In the replication cycle of nonsegmented negative-strand RNA viruses, the viral RNA-dependent RNA polymerase (L) recognizes a nucleoprotein (N)-enwrapped RNA template during the RNA polymerase reaction. The viral phosphoprotein (P) is a polymerase cofactor essential for this recognition. We report here the 2.3-Å-resolution crystal structure of the central domain (residues 107 to 177) of P from vesicular stomatitis virus. The fold of this domain consists of a ␤ hairpin, an ␣ helix, and another ␤ hairpin. The ␣ helix provides the stabilizing force for forming a homodimer, while the two ␤ hairpins add additional stabilization by forming a four-stranded ␤ sheet through domain swapping between two molecules. This central dimer positions the Nand C-terminal domains of P to interact with the N and L proteins, allowing the L protein to specifically recognize the nucleocapsid-RNA template and to progress along the template while concomitantly assembling N with nascent RNA. The interdimer interactions observed in the noncrystallographic packing may offer insight into the mechanism of the RNA polymerase processive reaction along the viral nucleocapsid-RNA template.Vesicular stomatitis virus (VSV) is an enveloped virus belonging to the family Rhabdoviridae in the order Mononegavirales. VSV contains a single, negative-sense RNA genome (2). As the prototype of nonsegmented negative-strand viruses, the 11,161-nucleotide-long VSV genome encodes five viral proteins: the glycoprotein (G), the matrix protein (M), the nucleoprotein (N), the RNA-dependent RNA polymerase large protein (L), and the phosphoprotein (P) (23). The G protein is anchored on the viral membrane surface and is responsible for virus entry into host cells (25). M is involved in VSV budding and assembly in the host cell (21). The nucleocapsid protein (N) enwraps the viral genome that is associated with L and P to form the ribonucleoprotein core complex (N-RNA) (3, 4).The N-RNA forms a supercoil structure beneath the viral envelope. Upon entering the host cell, the N-RNA is released and serves as the template for transcription and replication. The N-RNA template is recognized by the viral polymerase, L, and by the P protein that tethers L to the N-RNA template (13,14). During the early stage of the virus replication cycle, the L and P proteins, which are carried into the host by the virion, initiate transcription of mRNAs of each viral gene. The relative abundance is highest for the N protein, followed by the P protein, simply because they are located near the 3Ј end of the viral genome, while the L protein, which is nearest the 5Ј end of the viral genome, is produced in the least abundance (38). When sufficient amounts of the N and P proteins are produced, the polymerase, L, switches from transcribing mRNAs to replicating the viral genome. In genomic replication, a complementary positive-strand genome is first synthesized from the negative-strand genomic template and is then used as the template for producing more copies of the negative-strand genome (16). The ne...

Hainantoxin-IV (HNTX-IV) can specifically inhibit the neuronal tetrodotoxin-sensitive sodium channels and defines a new class of depressant spider toxin. The sequence of native HNTX-IV is ECLGFGKGCNPSNDQC-CKSSNLVCSRKHRWCKYEI-NH 2 . In the present study, to obtain further insight into the primary and tertiary structural requirements of neuronal sodium channel blockers, we determined the solution structure of HNTX-IV as a typical inhibitor cystine knot motif and synthesized four mutants designed based on the predicted sites followed by structural elucidation of two inactive mutants. Pharmacological studies indicated that the S12A and R26A mutants had activities near that of native HNTX-IV, while K27A and R29A demonstrated activities reduced by 2 orders of magnitude.1 H MR analysis showed the similar molecular conformations for native HNTX-IV and four synthetic mutants. Furthermore, in the determined structures of K27A and R29A, the side chains of residues 27 and 29 were located in the identical spatial position to those of native HNTX-IV.

