Glycogen is the major glucose reserve in eukaryotes, and defects in glycogen metabolism and structure lead to disease. Glycogenesis involves interaction of glycogenin (GN) with glycogen synthase (GS), where GS is activated by glucose-6-phosphate (G6P) and inactivated by phosphorylation. We describe the 2.6 angstrom resolution cryo-EM structure of phosphorylated human GS revealing an autoinhibited GS tetramer flanked by two GN dimers. Phosphorylated N- and C-termini from two GS protomers converge near the G6P-binding pocket and buttress against GS regulatory helices. This keeps GS in an inactive conformation mediated by phospho-Ser641 interactions with a composite "arginine cradle". Structure-guided mutagenesis perturbing interactions with phosphorylated tails led to increased basal/unstimulated GS activity. We propose that multivalent phosphorylation supports GS autoinhibition through interactions from a dynamic "spike" region, allowing a tuneable rheostat for regulating GS activity. This work therefore provides insights into glycogen synthesis regulation and facilitates studies of glycogen-related diseases. Glycogen is a major energy reserve in eukaryotes and is synthesised in part by glycogenin (GN) and glycogen synthase (GS). Here, authors describe the structural basis of GS regulation, specifically the mechanism of inactivation by phosphorylation.

The ecdysone receptor (EcR) possesses the remarkable capacity to adapt structurally to different types of ligands. EcR binds ecdysteroids, including 20-hydroxyecdysone (20E), as well as nonsteroidal synthetic agonists such as insecticidal dibenzoylhydrazines (DBHs). Here, we report the crystal structures of the ligand-binding domains of Heliothis virescens EcR/USP bound to the DBH agonist BYI09181 and to the imidazole-type compound BYI08346. The region delineated by helices H7 and H10 opens up to tightly fit a phenyl ring of the ligands to an extent that depends on the bulkiness of ring substituent. In the structure of 20E-bound EcR, this part of the ligand-binding pocket (LBP) contains a channel filled by water molecules that form an intricate hydrogen bond network between 20E and LBP. The water channel present in the nuclear receptor bound to its natural hormone acts as a critical molecular adaptation spring used to accommodate synthetic agonists inside its binding cavity.

R-type bacteriocins are minimal contractile nanomachines that hold promise as precision antibiotics 1 – 4 . Each bactericidal complex uses a collar to bridge a hollow tube with a contractile sheath loaded in a metastable state by a baseplate scaffold 1 , 2 . Fine-tuning of such nucleic acid-free protein machines for precision medicine calls for an atomic description of the entire complex and contraction mechanism, which is not available from baseplate structures of the (DNA-containing) T4 bacteriophage 5 . Here we report the atomic model of the complete R2 pyocin in its pre-contraction and post-contraction states, each containing 384 subunits of 11 unique atomic models of 10 gene products. Comparison of these structures suggests the following sequence of events during pyocin contraction: tail fibres trigger lateral dissociation of baseplate triplexes; the dissociation then initiates a cascade of events leading to sheath contraction; and this contraction converts chemical energy into mechanical force to drive the iron-tipped tube across the bacterial cell surface, killing the bacterium.

Pseudomonas phage LKA1 of the subfamily Autographivirinae encodes a tailspike protein (LKA1gp49) which binds and cleaves B-band LPS (O-specific antigen, OSA) of Pseudomonas aeruginosa PAO1. The crystal structure of LKA1gp49 catalytic domain consists of a beta-helix, an insertion domain and a C-terminal discoidin-like domain. The putative substrate binding and processing site is located on the face of the beta-helix whereas the C-terminal domain is likely involved in carbohydrates binding. NMR spectroscopy and mass spectrometry analyses of degraded LPS (OSA) fragments show an O5 serotype-specific polysaccharide lyase specificity. LKA1gp49 reduces virulence in an in vivo Galleria mellonella infection model and sensitizes P. aeruginosa to serum complement activity. This enzyme causes biofilm degradation and does not affect the activity of ciprofloxacin and gentamicin. This is the first comprehensive report on LPS-degrading lyase derived from a Pseudomonas phage. Biological properties reveal a potential towards its applications in antimicrobial design and as a microbiological or biotechnological tool.

Bacteriophages recognize and bind to their hosts with the help of receptor-binding proteins (RBPs) that emanate from the phage particle in the form of fibers or tailspikes. RBPs show a great variability in their shapes, sizes, and location on the particle. Some RBPs are known to depolymerize surface polysaccharides of the host while others show no enzymatic activity. Here we report that both RBPs of podovirus G7C - tailspikes gp63.1 and gp66 - are essential for infection of its natural host bacterium E. coli 4s that populates the equine intestinal tract. We characterize the structure and function of gp63.1 and show that unlike any previously described RPB, gp63.1 deacetylates surface polysaccharides of E. coli 4s leaving the backbone of the polysaccharide intact. We demonstrate that gp63.1 and gp66 form a stable complex, in which the N-terminal part of gp66 serves as an attachment site for gp63.1 and anchors the gp63.1-gp66 complex to the G7C tail. The esterase domain of gp63.1 as well as domains mediating the gp63.1-gp66 interaction is widespread among all three families of tailed bacteriophages.

