Structural characterisation of hemagglutinin from seven Influenza A H1N1 strains reveal diversity in the C05 antibody recognition site.

Influenza virus (IV) causes several outbreaks of the flu each year resulting in an economic burden to the healthcare system in the billions of dollars. Several influenza pandemics have occurred during the last century and estimated to have caused 100 million deaths. There are four genera of IV, A (IVA), B (IVB), C (IVC), and D (IVD), with IVA being the most virulent to the human population. Hemagglutinin (HA) is an IVA surface protein that allows the virus to attach to host cell receptors and enter the cell. Here we have characterised the high-resolution structures of seven IVA HAs, with one in complex with the anti-influenza head-binding antibody C05. Our analysis revealed conserved receptor binding residues in all structures, as seen in previously characterised IV HAs. Amino acid conservation is more prevalent on the stalk than the receptor binding domain (RBD; also called the head domain), allowing the virus to escape from antibodies targeting the RBD. The equivalent site of C05 antibody binding to A/Denver/57 HA appears hypervariable in the other H1N1 IV HAs. Modifications within this region appear to disrupt binding of the C05 antibody, as these HAs no longer bind the C05 antibody by analytical SEC. Our study brings new insights into the structural and functional recognition of IV HA proteins and can contribute to further development of anti-influenza vaccines.

Crystal structure of a putative short-chain dehydrogenase/reductase from Paraburkholderia xenovorans.

Paraburkholderia xenovorans degrades organic wastes, including polychlorinated biphenyls. The atomic structure of a putative dehydrogenase/reductase (SDR) from P. xenovorans (PxSDR) was determined in space group P21 at a resolution of 1.45 Å. PxSDR shares less than 37% sequence identity with any known structure and assembles as a prototypical SDR tetramer. As expected, there is some conformational flexibility and difference in the substrate-binding cavity, which explains the substrate specificity. Uniquely, the cofactor-binding cavity of PxSDR is not well conserved and differs from those of other SDRs. PxSDR has an additional seven amino acids that form an additional unique loop within the cofactor-binding cavity. Further studies are required to determine how these differences affect the enzymatic functions of the SDR.

Structures of glyceraldehyde 3-phosphate dehydrogenase in Neisseria gonorrhoeae and Chlamydia trachomatis.

Neisseria gonorrhoeae (Ng) and Chlamydia trachomatis (Ct) are the most commonly reported sexually transmitted bacteria worldwide and usually present as co-infections. Increasing resistance of Ng to currently recommended dual therapy of azithromycin and ceftriaxone presents therapeutic challenges for syndromic management of Ng-Ct co-infections. Development of a safe, effective, and inexpensive dual therapy for Ng-Ct co-infections is an effective strategy for the global control and prevention of these two most prevalent bacterial sexually transmitted infections. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a validated drug target with two approved drugs for indications other than antibacterials. Nonetheless, any new drugs targeting GAPDH in Ng and Ct must be specific inhibitors of bacterial GAPDH that do not inhibit human GAPDH, and structural information of Ng and Ct GAPDH will aid in finding such selective inhibitors. Here, we report the X-ray crystal structures of Ng and Ct GAPDH. Analysis of the structures demonstrates significant differences in amino acid residues in the active sites of human GAPDH from those of the two bacterial enzymes suggesting design of compounds to selectively inhibit Ng and Ct is possible. We also describe an efficient in vitro assay of recombinant GAPDH enzyme activity amenable to high-throughput drug screening to aid in identifying inhibitory compounds and begin to address selectivity.

Functional consequences of B-repeat sequence variation in the staphylococcal biofilm protein Aap: deciphering the assembly code.

Staphylococcus epidermidis is an opportunistic pathogen that can form robust biofilms that render the bacteria resistant to antibiotic action and immune responses. Intercellular adhesion in S. epidermidis biofilms is mediated by the cell wall-associated accumulation-associated protein (Aap), via zinc-mediated self-assembly of its B-repeat region. This region contains up to 17 nearly identical sequence repeats, with each repeat assumed to be functionally equivalent. However, Aap B-repeats exist as two subtypes, defined by a cluster of consensus or variant amino acids. These variable residues are positioned near the zinc-binding (and dimerization) site and the stability determinant for the B-repeat fold. We have characterized four B-repeat constructs to assess the functional relevance of the two Aap B-repeat subtypes. Analytical ultracentrifugation experiments demonstrated that constructs with the variant sequence show reduced or absent Zn2+-induced dimerization. Likewise, circular dichroism thermal denaturation experiments showed that the variant sequence could significantly stabilize the fold, depending on its location within the construct. Crystal structures of three of the constructs revealed that the side chains from the variant sequence form an extensive bonding network that can stabilize the fold. Furthermore, altered distribution of charged residues between consensus and variant sequences changes the electrostatic potential in the vicinity of the Zn2+-binding site, providing a mechanistic explanation for the loss of zinc-induced dimerization in the variant constructs. These data suggest an assembly code that defines preferred oligomerization modes of the B-repeat region of Aap and a slip-grip model for initial contact followed by firm intercellular adhesion during biofilm formation.

