clinically actionable variants in 18 genes unrelated to the referral disorder, although only 4 were ultimately considered reportable. Our results demonstrate the value of genome sequencing for routine clinical diagnosis but also highlight many outstanding challenges