Helical Stalk Research Articles

Cretaceous Fe-oxidising microbial mats in injection dykes in the Variscan gold-bearing metamorphic basement of the Tisza Mega-unit (SE Hungary)

Large quantities of Cretaceous Fe-oxidising bacteria (FeOB) are documented here for the first time in carbonate injection dykes. These injection dykes occur in fissures of a Palaeozoic metamorphic complex in the Geresd Hills, Hungary, and are made up of microlaminated haematitic carbonate that contain very well preserved helical FeOB stalk fossils similar to recent iron-oxidising Gallionella and Mariprofundus. The presence of filament clusters with semi-parallel textures suggests aerotactic bacterial cell movements. Fractal analysis of the filament clusters showed higher fractal dimension values (Df ∼ 1.9) compared to the typical fractal dimension values of the texturally similar, but inorganic Diffusion-Limited Aggregation (DLA) structures (Df ∼ 1.7). This finding is interpreted to suggest that the observed filamentous texture is of microbial origin. Cretaceous basaltic intrusions that penetrated the gold-bearing pyritic metamorphic basement launched a low-temperature flow of reductive iron-rich fluids. Well‑oxygenated bottom waters percolated through thin pelagic sediments and picked up ferrous iron from the pyritic metamorphic bedrock and made it available for the Fe-oxidising microbes. Reactivation of a major fault zone (Mecsekalja Zone) resulted in the development of extensional faults in the metamorphic basement beneath the seafloor. Pelagic carbonate sediments with FeOB mats were drawn into these fissures, forming a network of injection dykes. This study shows that fractal dimension analysis and directional analysis may be a helpful method to differentiate FeOB textures from inorganic textures in ancient rocks. The presented results are expected to provide useful analogues to aid the reconstruction of similar palaeoenvironments worldwide.

Legionella pneumophila macrophage infectivity potentiator protein appendage domains modulate protein dynamics and inhibitor binding

Macrophage infectivity potentiator (MIP) proteins are widespread in human pathogens including Legionella pneumophila, the causative agent of Legionnaires' disease and protozoans such as Trypanosoma cruzi. All MIP proteins contain a FKBP (FK506 binding protein)-like prolyl-cis/trans-isomerase domain that hence presents an attractive drug target. Some MIPs such as the Legionella pneumophila protein (LpMIP) have additional appendage domains of mostly unknown function. In full-length, homodimeric LpMIP, the N-terminal dimerization domain is linked to the FKBP-like domain via a long, free-standing stalk helix. Combining X-ray crystallography, NMR and EPR spectroscopy and SAXS, we elucidated the importance of the stalk helix for protein dynamics and inhibitor binding to the FKBP-like domain and bidirectional crosstalk between the different protein regions. The first comparison of a microbial MIP and a human FKBP in complex with the same synthetic inhibitor was made possible by high-resolution structures of LpMIP with a [4.3.1]-aza-bicyclic sulfonamide and provides a basis for designing pathogen-selective inhibitors. Through stereospecific methylation, the affinity of inhibitors to L. pneumophila and T. cruzi MIP was greatly improved. The resulting X-ray inhibitor-complex structures of LpMIP and TcMIP at 1.49 and 1.34 Å, respectively, provide a starting point for developing potent inhibitors against MIPs from multiple pathogenic microorganisms.

Structure and supramolecular organization of the canine distemper virus attachment glycoprotein

