Crystal structure of human peptidoglycan recognition protein S (PGRP-S) at 1.70 A resolution

Peptidoglycan recognition proteins (PGRPs) are pattern recognition receptors of the innate immune system that bind peptidoglycans (PGNs) of bacterial cell walls. These molecules, which are highly conserved from insects to mammals, contribute to host defense against infections by both Gram-positive and Gram-negative bacteria. Here, we present the crystal structure of human PGRP-S at 1.70A resolution. The overall structure of PGRP-S, which participates in intracellular killing of Gram-positive bacteria, is similar to that of other PGRPs, including Drosophila PGRP-LB and PGRP-SA and human PGRP-Ialpha. However, comparison with these PGRPs reveals important differences in both the PGN-binding site and a groove formed by the PGRP-specific segment on the opposite face of the molecule. This groove, which may constitute a binding site for effector or signaling proteins, is less hydrophobic and deeper in PGRP-S than in PGRP-IalphaC, whose PGRP-specific segments vary considerably in amino acid sequence. By docking a PGN ligand into the PGN-binding cleft of PGRP-S based on the known structure of a PGRP-Ialpha-PGN complex, we identified potential PGN-binding residues in PGRP-S. Differences in PGN-contacting residues and interactions suggest that, although PGRPs may engage PGNs in a similar mode, structural differences exist that likely regulate the affinity and fine specificity of PGN recognition.     

Crystal structure of a glutamate/aspartate binding protein complexed with a glutamate molecule: structural basis of ligand specificity at atomic resolution

The crystal structure of a periplasmic l-aspartate/l-glutamate binding protein (DEBP) from Shigella flexneri complexed with an l-glutamate molecule has been determined and refined to an atomic resolution of 1.0 Å. There are two DEBP molecules in the asymmetric unit. The refined model contains 4462 non-hydrogen protein atoms, 730 water molecules, 2 bound glutamate molecules, and 2 Tris molecules from the buffer used in crystallization. The final R_cryst and R_free factors are 13.61% and 16.89%, respectively. The structure has root-mean-square deviations of 0.016 Å from standard bond lengths and 2.35° from standard bond angles.The DEBP molecule is composed of two similarly folded domains separated by the ligand binding region. Both domains contain a central five-stranded β-sheet that is surrounded by several α-helices. The two domains are linked by two antiparallel β-strands. The overall shape of DEBP is that of an ellipsoid approximately 55 Å × 45 Å × 40 Å in size.The binding of ligand to DEBP is achieved mostly through hydrogen bonds between the glutamate and side-chain and main-chain groups of DEBP. Side chains of residues Arg24, Ser72, Arg75, Ser90, and His164 anchor the deprotonated γ-carboxylate group of the glutamate with six hydrogen bonds. Side chains of Arg75 and Arg90 form salt bridges with the deprotonated α-carboxylate group, while the main-chain amide groups of Thr92 and Thr140 form hydrogen bonds with the same group. The positively charged α-amino group of the l-glutamate forms salt bridge interaction with the side-chain carboxylate group of Asp182 and hydrogen bond interaction with main-chain carbonyl oxygen of Ser90. In addition to these hydrogen bond and electrostatic interactions, other interactions may also play important roles. For example, the two methylene groups from the glutamate form van der Waals interactions with hydrophobic side chains of DEBP.Comparisons with several other periplasmic amino acid binding proteins indicate that DEBP residues involved in the binding of α-amino and α-carboxylate groups of the ligand and the pattern of hydrogen bond formation between these groups are very well conserved, but the binding pocket around the ligand side chain is not, leading to the specificity of DEBP. We have identified structural features of DEBP that determine its ability of binding glutamate and aspartate, two molecules with different sizes, but discriminating against very similar glutamine and asparagine molecules.     

