Crystal structure of FliC flagellin from Pseudomonas aeruginosa and its implication in TLR5 binding and formation of the flagellar filament

Pseudomonas aeruginosa is one of leading opportunistic pathogens in humans and its movement is driven by a flagellar filament that is constituted through the polymerization of a single protein, FliC flagellin (paFliC). paFliC is an essential virulence factor for the colonization of P. aeruginosa. paFliC activates innate immune responses via its recognition by Toll-like receptor 5 (TLR5) and adaptive immunity in the host. Thus, paFliC has been a vaccine candidate to prevent P. aeruginosa infection, particularly for cystic fibrosis patients. To provide structural information on paFliC and its flagellar filament, we have determined the crystal structure of paFliC, which contains the conserved D1 and variable D2 domains, at 2.1 Å resolution. As observed for Salmonella FliC, the paFliC D1 domain is folded into a rod-shaped structure, and paFliC was demonstrated by gel filtration and native PAGE analyses to directly interact with TLR5. Moreover, a structural model of the paFliC-TLR5 complex suggests that paFliC D1 would provide major TLR5-binding sites, similar to Salmonella FliC. In contrast to the D1 domain, the paFliC D2 domain exhibits a unique structure of two β-sheets and one α-helix that has not been found in other flagellins. An in silico construction of a flagellar filament based on the packing of paFliC in the crystal suggests that the D2 domain would be exposed to solution and could play an important role in immunogenicity. Our biophysical and structure-based modeling study on paFliC, the paFliC-TLR5 complex, and the paFliC filament could contribute to the improvement of vaccine design to control P. aeruginosa infection.     

Use of β3-methionine as an amino acid substrate of Escherichia coli methionyl-tRNA synthetase

Polypeptides containing β-amino acids are attractive tools for the design of novel proteins having unique properties of medical or industrial interest. Incorporation of β-amino acids in vivo requires the development of efficient aminoacyl-tRNA synthetases specific of these non-canonical amino acids. Here, we have performed a detailed structural and biochemical study of the recognition and use of β³-Met by Escherichia coli methionyl-tRNA synthetase (MetRS). We show that MetRS binds β³-Met with a 24-fold lower affinity but catalyzes the esterification of the non-canonical amino acid onto tRNA with a rate lowered by three orders of magnitude. Accurate measurements of the catalytic parameters required careful consideration of the presence of contaminating α-Met in β³-Met commercial samples. The 1.45 Å crystal structure of the MetRS: β³-Met complex shows that β³-Met binds the enzyme essentially like α-Met, but the carboxylate moiety is mobile and not adequately positioned to react with ATP for aminoacyl adenylate formation. This study provides structural and biochemical bases for engineering MetRS with improved β³-Met aminoacylation capabilities.     

The Structure of Mycobacterium tuberculosis CYP125: MOLECULAR BASIS FOR CHOLESTEROL BINDING IN A P450 NEEDED FOR HOST INFECTION*

We report characterization and the crystal structure of the Mycobacterium tuberculosis cytochrome P450 CYP125, a P450 implicated in metabolism of host cholesterol and essential for establishing infection in mice. CYP125 is purified in a high spin form and undergoes both type I and II spectral shifts with various azole drugs. The 1.4-Å structure of ligand-free CYP125 reveals a “letterbox” active site cavity of dimensions appropriate for entry of a polycyclic sterol. A mixture of hexa-coordinate and penta-coordinate states could be discerned, with water binding as the 6th heme-ligand linked to conformation of the I-helix Val²⁶⁷ residue. Structures in complex with androstenedione and the antitubercular drug econazole reveal that binding of hydrophobic ligands occurs within the active site cavity. Due to the funnel shape of the active site near the heme, neither approaches the heme iron. A model of the cholesterol CYP125 complex shows that the alkyl side chain extends toward the heme iron, predicting hydroxylation of cholesterol C27. The alkyl chain is in close contact to Val²⁶⁷, suggesting a substrate binding-induced low- to high-spin transition coupled to reorientation of the latter residue. Reconstitution of CYP125 activity with a redox partner system revealed exclusively cholesterol 27-hydroxylation, consistent with structure and modeling. This activity may enable catabolism of host cholesterol or generation of immunomodulatory compounds that enable persistence in the host. This study reveals structural and catalytic properties of a potential M. tuberculosis drug target enzyme, and the likely mode by which the host-derived substrate is bound and hydroxylated.     

