Structural basis for the selective incorporation of an artificial nucleotide opposite a DNA adduct by a DNA polymerase.

The possibility to sequence cytotoxic O6-alkylG DNA adducts would greatly benefit research. Recently we reported a benzimidazole-derived nucleotide that is selectively incorporated opposite the damaged site by a mutated DNA polymerase. Here we provide the structural basis for this reaction which may spur future developments in DNA damage sequencing.

Better models by discarding data?

In macromolecular X-ray crystallography, typical data sets have substantial multiplicity. This can be used to calculate the consistency of repeated measurements and thereby assess data quality. Recently, the properties of a correlation coefficient, CC1/2, that can be used for this purpose were characterized and it was shown that CC1/2 has superior properties compared with `merging' R values. A derived quantity, CC*, links data and model quality. Using experimental data sets, the behaviour of CC1/2 and the more conventional indicators were compared in two situations of practical importance: merging data sets from different crystals and selectively rejecting weak observations or (merged) unique reflections from a data set. In these situations controlled `paired-refinement' tests show that even though discarding the weaker data leads to improvements in the merging R values, the refined models based on these data are of lower quality. These results show the folly of such data-filtering practices aimed at improving the merging R values. Interestingly, in all of these tests CC1/2 is the one data-quality indicator for which the behaviour accurately reflects which of the alternative data-handling strategies results in the best-quality refined model. Its properties in the presence of systematic error are documented and discussed.

Non-thermal microwave effects on protein dynamics? An X-ray diffraction study on tetragonal lysozyme crystals.

X-ray diffraction (XRD) was used to investigate the structural and dynamical effects of microwave fields on tetragonal single crystals of hen egg-white lysozyme at a resolution of 2.0 A. Using a modified slab-line waveguide allows on-line XRD to be carried out while the protein crystal is exposed to well defined microwave fields. High microwave power levels mainly lead to increased, but largely recoverable, lattice defects owing to the evaporation of crystal water. At lower microwave power levels, the presence of the microwave field results in localized reproducible changes in the mean-square displacements (B factors). At particular sites, it is found that the B factors even decrease with increasing microwave power. Most of these effects can be explained by a comparison of the data obtained under microwave irradiation with data obtained at elevated temperature which simulate heating owing to microwave absorption by unbound crystal water. The data show no indication of large microwave-driven displacements of structural subunits in the protein that would be expected if microwaves were to be absorbed resonantly by protein vibrations. Rather, the observed changes in the atomic mean-square displacements suggest that if microwaves couple non-thermally to globular proteins at hydration levels at which they still function, their effect on protein dynamics and structure is very small.

Crystallization and preliminary X-ray analysis of the trehalose/maltose ABC transporter MalFGK2 from Thermococcus litoralis.

Trehalose and maltose uptake in the hyperthermophilic archaeon Thermococcus litoralis is mediated by an ABC transport system. The heterotetrameric transport complex MalFGK(2), consisting of two membrane-spanning subunits and two copies of an ATP-binding cassette protein, has been crystallized. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 106.5, b = 150.5, c = 170.1 A, beta = 107.8 degrees. A native data set has been obtained at a resolution of 5 A.

Active transport of an antibiotic rifamycin derivative by the outer-membrane protein FhuA.

BACKGROUND: FhuA, an integral membrane protein of Escherichia coli, actively transports ferrichrome and the structurally related antibiotic albomycin across the outer membrane. The transport is coupled to the proton motive force, which energizes FhuA through the inner-membrane protein TonB. FhuA also transports the semisynthetic rifamycin derivative CGP 4832, although the chemical structure of this antibiotic differs markedly from that of ferric hydroxamates. RESULTS: X-ray crystallography revealed that rifamycin CGP 4832 occupies the same ligand binding site as ferrichrome and albomycin, thus demonstrating a surprising lack of selectivity. However, the binding of rifamycin CGP 4832 is deviant from the complexes of FhuA with hydroxamate-type ligands in that it does not result in the unwinding of the switch helix but only in its destabilization, as reflected by increased B factors. Unwinding of the switch helix is proposed to be required for efficient binding of TonB to FhuA and for coupling the proton motive force of the cytoplasmic membrane with energy-dependent ligand transport. The transport data from cells expressing mutant FhuA proteins indicated conserved structural and mechanistic requirements for the transport of both types of compounds. CONCLUSIONS: We conclude that the binding of rifamycin CGP 4832 destabilizes the switch helix and promotes the formation of a transport-competent FhuA-TonB complex, albeit with lower efficiency than ferrichrome. Active transport of this rifamycin derivative explains the 200-fold increase in potency as compared to rifamycin, which is not a FhuA-specific ligand and permeates across the cell envelope by passive diffusion only.