Towards structural elucidation of eukaryotic photosystem II: Purification, crystallization and preliminary X-ray diffraction analysis of photosystem II from a red alga

Crystal structure of photosystem II (PSII) has been reported from prokaryotic cyanobacteria but not from any eukaryotes. In the present study, we improved the purification procedure of PSII dimers from an acidophilic, thermophilic red alga Cyanidium caldarium, and crystallized them in two forms under different crystallization conditions. One had a space group of P222₁ with unit cell constants of a = 146.8 Å, b = 176.9 Å, and c = 353.7 Å, and the other one had a space group of P2₁2₁2₁ with unit cell constants of a = 209.2 Å, b = 237.5 Å, and c = 299.8 Å. The unit cell constants of both crystals and the space group of the first-type crystals are different from those of cyanobacterial crystals, which may reflect the structural differences between the red algal and cyanobacterial PSII, as the former contains a fourth extrinsic protein of 20 kDa. X-ray diffraction data were collected and processed to a 3.8 Å resolution with the first type crystal. For the second type crystal, a post-crystallization treatment of dehydration was employed to improve the resolution, resulting in a diffraction data of 3.5 Å resolution. Analysis of this type of crystal revealed that there are 2 PSII dimers in each asymmetric unit, giving rise to 16 PSII monomers in each unit cell, which contrasts to 4 dimers per unit cell in cyanobacterial crystals. The molecular packing of PSII within the unit cell was constructed with the molecular replacement method and compared with that of the cyanobacterial crystals.     

Divalent metal ion complexes of S100B in the absence and presence of pentamidine

As part of an effort to inhibit S100B, structures of pentamidine (Pnt) bound to Ca²⁺-loaded and Zn²⁺,Ca²⁺-loaded S100B were determined by X-ray crystallography at 2.15 Å (R_free = 0.266) and 1.85 Å (R_free = 0.243) resolution, respectively. These data were compared to X-ray structures solved in the absence of Pnt, including Ca²⁺-loaded S100B and Zn²⁺,Ca²⁺-loaded S100B determined here (1.88 Å; R_free = 0.267). In the presence and absence of Zn²⁺, electron density corresponding to two Pnt molecules per S100B subunit was mapped for both drug-bound structures. One Pnt binding site (site 1) was adjacent to a p53 peptide binding site on S100B (± Zn²⁺), and the second Pnt molecule was mapped to the dimer interface (site 2; ± Zn²⁺) and in a pocket near residues that define the Zn²⁺ binding site on S100B. In addition, a conformational change in S100B was observed upon the addition of Zn²⁺ to Ca²⁺-S100B, which changed the conformation and orientation of Pnt bound to sites 1 and 2 of Pnt-Zn²⁺,Ca²⁺-S100B when compared to Pnt-Ca²⁺-S100B. That Pnt can adapt to this Zn²⁺-dependent conformational change was unexpected and provides a new mode for S100B inhibition by this drug. These data will be useful for developing novel inhibitors of both Ca²⁺- and Ca²⁺,Zn²⁺-bound S100B.     

Unsupported intermolecular argentophilic interaction in the dimer of trinuclear silver(I) 3,5-diphenylpyrazolates

The reaction of a solution of sodium 3,5-diphenylpyrazolate, Na[Ph₂pz], with Ag(tht)NO₃ in dichloromethane affords thin needles of unsolvated and light-stable dimer of trimers [Ag₃(μ-3,5-Ph₂pz)₃]₂. The complex is characterized by X-ray crystallography and elemental analysis. The two trimers are rotated anti to each other. Three silver atoms bridged through exobidentate pyrazolate groups form a slightly puckered nine-membered ring with the shortest Ag?Ag intramolecular interaction in the metallocycle of 3.3571(8) Å. The other two silver centers are weakly interacting, Ag(3)?Ag(1) = 3.49 Å and Ag(3)?Ag(2) = 3.52 Å. The intermolecular interaction between the two trimers is Ag?Ag = 2.9712(14) Å. Packing diagram shows the dimer of trimer units are independent. Density Functional Theory calculations show that the M?M interaction is due to dispersion forces. [Ag₃(μ-3,5-Ph₂pz)₃]₂ crystallizes in the monoclinic space group C2/c with a = 22.169(4), b = 15.269(3), c = 22.482(5) Å, β = 103.69(3)° and V = 7394(3) ų.     