Structural basis for collagen recognition by the immune receptor OSCAR.

The osteoclast-associated receptor (OSCAR) is a collagen-binding immune receptor with important roles in dendritic cell maturation and activation of inflammatory monocytes as well as in osteoclastogenesis. The crystal structure of the OSCAR ectodomain is presented, both free and in complex with a consensus triple-helical peptide (THP). The structures revealed a collagen-binding site in each immunoglobulin-like domain (D1 and D2). The THP binds near a predicted collagen-binding groove in D1, but a more extensive interaction with D2 is facilitated by the unusually wide D1-D2 interdomain angle in OSCAR. Direct binding assays, combined with site-directed mutagenesis, confirm that the primary collagen-binding site in OSCAR resides in D2, in marked contrast to the related collagen receptors, glycoprotein VI (GPVI) and leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1). Monomeric OSCAR D1D2 binds to the consensus THP with a KD of 28 µM measured in solution, but shows a higher affinity (KD 1.5 µM) when binding to a solid-phase THP, most likely due to an avidity effect. These data suggest a 2-stage model for the interaction of OSCAR with a collagen fibril, with transient, low-affinity interactions initiated by the membrane-distal D1, followed by firm adhesion to the primary binding site in D2.

Antagonism screen for inhibitors of bacterial cell wall biogenesis uncovers an inhibitor of undecaprenyl diphosphate synthase.

Drug combinations are valuable tools for studying biological systems. Although much attention has been given to synergistic interactions in revealing connections between cellular processes, antagonistic interactions can also have tremendous value in elucidating genetic networks and mechanisms of drug action. Here, we exploit the power of antagonism in a high-throughput screen for molecules that suppress the activity of targocil, an inhibitor of the wall teichoic acid (WTA) flippase in Staphylococcus aureus. Well-characterized antagonism within the WTA biosynthetic pathway indicated that early steps would be sensitive to this screen; however, broader interactions with cell wall biogenesis components suggested that it might capture additional targets. A chemical screening effort using this approach identified clomiphene, a widely used fertility drug, as one such compound. Mechanistic characterization revealed the target was the undecaprenyl diphosphate synthase, an enzyme that catalyzes the synthesis of a polyisoprenoid essential for both peptidoglycan and WTA synthesis. The work sheds light on mechanisms contributing to the observed suppressive interactions of clomiphene and in turn reveals aspects of the biology that underlie cell wall synthesis in S. aureus. Further, this effort highlights the utility of antagonistic interactions both in high-throughput screening and in compound mode of action studies. Importantly, clomiphene represents a lead for antibacterial drug discovery.

Structure of EspB from the ESX-1 type VII secretion system and insights into its export mechanism.

Mycobacterium tuberculosis (Mtb) uses the ESX-1 type VII secretion system to export virulence proteins across its lipid-rich cell wall, which helps permeabilize the host's macrophage phagosomal membrane, facilitating the escape and cell-to-cell spread of Mtb. ESX-1 membranolytic activity depends on a set of specialized secreted Esp proteins, the structure and specific roles of which are not currently understood. Here, we report the X-ray and electron microscopic structures of the ESX-1-secreted EspB. We demonstrate that EspB adopts a PE/PPE-like fold that mediates oligomerization with apparent heptameric symmetry, generating a barrel-shaped structure with a central pore that we propose contributes to the macrophage killing functions of EspB. Our structural data also reveal unexpected direct interactions between the EspB bipartite secretion signal sequence elements that form a unified aromatic surface. These findings provide insight into how specialized proteins encoded within the ESX-1 locus are targeted for secretion, and for the first time indicate an oligomerization-dependent role for Esp virulence factors.

Structural basis for Zn2+-dependent intercellular adhesion in staphylococcal biofilms.

Staphylococcal bacteria, including Staphylococcus epidermidis and Staphylococcus aureus, cause chronic biofilm-related infections. The homologous proteins Aap and SasG mediate biofilm formation in S. epidermidis and S. aureus, respectively. The self-association of these proteins in the presence of Zn(2+) leads to the formation of extensive adhesive contacts between cells. This study reports the crystal structure of a Zn(2+) -bound construct from the self-associating region of Aap. Several unusual structural features include elongated ß-sheets that are solvent-exposed on both faces and the lack of a canonical hydrophobic core. Zn(2+)-dependent dimers are observed in three distinct crystal forms, formed via pleomorphic coordination of Zn(2+) in trans across the dimer interface. These structures illustrate how a long, flexible surface protein is able to form tight intercellular adhesion sites under adverse environmental conditions.