Canine distemper virus (CDV) is an enveloped RNA morbillivirus that triggers respiratory, enteric, and high incidence of severe neurological disorders. CDV induces devastating outbreaks in wild and endangered animals as well as in domestic dogs in countries associated with suboptimal vaccination programs. The receptor-binding tetrameric attachment (H)-protein is part of the morbilliviral cell entry machinery. Here, we present the cryo-electron microscopy (cryo-EM) structure and supramolecular organization of the tetrameric CDV H-protein ectodomain. The structure reveals that the morbilliviral H-protein is composed of three main domains: stalk, neck, and heads. The most unexpected feature was the inherent asymmetric architecture of the CDV H-tetramer being shaped by the neck, which folds into an almost 90° bent conformation with respect to the stalk. Consequently, two non-contacting receptor-binding H-head dimers, which are also tilted toward each other, are located on one side of an intertwined four helical bundle stalk domain. Positioning of the four protomer polypeptide chains within the neck domain is guided by a glycine residue (G158), which forms a hinge point exclusively in two protomer polypeptide chains. Molecular dynamics simulations validated the stability of the asymmetric structure under near physiological conditions and molecular docking showed that two receptor-binding sites are fully accessible. Thus, this spatial organization of the CDV H-tetramer would allow for concomitant protein interactions with the stalk and head domains without steric clashes. In summary, the structure of the CDV H-protein ectodomain provides new insights into the morbilliviral cell entry system and offers a blueprint for next-generation structure-based antiviral drug discovery.

The zinc-finger protein Red1 orchestrates MTREC submodules and binds the Mtl1 helicase arch domain

Cryptic unstable transcripts (CUTs) are rapidly degraded by the nuclear exosome in a process requiring the RNA helicase Mtr4 and specific adaptor complexes for RNA substrate recognition. The PAXT and MTREC complexes have recently been identified as homologous exosome adaptors in human and fission yeast, respectively. The eleven-subunit MTREC comprises the zinc-finger protein Red1 and the Mtr4 homologue Mtl1. Here, we use yeast two-hybrid and pull-down assays to derive a detailed interaction map. We show that Red1 bridges MTREC submodules and serves as the central scaffold. In the crystal structure of a minimal Mtl1/Red1 complex an unstructured region adjacent to the Red1 zinc-finger domain binds to both the Mtl1 KOW domain and stalk helices. This interaction extends the canonical interface seen in Mtr4-adaptor complexes. In vivo mutational analysis shows that this interface is essential for cell survival. Our results add to Mtr4 versatility and provide mechanistic insights into the MTREC complex.

Molecular cloning, GTP recognition mechanism and tissue-specific expression profiling of myxovirus resistance (Mx) protein in Labeo rohita (Hamilton) after Poly I:C induction

The myxovirus resistance (Mx) proteins belong to interferon-induced dynamin GTPase and play pivotal role in the inhibition of replication of numerous viruses. These antiviral proteins are released in usual or diseased condition to prevent the viral attack and to carry regular cellular activities like endocytosis and trafficking of nucleoproteins into the nucleus. The invasion of virus up-regulates the expression of Mx transcripts and double-stranded RNA mimic like polyinosinic polycytidyilic acid (Poly I:C). To understand the tissue-specific expression profiling and mechanism of GTP recognition of Mx protein from Labeo rohita (rohu), the full-length gene was cloned, sequenced and characterized through various Bioinformatics tools for the first time. The Mx cDNA was comprised of 2297 bp, and the open reading frame of 1938 bp encodes polypeptide of 631 amino acids. The coding sequence of Mx protein possess the signature motif of dynamin superfamily, LPRG(S/K)GIVTR, the tripartite guanosine-5/triphosphate (GTP)-binding motif (GXXXSGKS/T, DXXG and T/NKXD) and the leucine zipper motifs at the C-terminal end, well conserved in all interferon-induced Mx protein in vertebrates. Western blotting confirmed the molecular weight of Mx protein to be 72 kDa. After the intraperitoneal challenge of L. rohita with a Poly I:C, up-regulation of Mx protein was observed in brain, spleen, liver, kidney, intestine, heart, muscle, and gill. Ontogeny study displayed pronounced expression of Mx protein in all stages of the developmental of Rohu after Poly I:C induction. However a persistent expression of Mx transcript was also observed in Rohu egg as well as milt without induction with Poly I:C. Higher expression of Mx gene was observed on 96 h where it was 6.4 folds higher than the control. The computational modelling of Mx protein portrayed the tripartite N-terminal G-domain that binds to GTP, the bundle-signaling element (BSE) which interconnects the G-domain to the elongated stalk domain and C-terminal helical stalk domain. In agreement with the experimental studies, a series of conserved residues viz., Gln52, Ser53, Ser54, Leu68, Pro69, Gly71, Gly73, Thr76, Asp151, Gly154, Thr220, Lys221, Val251, Cys253, Arg254, and Gly255 were computed to be indispensable for tight anchoring of GTP within binding cavity of G-domain. The binding free energy calculation study depicted that the van der Waals and electrostatic terms contributs significantly to molecular recognition of GTP. Collectively, our study provides mechanistic insights into the tissue-specific expression profiling and GTP binding mechanism of Mx protein from Labeo rohita, which is expected to drive further research on several cellular events including viral resistance and endocytosis in the near future.