Structures of Glycinamide Ribonucleotide Transformylase (PurN) from Mycobacterium tuberculosis Reveal a Novel Dimer with Relevance to Drug Discovery

Enzymes from the de novo purine biosynthetic pathway have been exploited for the development of anti-cancer drugs, and represent novel targets for anti-bacterial drug development. In Mycobacterium tuberculosis, the cause of tuberculosis, this pathway has been identified as essential for growth and survival. The structure of M. tuberculosis PurN (MtPurN) has been determined in complex with magnesium and iodide at 1.30 Å resolution, and with cofactor analogue, 5-methyltetrahydrofolate (5MTHF) at 2.2 Å resolution. The structure shows a Rossmann-type fold that is very similar to the known structures of the human and E. coli PurN proteins. In contrast, MtPurN forms a dimer that is quite different from that formed by the Escherichia coli PurN, and which suggests a mechanism whereby communication could take place between the two active sites. Differences are seen in two active site loops and in the binding mode of the 5MTHF cofactor analogue between the two MtPurN molecules of the dimer. A binding site for halide ions is found in the dimer interface, and bound magnesium and iodide ions in the active site suggest sites that might be exploited in potential drug discovery strategies.     

Crystal Structure and Putative Mechanism of 3-Methylitaconate-Δ-isomerase from Eubacterium barkeri

3-Methylitaconate-Δ-isomerase (Mii) participates in the nicotinate fermentation pathway of the anaerobic soil bacterium Eubacterium barkeri (order Clostridiales) by catalyzing the reversible conversion of (R)-3-methylitaconate (2-methylene-3-methylsuccinate) to 2,3-dimethylmaleate. The enzyme is also able to catalyze the isomerization of itaconate (methylenesuccinate) to citraconate (methylmaleate) with ca 10-fold higher K_m but > 1000-fold lower k_cat. The gene mii from E. barkeri was cloned and expressed in Escherichia coli. The protein produced with a C-terminal Strep-tag exhibited the same specific activity as the wild-type enzyme. The crystal structure of Mii from E. barkeri has been solved at a resolution of 2.70 Å. The asymmetric unit of the P2₁2₁2₁ unit cell with parameters a = 53.1 Å, b = 142.3 Å, and c = 228.4 Å contains four molecules of Mii. The enzyme belongs to a group of isomerases with a common structural feature, the so-called diaminopimelate epimerase fold. The monomer of 380 amino acid residues has two topologically similar domains exhibiting an α/β-fold. The active site is situated in a cleft between these domains. The four Mii molecules are arranged as a tetramer with 222 symmetry for the N-terminal domains. The C-terminal domains have different relative positions with respect to the N-terminal domains resulting in a closed conformation for molecule A and two distinct open conformations for molecules B and D. The C-terminal domain of molecule C is disordered. The Mii active site contains the putative catalytic residues Lys62 and Cys96, for which mechanistic roles are proposed based on a docking experiment of the Mii substrate complex. The active sites of Mii and the closely related PrpF, most likely a methylaconitate Δ-isomerase, have been compared. The overall architecture including the active-site Lys62, Cys96, His300, and Ser17 (Mii numbering) is similar. This positioning of (R)-3-methylitaconate allows Cys96 (as thiolate) to deprotonate C-3 and (as thiol) to donate a proton to the methylene carbon atom of the resulting allylic carbanion. Interestingly, the active site of isopentenyl diphosphate isomerase type I also contains a cysteine that cooperates with glutamate rather than lysine. It has been proposed that the initial step in this enzyme is a protonation generating a tertiary carbocation intermediate.     

