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The antithrombin-heparin/heparan sulfate (H/HS) and thrombin-H/HS interactions are recognized as prototypic specific and non-specific glycosaminoglycan (GAG)-protein interactions, respectively. The fundamental structural basis for the origin of specificity, or lack thereof, in these interactions remains unclear. The availability of multiple co-crystal structures facilitates a structural analysis that challenges the long-held belief that the GAG binding sites in antithrombin and thrombin are essentially similar with high solvent exposure and shallow surface characteristics. Analyses of solvent accessibility and exposed surface areas, gyrational mobility, symmetry, cavity shape/size, conserved water molecules and crystallographic parameters were performed for 12 X-ray structures, which include 12 thrombin and 16 antithrombin chains. Novel calculations are described for gyrational mobility and prediction of water loci and conservation. The solvent accessibilities and gyrational mobilities of arginines and lysines in the binding sites of the two proteins reveal sharp contrasts. The distribution of positive charges shows considerable asymmetry in antithrombin, but substantial symmetry for thrombin. Cavity analyses suggest the presence of a reasonably sized bifurcated cavity in antithrombin that facilitates a firm 'hand-shake' with H/HS, but with thrombin, a weaker 'high-five'. Tightly bound water molecules were predicted to be localized in the pentasaccharide binding pocket of antithrombin, but absent in thrombin. Together, these differences in the binding sites explain the major H/HS recognition characteristics of the two prototypic proteins, thus affording an explanation of the specificity of binding. This provides a foundation for understanding specificity of interaction at an atomic level, which will greatly aid the design of natural or synthetic H/HS sequences that target proteins in a specific manner.

Matrix protein 1 (M1) of the influenza A virus plays multiple roles in virion assembly and infection. Interest in the pH dependence of M1's multiple functions led us to study the effect of subtle pH changes on M1 structure, resulting in the elucidation of a unique low-pH crystal structure of the N(1-165)-domain of A/WSN/33 (H1N1) M1 that has never been reported. Although the 2.2 Å crystal structure of M1 N-terminus shows a dimer with the two monomers interacting in a face-to-face fashion at low pH as observed earlier, a 44° rotation of the second monomer has led to a significantly different dimer interface that possibly affects dimer stability. More importantly, while one of the monomers is fully defined, the N-terminal half of the second monomer shows considerable disorder that appears inherent in the protein and is potentially physiologically relevant. Such disorder has not been observed in any other previously reported structure at either low or high pH conditions, despite similar crystallization pH conditions. By comparing our novel N(1-165)-domain structure with other low-pH or neutral-pH M1 structures, it appears that M1 can energetically access different monomer and dimer conformations, as well as oligomeric states, with varying degree of similarities. The study reported here provides further insights into M1 oligomerization that may be essential for viral propagation and infectivity.

Three Bacillus bacteriophage-derived endolysins, designated PlyP56, PlyN74, and PlyTB40, were identified, cloned, purified, and characterized for their antimicrobial properties. Sequence alignment reveals these endolysins have an N-terminal enzymatically active domain (EAD) linked to a C-terminal cell wall binding domain (CBD). PlyP56 has a Peptidase_M15_4/VanY superfamily EAD with a conserved metal binding motif and displays biological dependence on divalent ions for activity. In contrast, PlyN74 and PlyTB40 have T7 lysozyme-type Amidase_2 and carboxypeptidase T-type Amidase_3 EADs, respectively, which are members of the MurNAc-LAA superfamily, but are not homologs and thus do not have a shared protein fold. All three endolysins contain similar SH3-family CBDs. Although minor host range differences were noted, all three endolysins show relatively broad antimicrobial activity against members of the Bacillus cereus sensu lato group with the highest lytic activity against B. cereus ATCC 4342. Characterization studies determined the optimal lytic activity for these enzymes was at physiological pH (pH 7.0–8.0), over a broad temperature range (4–55 °C), and at low concentrations of NaCl (<50 mM). Direct comparison of lytic activity shows the PlyP56 enzyme to be twice as effective at lysing the cell wall peptidoglycan as PlyN74 or PlyTB40, suggesting PlyP56 is a good candidate for further antimicrobial development as well as bioengineering studies.