Crystal Structure of Aldehyde Dehydrogenase 16 Reveals Trans-Hierarchical Structural Similarity and a New Dimer

The aldehyde dehydrogenase (ALDH) superfamily is a vast group of enzymes that catalyze the NAD⁺-dependent oxidation of aldehydes to carboxylic acids. ALDH16 is perhaps the most enigmatic member of the superfamily, owing to its extra C-terminal domain of unknown function and the absence of the essential catalytic cysteine residue in certain non-bacterial ALDH16 sequences. Herein we report the first production of recombinant ALDH16, the first biochemical characterization of ALDH16, and the first crystal structure of ALDH16. Recombinant expression systems were generated for the bacterial ALDH16 from Loktanella sp. and human ALDH16A1. Four high-resolution crystal structures of Loktanella ALDH16 were determined. Loktanella ALDH16 is found to be a bona fide enzyme, exhibiting NAD⁺-binding, ALDH activity, and esterase activity. In contrast, human ALDH16A1 apparently lacks measurable aldehyde oxidation activity, suggesting that it is a pseudoenzyme, consistent with the absence of the catalytic Cys in its sequence. The fold of ALDH16 comprises three domains: NAD⁺-binding, catalytic, and C-terminal. The latter is unique to ALDH16 and features a Rossmann fold connected to a protruding β-flap. The tertiary structural interactions of the C-terminal domain mimic the quaternary structural interactions of the classic ALDH superfamily dimer, a phenomenon we call “trans-hierarchical structural similarity.” ALDH16 forms a unique dimer in solution, which mimics the classic ALDH superfamily dimer-of-dimer tetramer. Small-angle X-ray scattering shows that human ALDH16A1 has the same dimeric structure and fold as Loktanella ALDH16. We suggest that the Loktanella ALDH16 structure may be considered to be the archetype of the ALDH16 family.     

Unique Structure and Stability of HmuY,a Novel Heme-Binding Protein of Porphyromonas gingivalis

Infection, survival, and proliferation of pathogenic bacteria in humans depend on their capacity to impair host responses and acquire nutrients in a hostile environment. Among such nutrients is heme, a co-factor for oxygen storage, electron transport, photosynthesis, and redox biochemistry, which is indispensable for life. Porphyromonas gingivalis is the major human bacterial pathogen responsible for severe periodontitis. It recruits heme through HmuY, which sequesters heme from host carriers and delivers it to its cognate outer-membrane transporter, the TonB-dependent receptor HmuR. Here we report that heme binding does not significantly affect the secondary structure of HmuY. The crystal structure of heme-bound HmuY reveals a new all-β fold mimicking a right hand. The thumb and fingers pinch heme iron through two apical histidine residues, giving rise to highly symmetric octahedral iron co-ordination. The tetrameric quaternary arrangement of the protein found in the crystal structure is consistent with experiments in solution. It shows that thumbs and fingertips, and, by extension, the bound heme groups, are shielded from competing heme-binding proteins from the host. This may also facilitate heme transport to HmuR for internalization. HmuY, both in its apo- and in its heme-bound forms, is resistant to proteolytic digestion by trypsin and the major secreted proteases of P. gingivalis, gingipains K and R. It is also stable against thermal and chemical denaturation. In conclusion, these studies reveal novel molecular properties of HmuY that are consistent with its role as a putative virulence factor during bacterial infection.     