Primary resistance mechanism of the canine distemper virus fusion protein against a small-molecule membrane fusion inhibitor

Morbilliviruses (e.g. measles virus [MeV] or canine distemper virus [CDV]) employ the attachment (H) and fusion (F) envelope glycoproteins for cell entry. H protein engagement to a cognate receptor eventually leads to F-triggering. Upon activation, F proteins transit from a prefusion to a postfusion conformation; a refolding process that is associated with membrane merging. Small-molecule morbilliviral fusion inhibitors such as the compound 3G (a chemical analog in the AS-48 class) were previously generated and mechanistic studies revealed a stabilizing effect on morbilliviral prefusion F trimers. Here, we aimed at designing 3G-resistant CDV F mutants by introducing single cysteine residues at hydrophobic core positions of the helical stalk region. Covalently-linked F dimers were generated, which highlighted substantial conformational flexibility within the stalk to achieve those irregular F conformations. Our findings demonstrate that “top-stalk” CDV F cysteine mutants (F-V571C and F-L575C) remained functional and gained resistance to 3G. Conversely, although not all “bottom-stalk” F cysteine variants preserved proper bioactivity, those that remained functional exhibited 3G-sensitivity. According to the recently determined prefusion MeV F trimer/AS-48 co-crystal structure, CDV residues F-V571 and F-L575 may directly interact with 3G. A combination of conformation-specific anti-F antibodies and low-resolution electron microscopy structural analyses confirmed that 3G lost its stabilizing effect on “top-stalk” F cysteine mutants thus suggesting a primary resistance mechanism. Overall, our data suggest that the fusion inhibitor 3G stabilizes prefusion CDV F trimers by docking at the top of the stalk domain.

Dependence of In Situ Bacterial Fe(II)-Oxidation and Fe(III)-Precipitation on Sequential Reactive Transport

ABSTRACTA study was conducted to determine in situ rates of Fe(II) oxidation and Fe(III) precipitation along a 5.0 m reach of a ferruginous groundwater discharge zone under two distinct conditions; (i) the natural state featuring abundant flocculent mats of bacteriogenic iron oxides (BIOS) produced by Fe(II)-oxidizing bacteria, and (ii) after a manual washout of the streambed to remove the microbial mat. Examination of mat samples by differential interference contrast light microscopy revealed tangled meshworks of filamentous Leptothrix sheaths and helical Gallionella stalks intermixed with fine-grained hydrous ferric oxide (HFO) precipitates. The greatest accumulation of BIOS mat was 1.0 m downstream of the groundwater spring. Redox potential (Eh) increased sharply from 200 mV to over 300 mV over the last 2.0 m of the reach. Similarly, dissolved oxygen increased from < 10% saturation to almost 100% saturation over the last 2.0 m of the reach, whereas pH increased from 6.4 to 7.3. Pseudo-first-order rate constants determined on the basis of analytical solutions to sequential partial differential advection-dispersion-reaction equations for the linear Fe(II)→Fe(III)→HFO reaction network yielded in situ Fe(II) oxidation rate constants (kox) of 1.70 ± 0.20 min−1 in natural conditions and 0.48 ± 0.14 min−1 after washout. Corresponding Fe(III)-precipitation rates (kp) before and after washout were 3.45 ± 0.10 min−1 and 0.90 ± 0.01 min−1, respectively. These values for kox and kp are higher than estimates obtained from closed batch microcosm and laboratory experiments, underscoring the crucial dependence of in situ Fe(II) oxidation and Fe(III) precipitation rates on advective and dispersive mass transport. The results also highlight the influence that BIOS microbial mats exert on the reaction kinetics of the multiple heterogeneous reactions contributing not only to Fe(II)/Fe(III) redox transformations in groundwater discharge zones, but also the precipitation of HFO.