Opioid receptors mediate the actions of endogenous and exogenous opioids on many physiological processes, including the regulation of pain, respiratory drive, mood, and—in the case of κ-opioid receptor (κ-OR)—dysphoria and psychotomimesis. Here we report the crystal structure of the human κ-OR in complex with the selective antagonist JDTic, arranged in parallel dimers, at 2.9 Å resolution. The structure reveals important features of the ligand-binding pocket that contribute to the high affinity and subtype selectivity of JDTic for the human κ-OR. Modelling of other important κ-OR-selective ligands, including the morphinan-derived antagonists norbinaltorphimine and 5′-guanidinonaltrindole, and the diterpene agonist salvinorin A analogue RB-64, reveals both common and distinct features for binding these diverse chemotypes. Analysis of site-directed mutagenesis and ligand structure–activity relationships confirms the interactions observed in the crystal structure, thereby providing a molecular explanation for κ-OR subtype selectivity, and essential insights for the design of compounds with new pharmacological properties targeting the human κ-OR. The crystal structure of the human κ-opioid receptor in complex with an antagonist, JDTic, is determined, with potential importance for the design of new therapeutic agents. Where opiates hit home Four papers in this issue of Nature present the long-awaited high-resolution crystal structures of the four known opioid receptors in ligand-bound conformations. These G-protein-coupled receptors are the targets of a broad range of drugs, including painkillers, antidepressants, anti-anxiety agents and anti-addiction medications. Brian Kobilka’s group reports the crystal structure of the µ-opioid receptor bound to a morphinan antagonist and the δ-opioid receptor bound to naltrindole. Raymond Stevens’ group reports on the κ-opioid receptor bound to the selective antagonist JDTic, and the nociceptin/orphanin FQ receptor bound to a peptide mimetic. In an associated News and Views, Marta Filizola and Lakshmi Devi discuss the implications of these landmark papers for research on the mechanisms underlying receptor function and drug development.

The M gene segment of influenza A virus has been shown to be a contributing factor to the high growth phenotype. However, it remains largely unknown why matrix protein 1 (M1), the major structural protein encoded by M gene, exhibits pH-dependent conformational changes during virus replication. Understanding the mechanisms underlying efficient virus replication can help to develop strategies not only to combat influenza infections but also to improve vaccine supplies. M(NLS-88R) and M(NLS-88E) are two M1 mutants differing by only a single amino acid: G88R vs G88E. G88R but not G88E was the compensatory mutation naturally selected by the virus after its nuclear localization signal was disrupted. Our study shows that, compared with M(NLS-88E) M1, M(NLS-88R) M1 dissociated quickly from viral ribonucleoproteins (vRNPs) at higher pH and took less time to dissemble in vitro, despite forming thicker matrix layer and having stronger association with vRNP in assembled virions. Correspondingly, M(NLS-88R) replicated more efficiently and was genetically more stable than M(NLS-88E). Crystallographic analysis indicated that M(NLS-88R) M1, like wild-type M1, is able to switch from a face-to-back-oriented conformation to a face-to-face-oriented conformation when pH drops from neutral to acidic, whereas G88E mutation causes M(NLS-88E) M1 to be trapped in a face-to-face-arranged conformation regardless of environmental pH. Our results suggest that maintaining M1 pH-dependent conformational flexibility is critical for efficient virus replication, and position 88 is a key residue controlling M1 pH-dependent conformational changes. Our findings provide insights into developing M1-based antiviral agents. Emerging Microbes & Infections (2017) 6, e108; doi: 10.1038/emi.2017.96 ; published online 6 December 2017