Bis-methionyl coordination in the crystal structure of the heme-binding domain of the streptococcal cell surface protein Shp

Surface proteins Shr, Shp, and the ATP-binding cassette (ABC) transporter HtsABC are believed to make up the machinery for heme uptake in Streptococcus pyogenes. Shp transfers its heme to HtsA, the lipoprotein component of HtsABC, providing the only experimentally demonstrated example of direct heme transfer from a surface protein to an ABC transporter in Gram-positive bacteria. To understand the structural basis of heme transfer in this system, the heme-binding domain of Shp (Shp¹⁸⁰) was crystallized, and its structure determined to a resolution of 2.1 Å. Shp¹⁸⁰ exhibits an immunoglobulin-like β-sandwich fold that has been recently found in other pathogenic bacterial cell surface heme-binding proteins, suggesting that the mechanisms of heme acquisition are conserved. Shp shows minimal amino acid sequence identity to these heme-binding proteins and the structure of Shp¹⁸⁰ reveals a unique heme-iron coordination with the axial ligands being two methionine residues from the same Shp molecule. A negative electrostatic surface of protein structure surrounding the heme pocket may serve as a docking interface for heme transfer from the more basic outer cell wall heme receptor protein Shr. The crystal structure of Shp¹⁸⁰ reveals two exogenous, weakly bound hemins, which form a large interface between the two Shp¹⁸⁰ molecules in the asymmetric unit. These “extra” hemins form a stacked pair with a structure similar to that observed previously for free hemin dimers in aqueous solution. The propionates of the protein-bound heme coordinate to the iron atoms of the exogenous hemin dimer, contributing to the stability of the protein interface. Gel filtration and analytical ultracentrifugation studies indicate that both full-length Shp and Shp¹⁸⁰ are monomeric in dilute aqueous solution.     

The solution structure of desulforedoxin,a simple iron-sulfur protein

Desulforedoxin is a simple dimeric protein isolated from Desulfovibrio gigas containing a distorted rubredoxin-like center with one iron coordinated by four cysteinyl residues (7.9?kDa with a 36-amino-acid monomer). ¹H NMR spectra of the oxidized Dx(Fe³⁺) and reduced Dx(Fe²⁺) forms were analyzed. The spectra show substantial line broadening due to the paramagnetism of iron. However, very low-field-shifted resonances, assigned to Hβ protons, were observed in the reduced state and their temperature dependence analyzed. The active site of Dx was reconstituted with zinc, and its solution structure was determined using 2D NMR methods. This diamagnetic form gave high-resolution NMR data enabling the identification of all the amino acid spin systems. Sequential assignment and the determination of secondary structural elements was attempted using 2D NOESY experiments. However, because of the symmetrical dimer nature of the protein standard, NMR sequential assignment methods could not resolve all cross peaks due to inter- and intra-chain effects. The X-ray structure enabled the spatial relationship between the monomers to be obtained, and resolved the assignment problems. Secondary structural features could be identified from the NMR data; an antiparallel β-sheet running from D5 to V18 with a well-defined β-turn around cysteines C9 and C12. The section G22 to T25 is poorly defined by the NMR data and is followed by a turn around V27-C29. The C-terminus ends up near residues V6 and Y7. Distance geometry (DG) calculations allowed families of structures to be generated from the NMR data. A family of structures with a low target function violation for the Dx monomer and dimer were found to have secondary structural elements identical to those seen in the X-ray structure. The amide protons for G4, D5, G13, L11 NH and Q14 NHε amide protons, H-bonded in the X-ray structure, were not seen by NMR as slowly exchanging, while structural disorder at the N-terminus, for the backbone at E10 and for the section G22–T25, was observed. Comparison between the Fe and Zn forms of Dx suggests that metal substitution does not have an effect on the structure of the protein.     