Three-dimensional structure of the nonaheme cytochrome c from Desulfovibrio desulfuricans Essex in the Fe(III) state at 1.89 A resolution.

A nine heme group containing cytochrome c isolated from the soluble and membrane fractions of Desulfovibrio desulfuricans Essex, termed nonaheme cytochrome c, was crystallized, and the structure was solved using the multiple wavelength anomalous dispersion (MAD) phasing method. Refinement was carried out to a resolution of 1.89 A, and anisotropic temperature factors were addressed to the iron and sulfur atoms in the model. The structure revealed two cytochrome c(3) like domains with the typical arrangement of four heme centers. Both domains flanked an extra heme buried under the protein surface. This heme is held in position by loop extensions in each of the two domains. Although both the N- and C-terminal tetraheme domains exhibit a fold and heme arrangement very similar to that of cytochrome c(3), they differ considerably in their loop extensions and electrostatic surface. Analysis of the structure provides evidence for a different function of both domains, namely, anchoring the protein in a transmembranous complex with the N-terminal domain and formation of an electron-transfer complex with hydrogenase by the C-terminal domain.

The crystal structure of a liganded trehalose/maltose-binding protein from the hyperthermophilic Archaeon Thermococcus litoralis at 1.85 A.

We report the crystallization and structure determination at 1.85 A of the extracellular, membrane-anchored trehalose/maltose-binding protein (TMBP) in complex with its substrate trehalose. TMBP is the substrate recognition site of the high-affinity trehalose/maltose ABC transporter of the hyperthermophilic Archaeon Thermococcus litoralis. In vivo, this protein is anchored to the membrane, presumably via an N-terminal cysteine lipid modification. The crystallized protein was N-terminally truncated, resulting in a soluble protein exhibiting the same binding characteristics as the wild-type protein. The protein shows the characteristic features of a transport-related, substrate-binding protein and is structurally related to the maltose-binding protein (MBP) of Escherichia coli. It consists of two similar lobes, each formed by a parallel beta-sheet flanked by alpha-helices on both sides. Both are connected by a hinge region consisting of two antiparallel beta-strands and an alpha-helix. As in MBP, the substrate is bound in the cleft between the lobes by hydrogen bonds and hydrophobic interactions. However, compared to maltose binding in MBP, direct hydrogen bonding between the substrate and the protein prevails while apolar contacts are reduced. To elucidate factors contributing to thermostability, we compared TMBP with its mesophilic counterpart MBP and found differences known from similar investigations. Specifically, we find helices that are longer than their structurally equivalent counterparts, and fewer internal cavities.

Low-resolution data analysis for low-density lipoprotein particle.

The knowledge of the molecular structure of LDL, a large lipoprotein complex, is of great interest for medical investigations. Currently available LDL crystals do not diffract to high resolution and do not allow the application of standard crystallographic techniques. Additional difficulties arise because of a very dense crystal packing and the presence of several components with quite different mean densities. Several ab initio phasing methods previously reported by the authors have been successfully applied to find a crystallographic image of LDL at a resolution of 27 A. The most promising results have been obtained using direct phasing with a connectivity analysis of the electron-density maps. The current image makes it possible to discern a single particle covered by a layer of relatively high density that is asymmetrically distributed on the particle surface. It shows a partition of high and low densities inside the particle and, in particular, strips of varying density in the lipid core.

The x-ray structure of D-amino acid oxidase at very high resolution identifies the chemical mechanism of flavin-dependent substrate dehydrogenation.

Flavin is one of the most versatile redox cofactors in nature and is used by many enzymes to perform a multitude of chemical reactions. d-Amino acid oxidase (DAAO), a member of the flavoprotein oxidase family, is regarded as a key enzyme for the understanding of the mechanism underlying flavin catalysis. The very high-resolution structures of yeast DAAO complexed with d-alanine, d-trifluoroalanine, and l-lactate (1.20, 1.47, and 1.72 A) provide strong evidence for hydride transfer as the mechanism of dehydrogenation. This is inconsistent with the alternative carbanion mechanism originally favored for this type of enzymatic reaction. The step of hydride transfer can proceed without involvement of amino acid functional groups. These structures, together with results from site-directed mutagenesis, point to orbital orientation/steering as the major factor in catalysis. A diatomic species, proposed to be a peroxide, is found at the active center and on the Re-side of the flavin. These results are of general relevance for the mechanisms of flavoproteins and lead to the proposal of a common dehydrogenation mechanism for oxidases and dehydrogenases.