I n common with other heat-labile enterotoxins from Vibrio cholerae or Escherichia coli, the type IIb enterotoxin of E. coli (LT-IIb3 ) displays AB 5 oligomeric structure, in which an enzymatically toxic A subunit is linked to a pentameric ganglioside-binding (B 5 ) subunit (1, 2). The actual catalytic moiety is the A1 polypeptide, whereas the C-terminal A2 segment acts as a noncovalent linker into the central pore of the doughnut-shaped B pentamer (1). Although LT-IIb and related heat-labile enterotoxins are potent mucosal adjuvants, their intrinsic enterotoxicity precludes their use as adjuvants for human vaccines (3, 4). In our efforts to identify immunostimulatory activities mediated exclusively by the noncatalytic B pentameric subunits, we discovered that TLR2 is uniquely activated by the B pentamers of type II but not type I enterotoxins such as the cholera toxin (5). Subsequent work focusing on the 60-kDa B pentamer of LT-IIb (LT-IIb-B 5 ) revealed that its interaction with and activation of TLR2 in membrane lipid rafts is facilitated by binding to the GD1a ganglioside (6). Strikingly, the LT-IIb holotoxin does not bind or activate TLR2; this is attributable to the presence of the A subunit which, in either wild-type or catalytically inactive version, appears to interfere sterically with TLR2 binding (7). We moreover found that TLR1 is a signaling partner of TLR2 in response to LT-IIb-B 5 (6, 7).The TLR family of pattern recognition receptors fulfill their central role as sensors of infection by recognizing multiple microbial ligands (8, 9); this is particularly true for TLR2 which, in cooperation with TLR1 or TLR6, interacts with a variety of structurally diverse molecules (10 -12). The triacylated lipopeptide Pam 3 CysSerLys 4 (Pam 3 CSK 4 ) is a prototypical TLR2 ligand which interacts with a hydrophobic pocket in the convex region of TLR2, at the border of its central and C-terminal domains (13). The TLR2 pocket accommodates two of the acyl chains of the lipopeptide, whereas the third acyl chain is inserted into a similar hydrophobic channel in the TLR1 component of the TLR2/1 heterodimer (13). In addition to microbial lipopeptides/lipoproteins, TLR2 interacts with several microbial proteins that do not bear acyl chains (14 -19) and are unlikely to be accommodated within the TLR2/1 hydrophobic pockets due to size constraints. The issue of how microbial protein ligands interact with TLR2 or other TLRs was raised in a recent review (20) and, in the absence of specific crystallographic data, has remained a matter of conjecture. The objective of this study was to define the molecular basis of the LT-IIb-B 5 interaction with the TLR2/1 heterodimer, thereby offering an The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by National Institutes of Health R01 Grants AI052344 and AI056148 (to R.I.T.), DE013833 (to T...

In this work, various TiO 2 hollow structures, such as pseudocubes, ellipsoids, capsules and peanuts, have been synthesized through the following process: hydrothermal deposition of anatase TiO 2 on monodisperse Fe 2 O 3 microscale particles with different shapes (pseudocubes, ellipsoids, capsules and peanuts) and the sequential acid-dissolution of the Fe 2 O 3 cores. The morphologies of these TiO 2 hollow structures are similar to their Fe 2 O 3 templates. When all the obtained hollow TiO 2 structures are used as the anode material for lithium-ion batteries, they show higher discharge capacity as compared to TiO 2 solid microspheres.

In this work, hollow silica colloids with different shapes, such as pseudocubes, ellipsoids, capsules, and peanuts, have been synthesized through the following process: silica coating on the surface of hematite colloidal particles with different shapes (pseudocubes, ellipsoids, capsules, and peanuts) and the sequential acid dissolution of the hematite cores. The as-obtained hollow silica colloids with different shapes have uniform sizes, shapes, and shells.