Dynein's Direction-Dependent Microtubule-Binding Strength is Controlled via a Tension-Induced Sliding of Dynein's Stalk Helices Mediated by the Coiled-Coil Strut

Cytoskeletal motor protein motility requires coordination of ATPase and filament-binding cycles. Mechanical tension strongly influences these processes, and likely regulates motor stepping as external forces resist motor movement and intramolecular tension develops between motor domains. In cytoplasmic dynein, an AAA+ ATPase, applied tension affects microtubule (MT)-binding strength anisotropically -backward tension induces stronger binding- while in the absence of tension, reconfiguration of the coiled-coil ‘stalk’ (which connects the AAA+ and MT-binding domains) is known to alter MT affinity. Using optical tweezers, mutagenesis, and chemical cross-linking, we show that preventing relative motion of the stalk helices or deleting the ‘strut’ (which emerges from the AAA+ domain and contacts the stalk) both eliminate tension-based regulation of MT-binding strength. Thus, tension alters dynein's MT-binding strength by inducing sliding of the stalk helices, and the strut is a key mediator of this process.

Cytoplasmic dynein binding, run length, and velocity are guided by long-range electrostatic interactions.

Dyneins are important molecular motors involved in many essential biological processes, including cargo transport along microtubules, mitosis, and in cilia. Dynein motility involves the coupling of microtubule binding and unbinding to a change in the configuration of the linker domain induced by ATP hydrolysis, which occur some 25 nm apart. This leaves the accuracy of dynein stepping relatively inaccurate and susceptible to thermal noise. Using multi-scale modeling with a computational focusing technique, we demonstrate that the microtubule forms an electrostatic funnel that guides the dynein’s microtubule binding domain (MTBD) as it finally docks to the precise, keyed binding location on the microtubule. Furthermore, we demonstrate that electrostatic component of the MTBD’s binding free energy is linearly correlated with the velocity and run length of dynein, and we use this linearity to predict the effect of mutating each glutamic and aspartic acid located in MTBD domain to alanine. Lastly, we show that the binding of dynein to the microtubule is associated with conformational changes involving several helices, and we localize flexible hinge points within the stalk helices. Taken all together, we demonstrate that long range electrostatic interactions bring a level of precision to an otherwise noisy dynein stepping process.

Single-Particle Cryo-EM Studies of a 200-kDa Magnesium Ion Channel Reveal Large Structural Changes Upon Gating

Single-particle cryo-EM was used to explore the structure and conformational landscape of ∼200-kDa magnesium ion channel CorA. CorA is the major Mg2+-uptake system in prokaryotes and can functionally complement the eukaryotic mitochondrial Mrs2 channel, which is essential for normal mitochondrial activity, and whose abnormal expression has been associated with diseases ranging from cancer to demyelination of neuronal tissue. CorA is a homo-pentamer and is gated by intracellular Mg2+. X-ray crystallographic studies of CorA show similar, rather symmetric conformations under Mg2+-bound and Mg2+-free conditions, but EPR spectroscopic studies reveal large Mg2+-driven quaternary conformational changes. Here, we determined the cryo-EM structures of CorA in the presence and absence of Mg2+. The five-fold symmetric Mg2+-bound “closed” state of detergent-solubilized CorA at an overall 3.8-A resolution reveals side-chain densities as well as densities for several magnesium ions, and is similar to known X-ray structures with only minor differences in some of the transmembrane segments, the C-terminal domain, and the periplasmic loops. Cryo-EM of Mg2+-free “open” state CorA shows dramatic differences in the conformation of the channel with large asymmetric changes in the soluble domain of the pentamer. In the absence of bound Mg2+, four of the five subunits are displaced ∼10 to ∼25 A by hinge-like motions at the stalk helix with a bending of up to 35°. Extensive 3D-classification during data-processing resulted in at least two distinct “open” state CorA structures at ∼7-A. The asymmetric structures were supported by single-particle cryo-EM of CorA in lipid nanodiscs and other biochemical and biophysical studies. Our findings suggest an unprecedented gating mechanism where the reversible loss of five-fold symmetry is the key structural signature of the transition between a single “closed” state and an ensemble of low-Mg2+, “open” conformational states.