Crystal structures and mutational analysis of the arginine-, lysine-, histidine-binding protein ArtJ from Geobacillus stearothermophilus. Implications for interactions of ArtJ with its cognate ATP-binding cassette transporter, Art(MP)2

ArtJ is the substrate-binding component (receptor) of the ATP-binding cassette (ABC) transport system ArtJ-(MP)₂ from the thermophilic bacterium Geobacillus stearothermophilus that is specific for arginine, lysine, and histidine. The highest affinity is found for arginine (K_d = 0.039(±0.014) μM), while the affinities for lysine and histidine are about tenfold lower. We have determined the X-ray structures of ArtJ liganded with each of these substrates at resolutions of 1.79 Å (arginine), 1.79 Å (lysine), and 2.35 Å (histidine), respectively. As found for other solute receptors, the polypeptide chain is folded into two distinct domains (lobes) connected by a hinge. The interface between the lobes forms the substrate-binding pocket whose geometry is well preserved in all three ArtJ/amino acid complexes. Structure-derived mutational analyses indicated the crucial role of a region in the carboxy-terminal lobe of ArtJ in contacting the transport pore Art(MP)₂ and revealed the functional importance of Gln132 and Trp68. While variant Gln132Leu exhibited lower binding affinity for arginine but no binding of lysine and histidine, the variant Trp68Leu had lost binding activity for all three substrates. The results are discussed in comparison with known structures of homologous proteins from mesophilic bacteria.     

X-ray Crystal Structure of the Rotavirus Inner Capsid Particle at 3.8 Å Resolution

The rotavirus inner capsid particle, known as the “double-layered particle” (DLP), is the “payload” delivered into a cell in the process of viral infection. Its inner and outer protein layers, composed of viral protein (VP) 2 and VP6, respectively, package the 11 segments of the double-stranded RNA (dsRNA) of the viral genome, as well as about the same number of polymerase molecules (VP1) and capping-enzyme molecules (VP3). We have determined the crystal structure of the bovine rotavirus DLP. There is one full particle (outer diameter ∼ 700 Å) in the asymmetric unit of the P2₁2₁2₁ unit cell of dimensions a = 740 Å, b = 1198 Å, and c = 1345 Å. A three-dimensional reconstruction from electron cryomicroscopy was used as a molecular replacement model for initial phase determination to about 18.5 Å resolution, and the 60-fold redundancy of icosahedral particle symmetry allowed phases to be extended stepwise to the limiting resolution of the data (3.8 Å). The structure of a VP6 trimer (determined previously by others) fits the outer layer density with very little adjustment. The T = 13 triangulation number of that layer implies that there are four and one-third VP6 trimers per icosahedral asymmetric unit. The inner layer has 120 copies of VP2 and thus 2 copies per icosahedral asymmetric unit, designated VP2A and VP2B. Residues 101-880 fold into a relatively thin principal domain, comma-like in outline, shaped such that only rather modest distortions (concentrated at two “subdomain” boundaries) allow VP2A and VP2B to form a uniform layer with essentially no gaps at the subunit boundaries, except for a modest pore along the 5-fold axis. The VP2 principal domain resembles those of the corresponding shells and homologous proteins in other dsRNA viruses: λ1 in orthoreoviruses and VP3 in orbiviruses. Residues 1-80 of VP2A and VP2B fold together with four other such pairs into a “5-fold hub” that projects into the DLP interior along the 5-fold axis; residues 81-100 link the 10 polypeptide chains emerging from a 5-fold hub to the N-termini of their corresponding principal domains, clustered into a decameric assembly unit. The 5-fold hub appears to have several distinct functions. One function is to recruit a copy of VP1 (or of a VP1-VP3 complex), potentially along with a segment of plus-strand RNA, as a decamer of VP2 assembles. The second function is to serve as a shaft around which can coil a segment of dsRNA. The third function is to guide nascent mRNA, synthesized in the DLP interior by VP1 and 5′-capped by the action of VP3, out through a 5-fold exit channel. We propose a model for rotavirus particle assembly, based on known requirements for virion formation, together with the structure of the DLP and that of VP1, determined earlier.     