␣-Amylase inhibitor (AAI), a 32-residue miniprotein from the Mexican crop plant amaranth (Amaranthus hypochondriacus), is the smallest known ␣-amylase inhibitor and is specific for insect ␣-amylases (ChagollaLopez, A., Blanco-Labra, A., Patthy, A., Sanchez, R., and Pongor, S. (1994) J. Biol. Chem. 269, 23675-23680). Its disulfide topology was confirmed by Edman degradation, and its three-dimensional solution structure was determined by two-dimensional 1 H NMR spectroscopy at 500 MHz. Structural constraints (consisting of 348 nuclear Overhauser effect interproton distances, 8 backbone dihedral constraints, and 9 disulfide distance constraints) were used as an input to the X-PLOR program for simulated annealing and energy minimization calculations. The final set of 10 structures had a mean pairwise root mean square deviation of 0.32 Å for the backbone atoms and 1.04 Å for all heavy atoms. The structure of AAI consists of a short triple-stranded ␤-sheet stabilized by three disulfide bonds, forming a typical knottin or inhibitor cystine knot fold found in miniproteins, which binds various macromolecular ligands. When the first intercystine segment of AAI (sequence IPKWNR) was inserted into a homologous position of the spider toxin Huwentoxin I, the resulting chimera showed a significant inhibitory activity, suggesting that this segment takes part in enzyme binding. Plant seeds produce a large variety of enzyme inhibitors that are thought to provide protection against insects and microbial pathogens. As plant seed inhibitors are often species specific, i.e. they inhibit enzymes of a well defined group of pathogenic organisms but do not affect the mammalian counterpart, they make attractive candidates for conferring pest resistance to transgenic plants (for a review see Ref. 1).The ␣-amylase inhibitors vary considerably in their structures. Many of their structural relatives, e.g. proteinase inhibitors, osmotin, and salt-induced proteins (Table I), play roles in plant stress response. The smallest of the known ␣-amylase inhibitors, AAI, 1 is found in the seeds of Amaranthus hypochondriacus, a variety of the Mexican crop plant amaranth or Prince's feather (2). AAI is a 32-residue polypeptide with three disulfide bridges, which has no significant sequence similarity to other proteins in the data bases. It has a spurious sequence similarity to various members of the so-called knottin (3) or "inhibitor-type cystine knot" (4) family, which includes various proteinase inhibitors and toxins. AAI is species specific; it inhibits ␣-amylase of several pathogenic insect larvae (Tribolium castaneum, Prostaphanus truncatus, Periplaneta americana, and Tenebrio mollitor) but not the mammalian ␣-amylases.Here we report the three-dimensional structure of AAI as determined by NMR spectroscopy and show via amino acid replacement and chimera construction that a short segment of the first loop of AAI is involved in enzyme inhibition. EXPERIMENTAL PROCEDURES MaterialsAAI was prepared as described (5). Sephadex G-75 and DEAE-Sepharose CL6B ...

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PDB Reference: nicotinic acid mononucleotide adenylyltransferase, 3dv2, r3dv2sf.Nicotinic acid mononucleotide adenylyltransferase (NaMNAT; EC 2.7.7.18) is the penultimate enzyme in the biosynthesis of NAD + and catalyzes the adenylation of nicotinic acid mononucleotide (NaMN) by ATP to form nicotinic acid adenine dinucleotide (NaAD). This enzyme is regarded as a suitable candidate for antibacterial drug development; as such, Bacillus anthracis NaMNAT (BA NaMNAT) was heterologously expressed in Escherichia coli for the purpose of inhibitor discovery and crystallography. The crystal structure of BA NaMNAT was determined by molecular replacement, revealing two dimers per asymmetric unit, and was refined to an R factor and R free of 0.228 and 0.263, respectively, at 2.3 Å resolution. The structure is very similar to that of B. subtilis NaMNAT (BS NaMNAT), which is also a dimer, and another independently solved structure of BA NaMNATrecently released from the PDB along with two ligated forms. Comparison of these and other less related bacterial NaMNAT structures support the presence of considerable conformational heterogeneity and flexibility in three loops surrounding the substrate-binding area.