Cryo-EM Structures of the Magnesium Channel CorA Reveal Symmetry Break upon Gating

CorA, the major Mg(2+) uptake system in prokaryotes, is gated by intracellular Mg(2+) (KD ∼ 1-2 mM). X-ray crystallographic studies of CorA show similar conformations under Mg(2+)-bound and Mg(2+)-free conditions, but EPR spectroscopic studies reveal large Mg(2+)-driven quaternary conformational changes. Here, we determined cryo-EM structures of CorA in the Mg(2+)-bound closed conformation and in two open Mg(2+)-free states at resolutions of 3.8, 7.1, and 7.1 Å, respectively. In the absence of bound Mg(2+), four of the five subunits are displaced to variable extents (∼ 10-25 Å) by hinge-like motions as large as ∼ 35° at the stalk helix. The transition between a single 5-fold symmetric closed state and an ensemble of low Mg(2+), open, asymmetric conformational states is, thus, the key structural signature of CorA gating. This mechanism is likely to apply to other structurally similar divalent ion channels.

Ecophysiology of Zetaproteobacteria Associated with Shallow Hydrothermal Iron-Oxyhydroxide Deposits in Nagahama Bay of Satsuma Iwo-Jima, Japan.

Previous studies of microbial communities in deep-sea hydrothermal ferric deposits have demonstrated that members of Zetaproteobacteria play significant ecological roles in biogeochemical iron-cycling. However, the ecophysiological characteristics and interaction between other microbial members in the habitat still remain largely unknown. In this study, we investigated microbial communities in a core sample obtained from shallow hydrothermal iron-oxyhydroxide deposits at Nagahama Bay of Satsuma Iwo-Jima, Japan. Scanning electron microscopic observation showed numerous helical stalk structures, suggesting the occurrence of iron-oxidizing bacteria. Analysis of 16S rRNA gene sequences indicated the co-occurrence of iron-oxidizing Zetaproteobacteria and iron-reducing bacteria such as the genera Deferrisoma and Desulfobulbus with strong correlations on the sequence abundance. CARD-FISH indicated that the numbers of Zetaproteobacteria were not always consistent to the frequency of stalk structures. In the stalk-abundant layers with relatively small numbers of Zetaproteobacteria cells, accumulation of polyphosphate was observed inside Zetaproteobacteria cells, whereas no polyphosphate grains were observed in the topmost layers with fewer stalks and abundant Zetaproteobacteria cells. These results suggest that Zetaproteobacteria store intracellular polyphosphates during active iron oxidation that contributes to the mineralogical growth and biogeochemical iron cycling.

Fragmentation of Bacteriogenic Iron Oxides in Response to Hydrodynamic Shear Stress

A viscous shear stress of 1.97 Nm2 was applied to suspensions of freshwater, brackish and marine bacteriogenic iron oxides (BIOS) in shake flasks. Samples were taken at intervals over 480 minutes for scanning electron microscopy. The remains of three Fe2+-oxidizing bacteria (FeOB) genera were conspicuous biomass constituents in the BIOS samples; these included the filamentous extracellular sheaths of Leptothrix, as well as helical fibrous stalks of Gallionella and Mariprofundus. The applied hydrodynamic shear stress resulted in an exponential decrease in Leptothrix sheath and Gallionella-Mariprofundus stalk lengths; first-order breakage rate constants derived from the experimental measurements ranged from 0.009 to 0.023 min−1 and 0.014 to 0.021 min−1, respectively. Breakage half-times extended from 30 to 70 min. Shear strength values calculated from the time course data ranged from 27.8 to 29.6 Nm−2 for Leptothrix sheaths, and from 28.0 to 28.8 Nm−2 for Gallionella-Mariprofundus stalks. Computational analyses showed that breakage half-times decreased rapidly with increasing shear stress, implying that the accumulation of BIOS is constrained hydrodynamically to quiescent aqueous environments. These results imply that there is considerable potential for natural hydrodynamic fluctuations in basal shear stress levels to bring about resuspension and advective dispersal of BIOS. Immediate consequences anticipated from such events include enhanced particulate transport of BIOS-associated chemical species through aquatic systems, as well as reduced preservation potential and underrepresentation of BIOS bacteria structures as microfossils in the geological record.