Several systems, including contractile tail bacteriophages, the type VI secretion system and R-type pyocins, use a multiprotein tubular apparatus to attach to and penetrate host cell membranes. This macromolecular machine resembles a stretched, coiled spring (or sheath) wound around a rigid tube with a spike-shaped protein at its tip. A baseplate structure, which is arguably the most complex part of this assembly, relays the contraction signal to the sheath. Here we present the atomic structure of the approximately 6-megadalton bacteriophage T4 baseplate in its pre- and post-host attachment states and explain the events that lead to sheath contraction in atomic detail. We establish the identity and function of a minimal set of components that is conserved in all contractile injection systems and show that the triggering mechanism is universally conserved.

Surface-associated capsular polysaccharides (CPSs) protect bacteria against phage infection and enhance pathogenicity by interfering with the function of the host innate immune system. The CPS of enteropathogenic Escherichia coli K92 is a unique sialic acid polymer (polySia) with alternating α2,8- and α2,9-linkages. This CPS can be digested by the gene 143 encoded endosialidase of bacteriophage phi92. Here we report the crystal structure of the phi92 endosialidase in complex with a dimer of α2,9-linked sialic acid and analyze its catalytic functions. Unlike the well characterized and homologous endosialidase of phage K1F, the phi92 endosialidase is a bifunctional enzyme with high activity against α2,8- and low activity against α2,9-linkages in a polySia chain. Moreover, in contrast to the processive K1F endosialidase, the phi92 endosialidase degrades the polymer in a non-processive mode. Beyond describing the first endosialidase with α2,9-specificity, our data introduce a novel platform for studies of endosialidase regioselectivity and for engineering highly active α2,9-specific enzymes.

ABSTRACT Bacteriophage phi92 is a large, lytic myovirus isolated in 1983 from pathogenic Escherichia coli strains that carry a polysialic acid capsule. Here we report the genome organization of phi92, the cryoelectron microscopy reconstruction of its virion, and the reinvestigation of its host specificity. The genome consists of a linear, double-stranded 148,612-bp DNA sequence containing 248 potential open reading frames and 11 putative tRNA genes. Orthologs were found for 130 of the predicted proteins. Most of the virion proteins showed significant sequence similarities to proteins of myoviruses rv5 and PVP-SE1, indicating that phi92 is a new member of the novel genus of rv5-like phages. Reinvestigation of phi92 host specificity showed that the host range is not limited to polysialic acid-encapsulated Escherichia coli but includes most laboratory strains of Escherichia coli and many Salmonella strains. Structure analysis of the phi92 virion demonstrated the presence of four different types of tail fibers and/or tailspikes, which enable the phage to use attachment sites on encapsulated and nonencapsulated bacteria. With this report, we provide the first detailed description of a multivalent, multispecies phage armed with a host cell adsorption apparatus resembling a nanosized Swiss army knife. The genome, structure, and, in particular, the organization of the baseplate of phi92 demonstrate how a bacteriophage can evolve into a multi-pathogen-killing agent.

Bacteriophages with contractile tails and the bacterial type VI secretion system have been proposed to use a special protein to create an opening in the host cell membrane during infection. These proteins have a modular architecture but invariably contain an oligonucleotide/oligosaccharide-binding (OB-fold) domain and a long β-helical C-terminal domain, which initiates the contact with the host cell membrane. Using X-ray crystallography and electron microscopy, we report the atomic structure of the membrane-piercing proteins from bacteriophages P2 and ϕ92 and identify the residues that constitute the membrane-attacking apex. Both proteins form compact spikes with a ∼10Å diameter tip that is stabilized by a centrally positioned iron ion bound by six histidine residues. The accumulated data strongly suggest that, in the process of membrane penetration, the spikes are translocated through the lipid bilayer without undergoing major unfolding.

We have identified a small molecule (DD2) that inhibits an essential dimeric protease of the human Kaposi's sarcoma-associated herpesvirus (KSHV Pr).Proteolytic activity of KSHV Pr is disrupted by dissociation of dimeric state.Circular dichroism, chemical shift perturbation mapping and hydrogen exchange experiments indicate that a significant loss of structure at the C-terminal helices occurs concurrently with the dimer to monomer transition [1].Complementary methods including size exclusion chromatography and 2D NMR spectroscopy show that the monomeric protease is enriched in the presence of DD2, leading to loss of enzymatic activity. 1H -15 N HSQC titration studies mapped the inhibitor-binding site to the dimer interface and mutagenesis studies targeting this region confirmed this binding mode [2].As currently there are no specific treatments for herpesviral infections and with increased incidents of drug resistance, there is a need for the identification of new human herpesvirus therapeutics.The crystal structure of a monomeric form of KSHV Pr in complex with the small molecule inhibitor bound at the dimer interface was solved at 2.0 Å resolution.The final R factor /R free is 20.5%/24.3%,respectively.The complex was crystallized in the I222 space group.Two monomers of the truncated KSHV Pr were observed in the asymmetric unit with one DD2 molecule bound to monomer A, and two DD2 molecules bound to monomer B. The crystal structure is consistent with a mechanism where inhibitor binding prevents dimerization through the conformational selection of an inactive monomeric intermediate.This represents the first crystallographic structure of an inhibited monomeric Human Herpesvirus Protease to date.