Vibrio vulnificus secretes a broad-specificity metalloprotease capable of interfering with blood homeostasis through prothrombin activation and fibrinolysis

Vibrio vulnificus is a causative agent of serious food-borne diseases in humans related to the consumption of raw seafood. It secretes a metalloprotease that is associated with skin lesions and serious hemorrhagic complications. In this study, we purified and characterized an extracellular metalloprotease (designated as vEP) having prothrombin activation and fibrinolytic activities from V. vulnificus ATCC 29307. vEP could cleave various blood clotting-associated proteins such as prothrombin, plasminogen, fibrinogen, and factor Xa, and the cleavage could be stimulated by addition of 1 mM Mn2+ in the reaction. The cleavage of prothrombin produced active thrombin capable of converting fibrinogen to fibrin. The formation of active thrombin appeared to be transient, with further cleavage resulting in a loss of activity. The cleavage of plasminogen, however, did not produce an active plasmin. vEP could cleave all three major chains of fibrinogen without forming a clot. It could cleave fibrin polymer formed by thrombin as well as the cross-linked fibrin formed by factor XIIIa. In addition, vEP could also cleave plasma proteins such as bovine serum albumin and gamma globulin, and its broad specificity is reflected in the cleavage sites, which include Asp207-Phe208 and Thr272-Ala273 bonds in prothrombin and a Tyr80-Leu81 bond in plasminogen. Taken together, the data suggest that vEP is a broad-specificity protease that could function as a prothrombin activator and a fibrinolytic enzyme to interfere with blood homeostasis as part of the mechanism associated with the pathogenicity of V. vulnificus in humans and thereby facilitate the development of systemic infection.     

1H, 13C, 15N backbone and side chain NMR resonance assignments of the N-terminal NEAr iron transporter domain 1 (NEAT 1) of the hemoglobin receptor IsdB of Staphylococcus aureus

Staphylococcus aureus is an opportunistic pathogen that causes skin and severe infections in mammals. Critical to S. aureus growth is its ability to scavenge iron from host cells. To this effect, S. aureus has evolved a sophisticated pathway to acquire heme from hemoglobin (Hb) as a preferred iron source. The pathway is comprised of nine iron-regulated surface determinant (Isd) proteins involved in heme capture, transport, and degradation. A key protein of the heme acquisition pathway is the surface-anchored hemoglobin receptor protein IsdB, which is comprised of two NEAr transporter (NEAT) domains that act in concert to bind Hb and extract heme for subsequent transfer to downstream acquisition pathway proteins. Despite significant advances in the structural knowledge of other Isd proteins, the structural mechanisms and molecular basis of the IsdB-mediated heme acquisition process are not well understood. In order to provide more insights into the mode of function of IsdB, we have initiated NMR structural studies of the first NEAT domain of IsdB (IsdB^N1). Herein, we report the near complete ¹H, ¹³C and ¹⁵N resonance assignments of backbone and side chain atoms, and the secondary structural topology of the 148-residue IsdB NEAT 1 domain. The NMR results are consistent with the presence of eight β-strands and one α-helix characteristic of an immunoglobulin-like fold observed in other NEAT domain family proteins. This work provides a solid framework to obtain atomic-level insights toward understanding how IsdB mediates IsdB-Hb protein–protein interactions critical for heme capture and transfer.     

The N-terminal propeptide of Vibrio vulnificus extracellular metalloprotease is both an inhibitor of and a substrate for the enzyme

Vibrio vulnificus, a marine bacterium capable of causing wound infection and septicemia, secretes a 45-kDa metalloprotease (vEP) with many biological activities. The precursor of vEP consists of four regions: a signal peptide, an N-terminal propeptide (nPP), a C-terminal propeptide, and the mature protease. Two forms of vEP-vEP-45, which contains the mature protease plus the C-terminal propeptide, and vEP-34, which contains only the mature protease-were expressed in Escherichia coli and purified. vEP-45 and vEP-34 had similar activities with azocasein as a substrate, but vEP-34 had reduced activity toward insoluble proteins. The nPP of vEP was expressed as a His tag fusion protein, and its effect on vEP activity was investigated. nPP inhibited the activities of both vEP-45 and vEP-34 but not that of thermolysin, a different but related zinc-dependent protease. The inhibition of vEP by nPP was further examined using vEP-34 as a representative enzyme. The inhibition could be completely reversed under conditions of low enzyme and propeptide concentrations and with prolonged incubation, which resulted from the degradation of nPP by vEP. However, even at high nPP and vEP concentrations, inhibition of vEP by nPP at high temperatures was not effective, resulting in the degradation of both nPP and vEP. These results demonstrate that the nPP of vEP could bind to vEP and inhibit its activity, resulting in the degradation of the propeptide.     