Measles virus glycoprotein complexes preassemble intracellularly and relax during transport to the cell surface in preparation for fusion.

Measles virus (MeV), a morbillivirus within the paramyxovirus family, expresses two envelope glycoproteins. The attachment (H) protein mediates receptor binding, followed by triggering of the fusion (F) protein, which leads to merger of the viral envelope with target cell membranes. Receptor binding by members of related paramyxovirus genera rearranges the head domains of the attachment proteins, liberating an F-contact domain within the attachment protein helical stalk. However, morbillivirus glycoproteins first assemble intracellularly prior to receptor binding, raising the question of whether alternative protein-protein interfaces are involved or whether an entirely distinct triggering principle is employed. To test these possibilities, we generated headless H stem mutants of progressively shorter length. Conformationally restricted H stems remained capable of intracellular assembly with a standard F protein and a soluble MeV F mutant. Proteolytic maturation of F, but not the altered biochemical conditions at the cell surface, reduces the strength of glycoprotein interaction, readying the complexes for triggering. F mutants stabilized in the prefusion conformation interact with H intracellularly and at the cell surface, while destabilized F mutants interact only intracellularly, prior to F maturation. These results showcase an MeV entry machinery that functionally varies conserved motifs of the proposed paramyxovirus infection pathway. Intracellular and plasma membrane-resident MeV glycoprotein complexes employ the same protein-protein interface. F maturation prepares for complex separation after triggering, and the H head domains in prereceptor-bound conformation prevent premature stalk rearrangements and F activation. Intracellular preassembly affects MeV fusion profiles and may contribute to the high cell-to-cell fusion activity characteristic of the morbillivirus genus. Paramyxoviruses of the morbillivirus genus, such as measles, are highly contagious, major human and animal pathogens. MeV envelope glycoproteins preassemble intracellularly into tightly associated hetero-oligomers. To address whether preassembly reflects a unique measles virus entry strategy, we characterized the protein-protein interface of intracellular and surface-exposed fusion complexes and investigated the effect of the attachment protein head domains, glycoprotein maturation, and altered biochemical conditions at the cell surface on measles virus fusion complexes. Our results demonstrate that measles virus functionally varies conserved elements of the paramyxovirus entry pathway, providing a possible explanation for the high cell-to-cell fusion activity of morbilliviruses. Insight gained from these data affects the design of effective broad-spectrum paramyxovirus entry inhibitors.

An intramolecular lock facilitates folding and stabilizes the tertiary structure of Streptococcus mutans adhesin P1.

The cariogenic bacterium Streptococcus mutans uses adhesin P1 to adhere to tooth surfaces, extracellular matrix components, and other bacteria. A composite model of P1 based on partial crystal structures revealed an unusual complex architecture in which the protein forms an elongated hybrid alpha/polyproline type II helical stalk by folding back on itself to display a globular head at the apex and a globular C-terminal region at the base. The structure of P1's N terminus and the nature of its critical interaction with the C-terminal region remained unknown, however. We have cocrystallized a stable complex of recombinant N- and C-terminal fragments and here describe a previously unidentified topological fold in which these widely discontinuous domains are intimately associated. The structure reveals that the N terminus forms a stabilizing scaffold by wrapping behind the base of P1's elongated stalk and physically "locking" it into place. The structure is stabilized through a highly favorable ΔG(solvation) on complex formation, along with extensive hydrogen bonding. We confirm the functional relevance of this intramolecular interaction using differential scanning calorimetry and circular dichroism to show that disruption of the proper spacing of residues 989-1001 impedes folding and diminishes stability of the full-length molecule, including the stalk. Our findings clarify previously unexplained functional and antigenic properties of P1.