Lactose Binding to Galectin-1 Modulates Structural Dynamics, Increases Conformational Entropy, and Occurs with Apparent Negative Cooperativity

Galectins are a family of lectins with a conserved carbohydrate recognition domain that interacts with β-galactosides. By binding cell surface glycoconjugates, galectin-1 (gal-1) is involved in cell adhesion and migration processes and is an important regulator of tumor angiogenesis. Here, we used heteronuclear NMR spectroscopy and molecular modeling to investigate lactose binding to gal-1 and to derive solution NMR structures of gal-1 in the lactose-bound and unbound states. Structure analysis shows that the β-strands and loops around the lactose binding site, which are more open and dynamic in the unbound state, fold in around the bound lactose molecule, dampening internal motions at that site and increasing motions elsewhere throughout the protein to contribute entropically to the binding free energy. CD data support the view of an overall more open structure in the lactose-bound state. Analysis of heteronuclear single quantum coherence titration binding data indicates that lactose binds the two carbohydrate recognition domains of the gal-1 dimer with negative cooperativity, in that the first lactose molecule binds more strongly (K₁ = 21 ± 6 × 10³ M^− 1) than the second (K₂ = 4 ± 2 × 10³ M^− 1). Isothermal calorimetry data fit using a sequential binding model present a similar picture, yielding K₁ = 20 ± 10 × 10³ M^− 1 and K₂ = 1.67 ± 0.07 × 10³ M^− 1. Molecular dynamics simulations provide insight into structural dynamics of the half-loaded lactose state and, together with NMR data, suggest that lactose binding at one site transmits a signal through the β-sandwich and loops to the second binding site. Overall, our results provide new insight into gal-1 structure-function relationships and to protein-carbohydrate interactions in general.     

Structural role of bacterioruberin in the trimeric structure of archaerhodopsin-2

Archaerhodopsin-2 (aR2), a retinal protein-carotenoid complex found in the claret membrane of Halorubrum sp. aus-2, functions as a light-driven proton pump. In this study, the membrane fusion method was utilized to prepare trigonal P321 crystals (a = b = 98.2 Å, c = 56.2 Å) and hexagonal P6₃ crystals (a = b = 108.8 Å, c = 220.7 Å). The trigonal crystal is made up of stacked membranes in which the aR2 trimers are arranged on a honeycomb lattice. Similar membranous structures are found in the hexagonal crystal, but four membrane layers with different orientations are contained in the unit cell. In these crystals, the carotenoid bacterioruberin [5,32-bis(2-hydroxypropan-2-yl)-2,8,12,16,21,25,29,35-octamethylhexatriaconta-6,8,10,12,14,16,18,20,22,24,26,28,30-tridecaene-2,35-diol] binds to crevices between the subunits of the trimer. Its polyene chain is inclined from the membrane normal by an angle of about 20° and, on the cytoplasmic side, it is surrounded by helices AB and DE of neighbouring subunits. This peculiar binding mode suggests that bacterioruberin plays a striking structural role for the trimerization of aR2. When compared with the aR2 structure in another crystal form containing no bacterioruberin, the proton release channel takes a more closed conformation in the P321 or P6₃ crystal; i.e., the native conformation of protein is stabilized in the trimeric protein-bacterioruberin complex. Interestingly, most residues participating in the trimerization are not conserved in bacteriorhodopsin, a homologous protein capable of forming a trimeric structure in the absence of bacterioruberin. Despite a large alteration in the amino acid sequence, the shape of the intratrimer hydrophobic space filled by lipids is highly conserved between aR2 and bacteriorhodopsin. Since a transmembrane helix facing this space undergoes a large conformational change during the proton pumping cycle, it is feasible that trimerization is an important strategy to capture special lipid components that are relevant to the protein activity.     