Increased folding stability of TEM-1 beta-lactamase by in vitro selection

In vitro selections of stabilized proteins lead to more robust enzymes and, at the same time, yield novel insights into the principles of protein stability. We employed Proside, a method of in vitro selection, to find stabilized variants of TEM-1 β-lactamase from Escherichia coli. Proside links the increased protease resistance of stabilized proteins to the infectivity of a filamentous phage. Several libraries of TEM-1 β-lactamase variants were generated by error-prone PCR, and variants with increased protease resistance were obtained by raising temperature or guanidinium chloride concentration during proteolytic selections. Despite the small size of phage libraries, several strongly stabilizing mutations could be obtained, and a manual combination of the best shifted the profiles for thermal unfolding and temperature-dependent inactivation of β-lactamase by almost 20 °C to a higher temperature. The wild-type protein unfolds in two stages: from the native state via an intermediate of the molten-globule type to the unfolded form. In the course of the selections, the native protein was stabilized by 27 kJ mol^− 1 relative to the intermediate and the cooperativity of unfolding was strongly increased. Three of our stabilizing replacements (M182T, A224V, and R275L) had been identified independently in naturally occurring β-lactamase variants with extended substrate spectrum. In these variants, they acted as global suppressors of destabilizations caused by the mutations in the active site. The comparison between the crystal structure of our best variant and the crystal structure of the wild-type protein indicates that most of the selected mutations optimize helices and their packing. The stabilization by the E147G substitution is remarkable. It removes steric strain that originates from an overly tight packing of two helices in the wild-type protein. Such unfavorable van der Waals repulsions are not easily identified in crystal structures or by computational approaches, but they strongly reduce the conformational stability of a protein.     

Crystal and Solution Structures of Plasmodium falciparum Erythrocyte-binding Antigen 140 Reveal Determinants of Receptor Specificity during Erythrocyte Invasion

Erythrocyte-binding antigen 140 (PfEBA-140) is a critical Plasmodium falciparum erythrocyte invasion ligand that engages glycophorin C on host erythrocytes during malaria infection. The minimal receptor-binding region of PfEBA-140 contains two conserved Duffy binding-like (DBL) domains, a fold unique to Plasmodium species. Here, we present the crystal structure of the receptor-binding region of PfEBA-140 at 2.4 Å resolution. The two-domain binding region is present as a monomer in the asymmetric unit, and the structure reveals novel features in PfEBA-140 that are likely determinants of receptor specificity. Analysis by small-angle x-ray scattering demonstrated that the minimal binding region is monomeric in solution, consistent with the crystal structure. Erythrocyte binding assays showed that the full-length binding region containing the tandem DBL domains is required for erythrocyte engagement, suggesting that both domains contain critical receptor contact sites. The electrostatic surface of PfEBA-140 elucidates a basic patch that constitutes a putative high-affinity binding interface spanning both DBL domains. Mutation of residues within this interface results in severely diminished erythrocyte binding. This study provides insight into the structural basis and mechanism of PfEBA-140 receptor engagement and forms a basis for future studies of this critical interaction. In addition, the solution and crystal structures allow the first identification of likely determinants of erythrocyte receptor specificity for P. falciparum invasion ligands. A complete understanding of the PfEBA-140 erythrocyte invasion pathway will aid in the design of invasion inhibitory therapeutics and vaccines.     