Probing the functions of the paramyxovirus glycoproteins F and HN with a panel of synthetic antibodies.

Paramyxoviruses are enveloped negative-strand RNA viruses that are significant human and animal pathogens. Most paramyxoviruses infect host cells via the concerted action of a tetrameric attachment protein (variously called HN, H, or G) that binds either sialic acid or protein receptors on target cells and a trimeric fusion protein (F) that merges the viral envelope with the plasma membrane at neutral pH. F initially folds to a metastable prefusion conformation that becomes activated via a cleavage event during cellular trafficking. Upon receptor binding, the attachment protein, which consists of a globular head anchored to the membrane via a helical tetrameric stalk, triggers a major conformation change in F which results in fusion of virus and host cell membranes. We recently proposed a model for F activation in which the attachment protein head domains move following receptor binding to expose HN stalk residues critical for triggering F. To test the model in the context of wild-type viral glycoproteins, we used a restricted-diversity combinatorial Fab library and phage display to rapidly generate synthetic antibodies (sAbs) against multiple domains of the paramyxovirus parainfluenza 5 (PIV5) pre- and postfusion F and HN. As predicted by the model, sAbs that bind to the critical F-triggering region of the HN stalk do not disrupt receptor binding or neuraminidase (NA) activity but are potent inhibitors of fusion. An inhibitory prefusion F-specific sAb recognized a quaternary antigenic site and may inhibit fusion by preventing F refolding or by blocking the F-HN interaction. Importance: The paramyxovirus family of negative-strand RNA viruses cause significant disease in humans and animals. The viruses bind to cells via their receptor binding protein and then enter cells by fusion of their envelope with the host cell plasma membrane, a process mediated by a metastable viral fusion (F) protein. To understand the steps in viral membrane fusion, a library of synthetic antibodies to F protein and the receptor binding protein was generated in bacteriophage. These antibodies bound to different regions of the F protein and the receptor binding protein, and the location of antibody binding affected different processes in viral entry into cells.

Molecular mechanism of Mg2+-dependent gating in CorA

CorA is the major transport system responsible for Mg2+ uptake in bacteria and can functionally substitute for its homologue Mrs2p in the yeast inner mitochondrial membrane. Although several CorA crystal structures are available, the molecular mechanism of Mg2+ uptake remains to be established. Here we use EPR spectroscopy, electrophysiology and molecular dynamic simulations to show that CorA is regulated by cytoplasmic Mg2+ acting as a ligand and elucidate the basic conformational rearrangements responsible for Mg2+-dependent gating. Mg2+ unbinding at the divalent cation sensor triggers a conformational change that leads to the inward motion of the stalk helix, which propagates to the pore forming transmembrane helix TM1. Helical tilting and rotation in TM1 generates an iris-like motion that increases the diameter of the permeation pathway, triggering ion conduction. This work establishes the molecular basis of a Mg2+-driven negative feedback loop in CorA as the key physiological event controlling Mg2+ uptake and homeostasis in prokaryotes.

Iron cycling at corroding carbon steel surfaces

Surfaces of carbon steel (CS) exposed to mixed cultures of iron-oxidizing bacteria (FeOB) and dissimilatory iron-reducing bacteria (FeRB) in seawater media under aerobic conditions were rougher than surfaces of CS exposed to pure cultures of either type of microorganism. The roughened surface, demonstrated by profilometry, is an indication of loss of metal from the surface. In the presence of CS, aerobically grown FeOB produced tight, twisted helical stalks encrusted with iron oxides. When CS was exposed anaerobically in the presence of FeRB, some surface oxides were removed. However, when the same FeOB and FeRB were grown together in an aerobic medium, FeOB stalks were less encrusted with iron oxides and appeared less tightly coiled. These observations suggest that iron oxides on the stalks were reduced and solubilized by the FeRB. Roughened surfaces of CS and denuded stalks were replicated with culture combinations of different species of FeOB and FeRB under three experimental conditions. Measurements of electrochemical polarization resistance established different rates of corrosion of CS in aerobic and anaerobic media, but could not differentiate rate differences between sterile controls and inoculated exposures for a given bulk concentration of dissolved oxygen. Similarly, total iron in the electrolyte could not be used to differentiate treatments. The experiments demonstrate the potential for iron cycling (oxidation and reduction) on corroding CS in aerobic seawater media.