Shiga toxin binding to glycolipids and glycans.

BACKGROUND: Immunologically distinct forms of Shiga toxin (Stx1 and Stx2) display different potencies and disease outcomes, likely due to differences in host cell binding. The glycolipid globotriaosylceramide (Gb3) has been reported to be the receptor for both toxins. While there is considerable data to suggest that Gb3 can bind Stx1, binding of Stx2 to Gb3 is variable. METHODOLOGY: We used isothermal titration calorimetry (ITC) and enzyme-linked immunosorbent assay (ELISA) to examine binding of Stx1 and Stx2 to various glycans, glycosphingolipids, and glycosphingolipid mixtures in the presence or absence of membrane components, phosphatidylcholine, and cholesterol. We have also assessed the ability of glycolipids mixtures to neutralize Stx-mediated inhibition of protein synthesis in Vero kidney cells. RESULTS: By ITC, Stx1 bound both Pk (the trisaccharide on Gb3) and P (the tetrasaccharide on globotetraosylceramide, Gb4), while Stx2 did not bind to either glycan. Binding to neutral glycolipids individually and in combination was assessed by ELISA. Stx1 bound to glycolipids Gb3 and Gb4, and Gb3 mixed with other neural glycolipids, while Stx2 only bound to Gb3 mixtures. In the presence of phosphatidylcholine and cholesterol, both Stx1 and Stx2 bound well to Gb3 or Gb4 alone or mixed with other neutral glycolipids. Pre-incubation with Gb3 in the presence of phosphatidylcholine and cholesterol neutralized Stx1, but not Stx2 toxicity to Vero cells. CONCLUSIONS: Stx1 binds primarily to the glycan, but Stx2 binding is influenced by residues in the ceramide portion of Gb3 and the lipid environment. Nanomolar affinities were obtained for both toxins to immobilized glycolipids mixtures, while the effective dose for 50% inhibition (ED(50)) of protein synthesis was about 10(-11) M. The failure of preincubation with Gb3 to protect cells from Stx2 suggests that in addition to glycolipid expression, other cellular components contribute to toxin potency.

FERM domain phosphoinositide binding targets merlin to the membrane and is essential for its growth-suppressive function.

The neurofibromatosis type 2 tumor suppressor protein, merlin, is related to the ERM (ezrin, radixin, and moesin) family of plasma membrane-actin cytoskeleton linkers. For ezrin, phosphatidylinositol 4,5-bisphosphate (PIP(2)) binding to the amino-terminal FERM domain is required for its conformational activation, proper subcellular localization, and function, but less is known about the role of phosphoinositide binding for merlin. Current evidence indicates that association with the membrane is important for merlin to function as a growth regulator; however, the mechanisms by which merlin localizes to the membrane are less clear. Here, we report that merlin binds phosphoinositides, including PIP(2), via a conserved binding motif in its FERM domain. Abolition of FERM domain-mediated phosphoinositide binding of merlin displaces merlin from the membrane and releases it into the cytosol without altering the folding of merlin. Importantly, a merlin protein whose FERM domain cannot bind phosphoinositide is defective in growth suppression. Retargeting the mutant merlin into the membrane using a dual-acylated amino-terminal decapeptide from Fyn is sufficient to restore the growth-suppressive properties to the mutant merlin. Thus, FERM domain-mediated phosphoinositide binding and membrane association are critical for the growth-regulatory function of merlin.

A human cancer-predisposing polymorphism in Cdc25A is embryonic lethal in the mouse and promotes ASK-1 mediated apoptosis.

BACKGROUND: Failure to regulate the levels of Cdc25A phosphatase during the cell cycle or during a checkpoint response causes bypass of DNA damage and replication checkpoints resulting in genomic instability and cancer. During G1 and S and in cellular response to DNA damage, Cdc25A is targeted for degradation through the Skp1-cullin-ß-TrCP (SCFß-TrCP) complex. This complex binds to the Cdc25A DSG motif which contains serine residues at positions 82 and 88. Phosphorylation of one or both residues is necessary for the binding and degradation to occur. RESULTS: We now show that mutation of serine 88 to phenylalanine, which is a cancer-predisposing polymorphic variant in humans, leads to early embryonic lethality in mice. The mutant protein retains its phosphatase activity both in vitro and in cultured cells. It fails to interact with the apoptosis signal-regulating kinase 1 (ASK1), however, and therefore does not suppress ASK1-mediated apoptosis. CONCLUSIONS: These data suggest that the DSG motif, in addition to its function in Cdc25A-mediated degradation, plays a role in cell survival during early embyogenesis through suppression of ASK1-mediated apoptosis.

Thermodynamic analysis of metal ion-induced protein assembly.