Crystal structure of MalK, the ATPase subunit of the trehalose/maltose ABC transporter of the archaeon Thermococcus litoralis.

The members of the ABC transporter family transport a wide variety of molecules into or out of cells and cellular compartments. Apart from a translocation pore, each member possesses two similar nucleoside triphosphate-binding subunits or domains in order to couple the energy-providing reaction with transport. In the maltose transporter of several Gram-negative bacteria and the archaeon Thermo coccus litoralis, the nucleoside triphosphate-binding subunit contains a C-terminal regulatory domain. A dimer of the subunit is attached cytoplasmically to the translocation pore. Here we report the crystal structure of this dimer showing two bound pyrophosphate molecules at 1.9 A resolution. The dimer forms by association of the ATPase domains, with the two regulatory domains attached at opposite poles. Significant deviation from 2-fold symmetry is seen at the interface of the dimer and in the regions corresponding to those residues known to be in contact with the translocation pore. The structure and its relationship to function are discussed in the light of known mutations from the homologous Escherichia coli and Salmonella typhimurium proteins.

Crystal structure of Omp32, the anion-selective porin from Comamonas acidovorans, in complex with a periplasmic peptide at 2.1 A resolution.

BACKGROUND: Porins provide diffusion channels for salts and small organic molecules in the outer membrane of bacteria. In OmpF from Escherichia coli and related porins, an electrostatic field across the channel and a potential, originating from a surplus of negative charges, create moderate cation selectivity. Here, we investigate the strongly anion-selective porin Omp32 from Comamonas acidovorans, which is closely homologous to the porins of pathogenic Bordetella and Neisseria species. RESULTS: The crystal structure of Omp32 was determined to a resolution of 2.1 A using single isomorphous replacement with anomalous scattering (SIRAS). The porin consists of a 16-stranded beta barrel with eight external loops and seven periplasmic turns. Loops 3 and 8, together with a protrusion located within beta-strand 2, narrow the cross-section of the pore considerably. Arginine residues create a charge filter in the constriction zone and a positive surface potential at the external and periplasmic faces. One sulfate ion was bound to Arg38 in the channel constriction zone. A peptide of 5.8 kDa appeared bound to Omp32 in a 1:1 stoichiometry on the periplasmic side close to the symmetry axis of the trimer. Eight amino acids of this peptide could be identified, revealing specific interactions with beta-strand 1 of the porin. CONCLUSIONS: The Omp32 structure explains the strong anion selectivity of this porin. Selectivity is conferred by a positive potential, which is not attenuated by negative charges inside the channel, and by an extremely narrow constriction zone. Moreover, Omp32 represents the anchor molecule for a peptide which is homologous to proteins that link the outer membrane to the cell wall peptidoglycan.

The crystal structure of the ligand binding module of axonin-1/TAG-1 suggests a zipper mechanism for neural cell adhesion.

We have determined the crystal structure of the ligand binding fragment of the neural cell adhesion molecule axonin-1/TAG-1 comprising the first four immunoglobulin (Ig) domains. The overall structure of axonin-1(Ig1-4) is U-shaped due to contacts between domains 1 and 4 and domains 2 and 3. In the crystals, these molecules are aligned in a string with adjacent molecules oriented in an anti-parallel fashion and their C termini perpendicular to the string. This arrangement suggests that cell adhesion by homophilic axonin-1 interaction occurs by the formation of a linear zipper-like array in which the axonin-1 molecules are alternately provided by the two apposed membranes. In accordance with this model, mutations in a loop critical for the formation of the zipper resulted in the loss of the homophilic binding capacity of axonin-1.

Crystal structure of the antibiotic albomycin in complex with the outer membrane transporter FhuA.

One alternative method for drug delivery involves the use of siderophore-antibiotic conjugates. These compounds represent a specific means by which potent antimicrobial agents, covalently linked to iron-chelating siderophores, can be actively transported across the outer membrane of gram-negative bacteria. These "Trojan Horse" antibiotics may prove useful as an efficient means to combat multi-drug-resistant bacterial infections. Here we present the crystallographic structures of the natural siderophore-antibiotic conjugate albomycin and the siderophore phenylferricrocin, in complex with the active outer membrane transporter FhuA from Escherichia coli. To our knowledge, this represents the first structure of an antibiotic bound to its cognate transporter. Albomycins are broad-host range antibiotics that consist of a hydroxamate-type iron-chelating siderophore, and an antibiotically active, thioribosyl pyrimidine moiety. As observed with other hydroxamate-type siderophores, the three-dimensional structure of albomycin reveals an identical coordination geometry surrounding the ferric iron atom. Unexpectedly, this antibiotic assumes two conformational isomers in the binding site of FhuA, an extended and a compact form. The structural information derived from this study provides novel insights into the diverse array of antibiotic moieties that can be linked to the distal portion of iron-chelating siderophores and offers a structural platform for the rational design of hydroxamate-type siderophore-antibiotic conjugates.