The X-ray crystal structure of Shewanella oneidensis OmcA reveals new insight at the microbe–mineral interface

The X-ray crystal structure of Shewanella oneidensis OmcA, an extracellular decaheme cytochrome involved in mineral reduction, was solved to a resolution of 2.7 Å. The four OmcA molecules in the asymmetric unit are arranged so the minimum distance between heme 5 on adjacent OmcA monomers is 9 Å, indicative of a transient OmcA dimer capable of intermolecular electron transfer. A previously identified hematite binding motif was identified near heme 10, forming a hydroxylated surface that would bring a heme 10 electron egress site to ∼10 Å of a mineral surface.     

Design of protein-ligand binding based on the molecular-mechanics energy model

While the molecular-mechanics field has standardized on a few potential energy functions, computational protein design efforts are based on potentials that are unique to individual laboratories. Here we show that a standard molecular-mechanics potential energy function without any modifications can be used to engineer protein-ligand binding. A molecular-mechanics potential is used to reconstruct the coordinates of various binding sites with an average root-mean-square error of 0.61 Å and to reproduce known ligand-induced side-chain conformational shifts. Within a series of 34 mutants, the calculation can always distinguish between weak (K_d > 1 mM) and tight (K_d < 10 μM) binding sequences. Starting from partial coordinates of the ribose-binding protein lacking the ligand and the 10 primary contact residues, the molecular-mechanics potential is used to redesign a ribose-binding site. Out of a search space of 2 × 10¹² sequences, the calculation selects a point mutant of the native protein as the top solution (experimental K_d = 17 μM) and the native protein as the second best solution (experimental K_d = 210 nM). The quality of the predictions depends on the accuracy of the generalized Born electrostatics model, treatment of protonation equilibria, high-resolution rotamer sampling, a final local energy minimization step, and explicit modeling of the bound, unbound, and unfolded states. The application of unmodified molecular-mechanics potentials to protein design links two fields in a mutually beneficial way. Design provides a new avenue for testing molecular-mechanics energy functions, and future improvements in these energy functions will presumably lead to more accurate design results.     

The three-dimensional structure of genomic RNA in bacteriophage MS2: implications for assembly

Using cryo-electron microscopy, single particle image processing and three-dimensional reconstruction with icosahedral averaging, we have determined the three-dimensional solution structure of bacteriophage MS2 capsids reassembled from recombinant protein in the presence of short oligonucleotides. We have also significantly extended the resolution of the previously reported structure of the wild-type MS2 virion. The structures of recombinant MS2 capsids reveal clear density for bound RNA beneath the coat protein binding sites on the inner surface of the T = 3 MS2 capsid, and show that a short extension of the minimal assembly initiation sequence that promotes an increase in the efficiency of assembly, interacts with the protein capsid forming a network of bound RNA. The structure of the wild-type MS2 virion at ∼9 Å resolution reveals icosahedrally ordered density encompassing ∼90% of the single-stranded RNA genome. The genome in the wild-type virion is arranged as two concentric shells of density, connected along the 5-fold symmetry axes of the particle. This novel RNA fold provides new constraints for models of viral assembly.     