A large number of biological systems are regulated by metal ion-induced protein assembly. This phenomenon can play a critical role in governing protein function and triggering downstream biological responses. We discuss the basic thermodynamic principles of linked equilibria that pertain to metal ion-induced dimerization and describe experimental approaches useful for studying such systems. The most informative techniques for studying these systems are sedimentation velocity and sedimentation equilibrium analytical ultracentrifugation, although a wide range of other spectroscopic, chromatographic, or qualitative approaches can provide a wealth of useful information. These experimental procedures are illustrated with examples from two systems currently under study: zinc-induced assembly of a staphylococcal protein responsible for intercellular adhesion in bacterial biofilms and calcium-induced dimerization of a human nucleotidase.

Molecular basis of differential B-pentamer stability of Shiga toxins 1 and 2.

Escherichia coli strain O157:H7 is a major cause of food poisoning that can result in severe diarrhea and, in some cases, renal failure. The pathogenesis of E. coli O157:H7 is in large part due to the production of Shiga toxin (Stx), an AB(5) toxin that consists of a ribosomal RNA-cleaving A-subunit surrounded by a pentamer of receptor-binding B subunits. There are two major isoforms, Stx1 and Stx2, which differ dramatically in potency despite having 57% sequence identity. Animal studies and epidemiological studies show Stx2 is associated with more severe disease. Although the molecular basis of this difference is unknown, data suggest it is associated with the B-subunit. Mass spectrometry studies have suggested differential B-pentamer stability between Stx1 and Stx2. We have examined the relative stability of the B-pentamers in solution. Analytical ultracentrifugation using purified B-subunits demonstrates that Stx2B, the more deadly isoform, shows decreased pentamer stability compared to Stx1B (EC(50) = 2.3 µM vs. EC(50) = 0.043 µM for Stx1B). X-ray crystal structures of Stx1B and Stx2B identified a glutamine in Stx2 (versus leucine in Stx1) within the otherwise strongly hydrophobic interface between B-subunits. Interchanging these residues switches the stability phenotype of the B-pentamers of Stx1 and Stx2, as demonstrated by analytical ultracentrifugation and circular dichroism. These studies demonstrate a profound difference in stability of the B-pentamers in Stx1 and Stx2, illustrate the mechanistic basis for this differential stability, and provide novel reagents to test the basis for differential pathogenicity of these toxins.

A zinc-dependent adhesion module is responsible for intercellular adhesion in staphylococcal biofilms.

Hospital-acquired bacterial infections are an increasingly important cause of morbidity and mortality worldwide. Staphylococcal species are responsible for the majority of hospital-acquired infections, which are often complicated by the ability of staphylococci to grow as biofilms. Biofilm formation by Staphylococcus epidermidis and Staphylococcus aureus requires cell-surface proteins (Aap and SasG) containing sequence repeats known as G5 domains; however, the precise role of these proteins in biofilm formation is unclear. We show here, using analytical ultracentrifugation (AUC) and circular dichroism (CD), that G5 domains from Aap are zinc (Zn(2+))-dependent adhesion modules analogous to mammalian cadherin domains. The G5 domain dimerizes in the presence of Zn(2+), incorporating 2-3 Zn(2+) ions in the dimer interface. Tandem G5 domains associate in a modular fashion, suggesting a "zinc zipper" mechanism for G5 domain-based intercellular adhesion in staphylococcal biofilms. We demonstrate, using a biofilm plate assay, that Zn(2+) chelation specifically prevents biofilm formation by S. epidermidis and methicillin-resistant S. aureus (MRSA). Furthermore, individual soluble G5 domains inhibit biofilm formation in a dose-dependent manner. Thus, the complex three-dimensional architecture of staphylococcal biofilms results from the self-association of a single type of protein domain. Surface proteins with tandem G5 domains are also found in other bacterial species, suggesting that this mechanism for intercellular adhesion in biofilms may be conserved among staphylococci and other Gram-positive bacteria. Zn(2+) chelation represents a potential therapeutic approach for combating biofilm growth in a wide range of bacterial biofilm-related infections.

Dissolution and regeneration of Bombyx mori silk fibroin using ionic liquids.

In this work, the suitability of imidazolium-based ionic liquid solvents is investigated for the dissolution and regeneration of silkworm (Bombyx mori) silk. Within an ionic liquid the anion plays a larger role in dictating the ultimate solubility of the silk. The dissolution of the silk in the ionic liquid is confirmed using wide-angle X-ray scattering. The dissolved silk is also processed into 100 mum-thick, two-dimensional films, and the structure of these films is examined. The rinse solvent, acetonitrile or methanol, has a profound impact on both the topography of the films and the secondary structure of the silk protein. The image depicts a silkworm cocoon dissolved in 1-butyl-3-methylimidazolium chloride and then regenerated as a film with birefringence.