A conserved structural motif for lipopolysaccharide recognition by procaryotic and eucaryotic proteins.

BACKGROUND: Lipopolysaccharide (LPS), a lipoglycan from the outer membrane of Gram-negative bacteria, is an immunomodulatory molecule that stimulates the innate immune response. High levels of LPS cause excessive release of inflammatory mediators and are responsible for the septic shock syndrome. The interaction of LPS with its cognate binding proteins has not, as yet, been structurally elucidated. RESULTS: The X-ray crystallographic structure of LPS in complex with the integral outer membrane protein FhuA from Escherichia coli K-12 is reported. It is in accord with data obtained using mass spectroscopy and nuclear magnetic resonance. Most of the important hydrogen-bonding or electrostatic interactions with LPS are provided by eight positively charged residues of FhuA. Residues in a similar three-dimensional arrangement were searched for in all structurally known proteins using a fast template-matching algorithm, and a subset of four residues was identified that is common to known LPS-binding proteins. CONCLUSIONS: These four residues, three of which form specific interactions with lipid A, appear to provide the structural basis of pattern recognition in the innate immune response. Their arrangement can serve to identify LPS-binding sites on proteins known to interact with LPS, and could serve as a template for molecular modeling of a LPS scavenger designed to reduce the septic shock syndrome.

Förster excitation energy transfer in peridinin-chlorophyll-a-protein.

Time-resolved fluorescence anisotropy spectroscopy has been used to study the chlorophyll a (Chl a) to Chl a excitation energy transfer in the water-soluble peridinin-chlorophyll a-protein (PCP) of the dinoflagellate Amphidinium carterae. Monomeric PCP binds eight peridinins and two Chl a. The trimeric structure of PCP, resolved at 2 A (, Science. 272:1788-1791), allows accurate calculations of energy transfer times by use of the Förster equation. The anisotropy decay time constants of 6.8 +/- 0.8 ps (tau(1)) and 350 +/- 15 ps (tau(2)) are respectively assigned to intra- and intermonomeric excitation equilibration times. Using the ratio tau(1)/tau(2) and the amplitude of the anisotropy, the best fit of the experimental data is achieved when the Q(y) transition dipole moment is rotated by 2-7 degrees with respect to the y axis in the plane of the Chl a molecule. In contrast to the conclusion of, Biochemistry. 23:1564-1571) that the refractive index (n) in the Förster equation should be equal to that of the solvent, n can be estimated to be 1.6 +/- 0.1, which is larger than that of the solvent (water). Based on our observations we predict that the relatively slow intermonomeric energy transfer in vivo is overruled by faster energy transfer from a PCP monomer to, e.g., the light-harvesting a/c complex.

Crystal structure of human beta2-glycoprotein I: implications for phospholipid binding and the antiphospholipid syndrome.

The high affinity of human plasma beta2-glycoprotein I (beta(2)GPI), also known as apolipoprotein-H (ApoH), for negatively charged phospholipids determines its implication in a variety of physiological pathways, including blood coagulation and the immune response. beta(2)GPI is considered to be a cofactor for the binding of serum autoantibodies from antiphospholipid syndrome (APS) and correlated with thrombosis, lupus erythematosus and recurrent fetal loss. We solved the beta(2)GPI structure from a crystal form with 84% solvent and present a model containing all 326 amino acid residues and four glycans. The structure reveals four complement control protein modules and a distinctly folding fifth C-terminal domain arranged like beads on a string to form an elongated J-shaped molecule. Domain V folds into a central beta-spiral of four antiparallel beta-sheets with two small helices and an extended C-terminal loop region. It carries a distinct positive charge and the sequence motif CKNKEKKC close to the hydrophobic loop composed of residues LAFW (313-316), resulting in an excellent counterpart for interactions with negatively charged amphiphilic substances. The beta(2)GPI structure reveals potential autoantibody-binding sites and supports mutagenesis studies where Trp316 and CKNKEKKC have been found to be essential for the phospholipid-binding capacity of beta(2)GPI.

Crystal structure of a phycourobilin-containing phycoerythrin at 1.90-A resolution.