186: Role of GTP-mediated structural changes of MxA on its antiviral function against influenza A

Mx proteins are interferon type I induced effector proteins and members of the family of dynamin-like GTPases. Human MxA protein exerts antiviral activity against a broad range of viruses including influenza A. The GTPase activity of MxA is essential for its proper antiviral function. Based on extensive mutational biochemical analyses we proposed a model where Mx proteins can assemble into highly ordered oligomers but exert their antiviral activity as monomers. Moreover, we have shown that human MxA physically interacts with the cellular DExD/h-box RNA helicases UAP56 and URH49 that are required for efficient replication of influenza A virus. Recently Gao and coworkers determined the crystal structure of MxA, revealing a globular GTP-binding domain and a four helical bundle stalk domain. In vitro, MxA multimerizes into large ring-like structures, involving three interaction interfaces. Mutations in the interfaces of MxA interfere with the oligomer formation, but, depending on the mutation, some MxA variants are still able to form monomers, dimers or tetramers. To date it is still a matter of debate whether MxA exerts antiviral activity in form of monomers, dimers and/or higher oligomeric structures. In order to assess whether GTP-binding has an effect on the stoichiometry of MxA we purified recombinant wildtype MxA and various mutants thereof and assessed the number of MxA protomers in the presence or absence GTP-γS by multi angle laser light scattering (MALLS). Surprisingly, we observed that in the presence of GTP-γS wildtype MxA converted from oligomers to tetramers and dimers. Similarly, addition of GTP-γS to the tetrameric mutant MxA (R640A) resulted in its dimerization. Furthermore, dimeric and monomeric MxA variants did not alter their stoichiometry upon addition of GTP-γS. Further, we evaluated the antiviral activity of wild type MxA and MxA variants employing an influenza A minireplicon system and influenza A virus infection of MxA expressing cells. The data clearly indicated that certain tetrameric, dimeric or monomeric variants of MxA were still able to efficiently inhibit virus replication while MxA mutants deficient for GTP binding were inactive. We currently test whether binding of UAP56 by MxA is required for its antiviral activity.

Structure of the parainfluenza virus 5 (PIV5) hemagglutinin-neuraminidase (HN) ectodomain.

Paramyxoviruses cause a wide variety of human and animal diseases. They infect host cells using the coordinated action of two surface glycoproteins, the receptor binding protein (HN, H, or G) and the fusion protein (F). HN binds sialic acid on host cells (hemagglutinin activity) and hydrolyzes these receptors during viral egress (neuraminidase activity, NA). Additionally, receptor binding is thought to induce a conformational change in HN that subsequently triggers major refolding in homotypic F, resulting in fusion of virus and target cell membranes. HN is an oligomeric type II transmembrane protein with a short cytoplasmic domain and a large ectodomain comprising a long helical stalk and large globular head domain containing the enzymatic functions (NA domain). Extensive biochemical characterization has revealed that HN-stalk residues determine F specificity and activation. However, the F/HN interaction and the mechanisms whereby receptor binding regulates F activation are poorly defined. Recently, a structure of Newcastle disease virus (NDV) HN ectodomain revealed the heads (NA domains) in a “4-heads-down” conformation whereby two of the heads form a symmetrical interaction with two sides of the stalk. The interface includes stalk residues implicated in triggering F, and the heads sterically shield these residues from interaction with F (at least on two sides). Here we report the x-ray crystal structure of parainfluenza virus 5 (PIV5) HN ectodomain in a “2-heads-up/2-heads-down” conformation where two heads (covalent dimers) are in the “down position,” forming a similar interface as observed in the NDV HN ectodomain structure, and two heads are in an “up position.” The structure supports a model in which the heads of HN transition from down to up upon receptor binding thereby releasing steric constraints and facilitating the interaction between critical HN-stalk residues and F.