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

S. aureus is a pathogenic bacterium that requires iron to carry out vital metabolic functions and cause disease. The most abundant reservoir of iron inside the human host is heme, which is the cofactor of hemoglobin. To acquire iron from hemoglobin, S. aureus utilizes an elaborate system known as the iron-regulated surface determinant (Isd) system¹. Components of the Isd system first bind host hemoglobin, then extract and import heme, and finally liberate iron from heme in the bacterial cytoplasm^2,3. This pathway has been dissected through numerous in vitro studies^4-9. Further, the contribution of the Isd system to infection has been repeatedly demonstrated in mouse models^8,10-14. Establishing the contribution of the Isd system to hemoglobin-derived iron acquisition and growth has proven to be more challenging. Growth assays using hemoglobin as a sole iron source are complicated by the instability of commercially available hemoglobin, contaminating free iron in the growth medium, and toxicity associated with iron chelators. Here we present a method that overcomes these limitations. High quality hemoglobin is prepared from fresh blood and is stored in liquid nitrogen. Purified hemoglobin is supplemented into iron-deplete medium mimicking the iron-poor environment encountered by pathogens inside the vertebrate host. By starving S. aureus of free iron and supplementing with a minimally manipulated form of hemoglobin we induce growth in a manner that is entirely dependent on the ability to bind hemoglobin, extract heme, pass heme through the bacterial cell envelope and degrade heme in the cytoplasm. This assay will be useful for researchers seeking to elucidate the mechanisms of hemoglobin-/heme-derived iron acquisition in S. aureus and possibly other bacterial pathogens.     

Horse Prion Protein NMR Structure and Comparisons with Related Variants of the Mouse Prion Protein

The NMR structure of the horse (Equus caballus) cellular prion protein at 25 °C exhibits the typical PrP^C [cellular form of prion protein (PrP)] global architecture, but in contrast to most other mammalian PrP^Cs, it contains a well-structured loop connecting the β2 strand with the α2 helix. Comparison with designed variants of the mouse prion protein resulted in the identification of a single amino acid exchange within the loop, D167S, which correlates with the high structural order of this loop in the solution structure at 25 °C and is unique to the PrP sequences of equine species. The β2-α2 loop and the α3 helix form a protein surface epitope that has been proposed to be the recognition area for a hypothetical chaperone, “protein X,” which would promote conversion of PrP^C into the disease-related scrapie form and thus mediate intermolecular interactions related to the transmission barrier for transmissible spongiform encephalopathies (TSEs) between different species. The present results are evaluated in light of recent indications from in vivo experiments that the local β2-α2 loop structure affects the susceptibility of transgenic mice to TSEs and the fact that there are no reports on TSE in horses.     

Activation of the Legionella pneumophila LegK7 Effector Kinase by the Host MOB1 Protein

Legionella pneumophila infects alveolar macrophages and can cause life-threatening pneumonia in humans. Upon internalization into the host cell, L. pneumophila injects numerous effector proteins into the host cytoplasm as a part of its pathogenesis. LegK7 is an effector kinase of L. pneumophila that functionally mimics the eukaryotic Mst kinase and phosphorylates the host MOB1 protein to exploit the Hippo pathway. To elucidate the LegK7 activation mechanism, we determined the apo structure of LegK7 in an inactive form and performed a comparative analysis of LegK7 structures. LegK7 is a non-RD kinase that contains an activation segment that is ordered, irrespective of stimulation, through a unique β-hairpin-containing segment, and it does not require phosphorylation of the activation segment for activation. Instead, bacterial LegK7 becomes an active kinase via its heterologous molecular interaction with the host MOB1 protein. MOB1 binding triggers reorientation of the two lobes of the kinase domain, as well as a structural change in the interlobe hinge region in LegK7, consequently reshaping the LegK7 structure into an ATP binding-compatible closed conformation. Furthermore, we reveal that LegK7 is an atypical kinase that contains an N-terminal capping domain and a hydrophilic interlobe linker motif, which play key roles in the MOB1-induced activation of LegK7.     