Aim: The σ1 receptor is a druggable target involved in many physiological processes and diseases. To clarify its physiology and derive therapeutic benefit, nine analogs based on the σ1 antagonist PB212 were synthesized replacing the 4-methylpiperidine with basic moieties of varying size and degree of conformational freedom. Results & methodology: 3-Phenylpyrrolidine, 4-phenylpiperidine or granatane derivatives displayed the highest affinity (Ki.#x00A0;= 0.12, 0.31 or 1.03 nM). Calcium flux assays in MCF7σ1 cells indicated that the highest σ1 receptor affinity are σ1 antagonists. Molecular models provided a structural basis for understanding the σ1 affinity and functional activity of the analogs and incorporated Glennon's σ1 pharmacophore model. Conclusion: Herein, we identify new compounds exploitable as therapeutic drug leads or as tools to study σ1 receptor physiology.Aim: The σ1 receptor is a druggable target involved in many physiological processes and diseases. To clarify its physiology and derive therapeutic benefit, nine analogs based on the σ1 antagonist PB212 were synthesized replacing the 4-methylpiperidine with basic moieties of varying size and degree of conformational freedom. Results & methodology: 3-Phenylpyrrolidine, 4-phenylpiperidine or granatane derivatives displayed the highest affinity (Ki.#x00A0;= 0.12, 0.31 or 1.03 nM). Calcium flux assays in MCF7σ1 cells indicated that the highest σ1 receptor affinity are σ1 antagonists. Molecular models provided a structural basis for understanding the σ1 affinity and functional activity of the analogs and incorporated Glennon's σ1 pharmacophore model. Conclusion: Herein, we identify new compounds exploitable as therapeutic drug leads or as tools to study σ1 receptor physiology.

The matrix protein 1 (M1) of influenza A virus (IAV) exists as a three-dimensional oligomeric structure in mature virions with high sequence conservation across different IAV subtypes, which makes it a potential broad spectrum antiviral target. We hypothesized that impairing self-association of M1 through a small molecule ‘wedge’, which avidly binds to an M1-M1 interface, would result in a completely new class of anti-influenza agents. To establish this proof-of-principle, we performed virtual screening on a library of >70,000 commercially available small molecules that resulted in several plausible ‘wedges’. Biophysical studies showed that the best molecule bound the M1 protein potently and weakened M1-M1 self-association. Most importantly, the agent reduced the thickness of the M1 layer in mature virions and inhibited in ovo propagation of multiple IAV strains including H1N1, pandemic H1N1, H3N2 and H5N1, which supports the “wedge” hypothesis. These results demonstrate that M1 is a promising druggable target for the discovery of a completely new line of broad spectrum anti-IAV agents.

Studies have shown that the TOM1 family of proteins, including TOM1 and TOM1L1, are actively involved in endosomal trafficking and function in the immune response. However, much less is known about the function of TOM1L2. To understand the biological importance of TOM1L2 and the potential significance of its cellular role, we created and evaluated Tom1l2 gene-trapped mice with reduced Tom1l2 expression. Mice hypomorphic for Tom1l2 exhibited numerous infections and tumors compared to wild-type littermates. Associated with this increased risk for infection and tumor formation, apparently healthy Tom1l2 hypomorphs also had splenomegaly, elevated B- and T-cell counts, and an impaired humoral response, although at a reduced penetrance. Furthermore, cellular localization studies showed that a Tom1l2-GFP fusion protein colocalizes with Golgi compartments, supporting the role of Tom1l2 in cellular trafficking, while molecular modeling and bioinformatic analysis of Tom1l2 illustrated a structural basis for a functional role in trafficking. These results indicate a role for Tom1l2 in the immune response and possibly in tumor suppression.

ABSTRACTWe report here the genome sequences of two novel Bacillus cereus group-infecting bacteriophages, Janet and OTooleKemple52. These bacteriophages are double-stranded DNA-containing Myoviridae isolated from soil samples. While their genomes share a high degree of sequence identity with one another, their host preferences are unique.

Opioid receptors mediate the actions of endogenous and exogenous opioids on many physiological processes, including the regulation of pain, respiratory drive, mood, and--in the case of κ-opioid receptor (κ-OR)--dysphoria and psychotomimesis. Here we report the crystal structure of the human κ-OR in complex with the selective antagonist JDTic, arranged in parallel dimers, at 2.9 A resolution. The structure reveals important features of the ligand-binding pocket that contribute to the high affinity and subtype selectivity of JDTic for the human κ-OR. Modelling of other important κ-OR-selective ligands, including the morphinan-derived antagonists norbinaltorphimine and 5'-guanidinonaltrindole, and the diterpene agonist salvinorin A analogue RB-64, reveals both common and distinct features for binding these diverse chemotypes. Analysis of site-directed mutagenesis and ligand structure-activity relationships confirms the interactions observed in the crystal structure, thereby providing a molecular explanation for κ-OR subtype selectivity, and essential insights for the design of compounds with new pharmacological properties targeting the human κ-OR.