The structure of R-phycoerythrin (R-PE) from the red alga Griffithsia monilis was solved at 1.90-A resolution by molecular replacement, using the atomic coordinates of cyanobacterial phycocyanin from Fremyella diplosiphon as a model. The crystallographic R factor for the final model is 17.5% (Rfree 22.7%) for reflections in the range 100-1.90 A. The model consists of an (alphabeta)2 dimer with an internal noncrystallographic dyad and a fragment of the gamma-polypeptide. The alpha-polypeptide (164 amino acid residues) has two covalently bound phycoerythrobilins at positions alpha82 and alpha139. The beta-polypeptide (177 residues) has two phycoerythrobilins bound to residues beta82 and beta158 and one phycourobilin covalently attached to rings A and D at residues beta50 and beta61, respectively. The electron density of the gamma-polypeptide is mostly averaged out by threefold crystallographic symmetry, but a dipeptide (Gly-Tyr) and one single Tyr could be modeled. These two tyrosine residues of the gamma-polypeptide are in close proximity to the phycoerythrobilins at position beta82 of two symmetry-related beta-polypeptides and are related by the same noncrystallographic dyad as the (alphabeta)2 dimer. Possible energy transfer pathways are discussed briefly.

Siderophore-mediated iron transport: crystal structure of FhuA with bound lipopolysaccharide.

FhuA, the receptor for ferrichrome-iron in Escherichia coli, is a member of a family of integral outer membrane proteins, which, together with the energy-transducing protein TonB, mediate the active transport of ferric siderophores across the outer membrane of Gram-negative bacteria. The three-dimensional structure of FhuA is presented here in two conformations: with and without ferrichrome-iron at resolutions of 2.7 and 2.5 angstroms, respectively. FhuA is a beta barrel composed of 22 antiparallel beta strands. In contrast to the typical trimeric arrangement found in porins, FhuA is monomeric. Located within the beta barrel is a structurally distinct domain, the "cork," which mainly consists of a four-stranded beta sheet and four short alpha helices. A single lipopolysaccharide molecule is noncovalently associated with the membrane-embedded region of the protein. Upon binding of ferrichrome-iron, conformational changes are transduced to the periplasmic pocket of FhuA, signaling the ligand-loaded status of the receptor. Sequence homologies and mutagenesis data are used to propose a structural mechanism for TonB-dependent siderophore-mediated transport across the outer membrane.

Crystal structure of the effector-binding domain of the trehalose-repressor of Escherichia coli, a member of the LacI family, in its complexes with inducer trehalose-6-phosphate and noninducer trehalose.

The crystal structure of the Escherichia coli trehalose repressor (TreR) in a complex with its inducer trehalose-6-phosphate was determined by the method of multiple isomorphous replacement (MIR) at 2.5 A resolution, followed by the structure determination of TreR in a complex with its noninducer trehalose at 3.1 A resolution. The model consists of residues 61 to 315 comprising the effector binding domain, which forms a dimer as in other members of the LacI family. This domain is composed of two similar subdomains each consisting of a central beta-sheet sandwiched between alpha-helices. The effector binding pocket is at the interface of these subdomains. In spite of different physiological functions, the crystal structures of the two complexes of TreR turned out to be virtually identical to each other with the conformation being similar to those of the effector binding domains of the LacI and PurR in complex with their effector molecules. According to the crystal structure, the noninducer trehalose binds to a similar site as the trehalose portion of trehalose-6-phosphate. The binding affinity for the former is lower than for the latter. The noninducer trehalose thus binds competitively to the repressor. Unlike the phosphorylated inducer molecule, it is incapable of blocking the binding of the repressor headpiece to its operator DNA. The ratio of the concentrations of trehalose-6-phosphate and trehalose thus is used to switch between the two alternative metabolic uses of trehalose as an osmoprotectant and as a carbon source.

Prediction by a neural network of outer membrane beta-strand protein topology.

An artificial neural network (NN) was trained to predict the topology of bacterial outer membrane (OM) beta-strand proteins. Specifically, the NN predicts the z-coordinate of Calpha atoms in a coordinate frame with the outer membrane in the xy-plane, such that low z-values indicate periplasmic turns, medium z-values indicate transmembrane beta-strands, and high z-values indicate extracellular loops. To obtain a training set, seven OM proteins (porins) with structures known to high resolution were aligned with their pores along the z-axis. The relationship between Calpha z-values and topology was thereby established. To predict the topology of other OM proteins, all seven porins were used for the training set. Z-values (topologies) were predicted for two porins with hitherto unknown structure and for OM proteins not belonging to the porin family, all with insignificant sequence homology to the training set. The results of topology prediction compare favorably with experimental topology data.