Dynamics and Stability in Maturation of a T = 4 Virus

Nudaurelia capensis ω virus is a T = 4, icosahedral virus with a bipartite, positive-sense RNA genome. Expression of the coat protein gene in a baculovirus system was previously shown to result in the formation of procapsids when purified at pH 7.6. Procapsids are round, porous particles (480 Å diameter) and have T = 4 quasi-symmetry. Reduction of pH from 7.6 to 5.0 resulted in virus-like particles (VLP_5.0) that are morphologically identical with authentic virions, with an icosahedral-shaped capsid and a maximum dimension of 410 Å. VLP_5.0 undergoes a maturation cleavage between residues N570 and F571, creating the covalently independent γ peptide (residues 571-641) that remains associated with the particle. This cleavage also occurs in authentic virions, and in each case, it renders the morphological change irreversible (i.e., capsids do not expand when the pH is raised back to 7.6). However, a non-cleavable mutant, N570T, undergoes the transition reversibly (NT_7.6 ↔ NT_5.0). We used electron cryo-microscopy and three-dimensional image reconstruction to study the icosahedral structures of NT_7.6, NT_5.0, and VLP_5.0 at about 8, 6, and 6 Å resolution, respectively. We employed the 2. 8-Å X-ray model of the mature virus, determined at pH 7.0 (XR_7.0), to establish (1) how and why procapsid and capsid structures differ, (2) why lowering pH drives the transition, and (3) why the non-cleaving NT_5.0 is reversible. We show that procapsid assembly minimizes the differences in quaternary interactions in the particle. The two classes of 2-fold contacts in the T = 4 surface lattice are virtually identical, both mediated by similarly positioned but dynamic γ peptides. Furthermore, quasi and icosahedral 3-fold interactions are indistinguishable. Maturation results from neutralizing the repulsive negative charge at subunit interfaces with significant differentiation of quaternary interactions (one 2-fold becomes flat, mediated by a γ peptide, while the other is bent with the γ peptide disordered) and dramatic stabilization of the particle. The γ peptide at the flat contact remains dynamic when cleavage cannot occur (NT_5.0) but becomes totally immobilized by noncovalent interactions after cleavage (VLP_5.0).     

An RNA Structural Switch Regulates Diploid Genome Packaging by Moloney Murine Leukemia Virus

Retroviruses selectively package two copies of their RNA genomes via mechanisms that have yet to be fully deciphered. Recent studies with small fragments of the Moloney murine leukemia virus (MoMuLV) genome suggested that selection may be mediated by an RNA switch mechanism, in which conserved UCUG elements that are sequestered by base-pairing in the monomeric RNA become exposed upon dimerization to allow binding to the cognate nucleocapsid (NC) domains of the viral Gag proteins. Here we show that a large fragment of the MoMuLV 5′ untranslated region that contains all residues necessary for efficient RNA packaging (Ψ^WT; residues 147-623) also exhibits a dimerization-dependent affinity for NC, with the native dimer ([Ψ^WT]₂) binding 12 ± 2 NC molecules with high affinity (K_d = 17 ± 7 nM) and with the monomer, stabilized by substitution of dimer-promoting loop residues with hairpin-stabilizing sequences (Ψ^M), binding 1-2 NC molecules. Identical dimer-inhibiting mutations in MoMuLV-based vectors significantly inhibit genome packaging in vivo (∼ 100-fold decrease), whereas a large deletion of nearly 200 nucleotides just upstream of the gag start codon has minimal effects. Our findings support the proposed RNA switch mechanism and further suggest that virus assembly may be initiated by a complex comprising as few as 12 Gag molecules bound to a dimeric packaging signal.     

X-ray structure of a galactose-specific lectin from Spatholobous parviflorous

A galactose-specific seed lectin from Spatholobous parviflorus (SPL) has been purified, crystallized and its X-ray structure solved. It is the first lectin purified and crystallized from the genus Spatholobus (family: Fabaceae). The crystals belong to the space group P1, with a = 60.792 Å, b = 60.998 Å, c = 78.179 Å, α = 78.68°, β = 88.62°, γ = 104.32°. The data were collected at 2.04 Å resolution under cryocondition, on a MAR image-plate detector system, mounted on a rotating anode X-ray generator. The coordinates of Dolichos biflorus lectin (1lu1) were successfully used for the structure solution by molecular replacement method. The primary structure of the SPL was not known earlier and it was unambiguously visible in the electron density. S. parviflorus lectin is a hetero-dimeric-tetramer with two alpha and two beta chains of 251 and 239 residues respectively. SPL has two metal ions, Ca²⁺ and Mn²⁺, bound to a loop region of each chain. The SPL monomers are in jelly roll form.     