I will summarize the limits of current technology for the crystallographic analysis of viruses and explore how far these limits might be pushed over the next few years, with some examples from current projects.I will also indicate with examples how the structural information we have might have an increasing impact on human and animal health as we start to integrate structural knowledge into the process of vaccine optimization.

The ligand-binding properties of recombinant ecdysone receptor EcR-USP heterodimeric ligand-binding domains (LBDs) from four insect orders are described for a range of ecdysteroids and for a dibenzoylhydrazine (DBH) insecticide (tebufenozide). Much of the order selectivity of the insecticide in the field is reproduced by the affinity of tebufenozide for the recombinant LBDs in the laboratory. Crystal structures are presented for the LBDs of ecdysone receptors from the pest insects Heliothis virescens, Bemisia tabaci and Tribolium castaneum in complex with ponasterone A, as well as of the H. virescens LBD in complex with 20-hydroxyecdysone and BYI06830 (a DBH insecticide). Comparison of ecdysteroid- and BYI06830-bound structures of the H. virescens LBD illustrates the way in which this remarkable protein can adapt its binding pocket to very different ligand chemistries. Finally, comparison of the ligand-binding pockets of H. virescens, B. tabaci and T. castaneum ecdysone receptors begins to provide insights at an atomic level of detail into the insect order selectivity of the DBH insecticides.

The insect steroid hormone 20-hydroxyecdysone (20E) binds to its cognate nuclear receptor composed of the ecdysone receptor (EcR) and Ultraspiracle (USP) and triggers the main developmental transitions, in particular molting and metamorphosis. We present the crystal structure of the ligand-binding domains of EcR/USP in complex with 20E at 2.4A resolution and compare it with published structures of EcR/USP bound to ponasterone A (ponA). ponA is essentially identical to 20E but lacks the 25-OH group of 20E. The structure of 20E-bound EcR indicates that an additional hydrogen bond is formed compared with the ponA-bound receptor, yet, paradoxically, ponA has a significantly higher affinity for EcR than 20E. Theoretical studies based on docking and free energy methods lead to a rationale for understanding the difference in binding affinities between 20E and ponA. Results of the calculations indicate that the favorable contribution from the extra H-bond made by 25-OH of 20E is counterbalanced by its larger desolvation cost compared with that of ponA. The contribution of 25-OH to the binding affinity is further compared with those of 20- and 22-OH groups. Ligands that lack the 20- or 22-OH group are indeed known to bind less favorably to EcR than 20E, an effect opposite to that observed for ponA. The results indicate that their respective contributions to receptor-ligand complex stability reside mostly in their different contributions to solvation/desolvation. Together, the data demonstrate the critical role of ligand desolvation in determining binding affinity, with general implications for the binding of hormones to their cognate nuclear receptors.

Ma these est dediee a la resolution de structures du recepteur a l'ecdysone (EcR) complexe a de nouveaux agonistes synthetiques, et d’assister dans la recherche et le developpement de ces insecticides. Cinq nouvelles structures de l'EcR ont ete resolues, en complexe avec ces ligands induisant une adaptation structurale du recepteur vis-a-vis du ligand. Trois conformations differentes de HvEcR sont induites. La resolution de la structure de l'EcR en complexe avec 20E a egalement ete entreprise. En effet, les precedentes structures de l'EcR ont ete resolues en complexe avec la ponasterone A (ponA). De plus, malgre une structure similaire a ponA, 20E presente un groupement OH supplementaire formant une liaison H lors de la fixation a l'EcR. Etonnament, on observe une plus faible energie de liaison. Des calculs d’energie libre ont permis de determiner que le gain d’energie libre totale du a cette liaison est inferieur au cout de desolvatation du 20E par rapport a celui de la ponA.

The ligand-binding properties of recombinant ecdysone receptor EcRUSP heterodimeric ligand-binding domains (LBDs) from four insect orders are described for a range of ecdysteroids and for a dibenzoylhydrazine (DBH) insecticide (tebufenozide). Much of the order selectivity of the insecticide in the field is reproduced by the affinity of tebufenozide for the recombinant LBDs in the laboratory. Crystal structures are presented for the LBDs of ecdysone receptors from the pest insects Heliothis virescens, Bemisia tabaci and Tribolium castaneum in complex with ponasterone A, as well as of the H. virescens LBD in complex with 20-hydroxyecdysone and BYI06830 (a DBH insecticide). Comparison of ecdysteroidand BYI06830-bound structures of the H. virescens LBD illustrates the way in which this remarkable protein can adapt its binding pocket to very different ligand chemistries. Finally, comparison of the ligand-binding pockets of H. virescens, B. tabaci and T. castaneum ecdysone receptors begins to provide insights at an atomic level of detail into the insect order selectivity of the DBH insecticides.