Multiple proteins of White spot syndrome virus involved in recognition of β-integrin

The recognition and attachment of virus to its host cell surface is a critical step for viral infection. Recent research revealed that β-integrin was involved in White spot syndrome virus (WSSV) infection. In this study, the interaction of β-integrin with structure proteins of WSSV and motifs involved in WSSV infection was examined. The results showed that envelope proteins VP26, VP31, VP37, VP90 and nucleocapsid protein VP136 interacted with LvInt. RGD-, YGL- and LDV-related peptide functioned as motifs of WSSV proteins binding with β-integrin. The β-integrin ligand of RGDT had better blocking effect compared with that of YGL- and LDV-related peptides. In vivo assay indicated that RGD-, LDV- and YGL-related peptides could partially block WSSV infection. These data collectively indicate that multiple proteins were involved in recognition of β-integrin. Identification of proteins in WSSV that are associated with β-integrin will assist development of new agents for effective control of the white spot syndrome.     

Substrate cleavage pattern, biophysical characterization and low-resolution structure of a novel hyperthermostable arabinanase from Thermotoga petrophila

Arabinan is a plant structural polysaccharide degraded by two enzymes; α-l-arabinofuranosidase and endo-1,5-α-l-arabinanase. These enzymes are highly diversified in nature, however, little is known about their biochemical and biophysical properties. We have characterized a novel arabinanase (AbnA) isolated from Thermotoga petrophila with unique thermostable properties such as the insignificant decrease of residual activity after incubation up to 90 °C. We determined the AbnA mode of operation through capillary zone electrophoresis, which accumulates arabinotriose and arabinobiose as end products after hydrolysis of arabinan-containing polysaccharides. Spectroscopic analyses by Far-UV circular dichroism and intrinsic tryptophan fluorescence emission demonstrated that AbnA is folded and formed mainly by β-sheet structural elements. In silico molecular modeling showed that the AbnA structure encompasses a five-bladed β-propeller catalytic core juxtaposed by distorted up-and-down β-barrel domain. The low-resolution structure determined by small angle X-ray scattering indicated that AbnA is monomeric in solution and its molecular shape is in full agreement with the model.     

Competing pathways control host resistance to virus via tRNA modification and programmed ribosomal frameshifting

Viral infection depends on a complex interplay between host and viral factors. Here, we link host susceptibility to viral infection to a network encompassing sulfur metabolism, tRNA modification, competitive binding, and programmed ribosomal frameshifting (PRF). We first demonstrate that the iron‐sulfur cluster biosynthesis pathway in Escherichia coli exerts a protective effect during lambda phage infection, while a tRNA thiolation pathway enhances viral infection. We show that tRNA^Lys uridine 34 modification inhibits PRF to influence the ratio of lambda phage proteins gpG and gpGT. Computational modeling and experiments suggest that the role of the iron‐sulfur cluster biosynthesis pathway in infection is indirect, via competitive binding of the shared sulfur donor IscS. Based on the universality of many key components of this network, in both the host and the virus, we anticipate that these findings may have broad relevance to understanding other infections, including viral infection of humans.     

Staphylococcus aureus Uses a Novel Multidomain Receptor to Break Apart Human Hemoglobin and Steal Its Heme

Staphylococcus aureus is a leading cause of life-threatening infections in the United States. It requires iron to grow, which must be actively procured from its host to successfully mount an infection. Heme-iron within hemoglobin (Hb) is the most abundant source of iron in the human body and is captured by S. aureus using two closely related receptors, IsdH and IsdB. Here we demonstrate that each receptor captures heme using two conserved near iron transporter (NEAT) domains that function synergistically. NMR studies of the 39-kDa conserved unit from IsdH (IsdH^N2N3, Ala³²⁶–Asp⁶⁶⁰) reveals that it adopts an elongated dumbbell-shaped structure in which its NEAT domains are properly positioned by a helical linker domain, whose three-dimensional structure is determined here in detail. Electrospray ionization mass spectrometry and heme transfer measurements indicate that IsdH^N2N3 extracts heme from Hb via an ordered process in which the receptor promotes heme release by inducing steric strain that dissociates the Hb tetramer. Other clinically significant Gram-positive pathogens capture Hb using receptors that contain multiple NEAT domains, suggesting that they use a conserved mechanism.