Structures and electrochemistry of monomeric and dimeric CpCo(dithiolene) complexes with substituted benzene-1,2-dithiolate ligand

The molecular structures of [CpCo(tdt)] and [CpCo(Cl₃bdt)] were determined by X-ray diffraction studies. [CpCo(tdt)] was monomeric in the crystal, coordinatively unsaturated with 16-electrons and had a two-legged piano-stool geometry. [CpCo(Cl₃bdt)] was dimeric, coordinatively saturated with 18-electrons and became a three-legged piano-stool geometry. There were two crystallographically independent molecules in [CpCo(tdt)] and both molecules were associated with intermolecular Cp?Cp face-to-face interaction. Some intermolecular Co?S interactions were also observed between the crystallographically identical molecules of [CpCo(tdt)]. The cyclic voltammograms of [CpCo(tdt)] and [CpCo(Cl₂bdt)] exhibited single oxidation waves, but those of [CpCo(Cl₃bdt)] and [CpCo(Cl₄bdt)] showed two oxidation waves due to both the monomer and dimer in the solutions. Electrochemical oxidations of these monomers occurred dimerizations by EC reactions in the solutions, and the oxidized dimers could be rereduced to form the original monomers by EC reactions. Spectroelectrochemical data using OTTLE supported the reversible ECEC reactions. Abbreviations of dithiolene ligands are as follows: tdt = toluene-3,4-dithiolate, Cl₂bdt = 3,6-dichlorobenzene-1,2-dithiolate, Cl₃bdt = 3,4,6-trichlorobenzene-1,2-dithiolate, and Cl₄bdt = tetrachlorobenzene-1,2-dithiolate.     

The 1.2 Å crystal structure of an E. coli tRNA acceptor stem microhelix reveals two magnesium binding sites

tRNA identity elements assure the correct aminoacylation of tRNAs by the cognate aminoacyl-tRNA synthetases. tRNA^Ser belongs to the so-called class II system, in which the identity elements are rather simple and are mostly located in the acceptor stem region, in contrast to ‘class I’, where tRNA determinants are more complex and are located within different regions of the tRNA.The structure of an Escherichia coli tRNA^Ser acceptor stem microhelix was solved by high resolution X-ray structure analysis. The RNA crystallizes in the space group C2, with one molecule per asymmetric unit and with the cell constants a = 35.79, b = 39.13, c = 31.37 Å, and β = 111.1°. A defined hydration pattern of 97 water molecules surrounds the tRNA^Ser acceptor stem microhelix. Additionally, two magnesium binding sites were detected in the tRNA^Ser aminoacyl stem.     

The X-ray structure of zebrafish (Danio rerio) ileal bile acid-binding protein reveals the presence of binding sites on the surface of the protein molecule

The ileal bile acid-binding proteins (I-BABPs), also called ileal lipid-binding proteins or gastrotropins, belong to the family of the fatty acid-binding proteins and play an important role in the solubilization and transport of bile acids in the enterocyte. This article describes the expression, purification, crystallization, and three-dimensional structure determination of zebrafish (Danio rerio) I-BABP both in its apo form and bound to cholic acid. This is the first X-ray structure of an I-BABP. The structure of the apoprotein was determined to a resolution of 1.6 Å, and two different monoclinic crystal forms of the holoprotein were solved and refined to 2.2 Å resolution. Three protein molecules are present in the asymmetric unit of one of the co-crystal forms and two in the other, and therefore, the results of this study refer to observations made on five different protein molecules in the crystalline state. In every case, two cholate ligands were found bound in approximately the same position in the internal cavity of the protein molecules, but an unexpected result is the presence of clear and unambiguous electron density for several cholate molecules bound on hydrophobic patches on the surface of all the five independent protein molecules examined. Isothermal titration calorimetry was used for the thermodynamic characterization of the binding mechanism and has yielded results that are consistent with the X-ray data. Ligand binding is described in detail, and the conformational changes undergone by the protein molecule in the apo-to-holo transition are examined by superposition of the apo- and holoprotein models. The structure of the holoprotein is also compared with that of the liver BABP from the same species and those of other I-BABPs determined by NMR.