Pharmacometabolomic mapping of early biochemical changes induced by sertraline and placebo.

In this study, we characterized early biochemical changes associated with sertraline and placebo administration and changes associated with a reduction in depressive symptoms in patients with major depressive disorder (MDD). MDD patients received sertraline or placebo in a double-blind 4-week trial; baseline, 1 week, and 4 weeks serum samples were profiled using a gas chromatography time of flight mass spectrometry metabolomics platform. Intermediates of TCA and urea cycles, fatty acids and intermediates of lipid biosynthesis, amino acids, sugars and gut-derived metabolites were changed after 1 and 4 weeks of treatment. Some of the changes were common to the sertraline- and placebo-treated groups. Changes after 4 weeks of treatment in both groups were more extensive. Pathway analysis in the sertraline group suggested an effect of drug on ABC and solute transporters, fatty acid receptors and transporters, G signaling molecules and regulation of lipid metabolism. Correlation between biochemical changes and treatment outcomes in the sertraline group suggested a strong association with changes in levels of branched chain amino acids (BCAAs), lower BCAAs levels correlated with better treatment outcomes; pathway analysis in this group revealed that methionine and tyrosine correlated with BCAAs. Lower levels of lactic acid, higher levels of TCA/urea cycle intermediates, and 3-hydroxybutanoic acid correlated with better treatment outcomes in placebo group. Results of this study indicate that biochemical changes induced by drug continue to evolve over 4 weeks of treatment and that might explain partially delayed response. Response to drug and response to placebo share common pathways but some pathways are more affected by drug treatment. BCAAs seem to be implicated in mechanisms of recovery from a depressed state following sertraline treatment.

Virus maturation involving large subunit rotations and local refolding.

Large-scale conformational changes transform viral precursors into infectious virions. The structure of bacteriophage HK97 capsid, Head-II, was recently solved by crystallography, revealing a catenated cross-linked topology. We have visualized its precursor, Prohead-II, by cryoelectron microscopy and modeled the conformational change by appropriately adapting Head-II. Rigid-body rotations ( approximately 40 degrees) cause switching to an entirely different set of interactions; in addition, two motifs undergo refolding. These changes stabilize the capsid by increasing the surface area buried at interfaces and bringing the cross-link-forming residues, initially approximately 40 angstroms apart, close together. The inner surface of Prohead-II is negatively charged, suggesting that the transition is triggered electrostatically by DNA packaging.

Topologically linked protein rings in the bacteriophage HK97 capsid.

The crystal structure of the double-stranded DNA bacteriophage HK97 mature empty capsid was determined at 3.6 angstrom resolution. The 660 angstrom diameter icosahedral particle contains 420 subunits with a new fold. The final capsid maturation step is an autocatalytic reaction that creates 420 isopeptide bonds between proteins. Each subunit is joined to two of its neighbors by ligation of the side-chain lysine 169 to asparagine 356. This generates 12 pentameric and 60 hexameric rings of covalently joined subunits that loop through each other, creating protein chainmail: topologically linked protein catenanes arranged with icosahedral symmetry. Catenanes have not been previously observed in proteins and provide a stabilization mechanism for the very thin HK97 capsid.

Increased resolution data from a large unit cell crystal collected at a third-generation synchrotron X-ray source.

A third-generation synchrotron source was used to collect data from crystals with a very large unit cell. There was an increase in the effective resolution of the data from 5 to 3.5 A. Data were collected on crystals of HK97 mature empty capsids, space group P2(1), with unit-cell parameters a = 580, b = 625, c = 790 A, beta = 90.0 degrees. Like other crystals with very large unit-cell dimensions, the intensity falls off rapidly as a function of resolution, with a precipitous drop beginning at 9 A resolution. Synchrotron data from these crystals were previously observed at the CHESS F1 beamline to about 3.5 A resolution, but the intensities could not be accurately measured beyond 5 A. In experiments conducted at the Advanced Photon Source (APS) beamline 14-BM-C, data from identical crystals could be processed to a resolution of 3.5 A. The lifetime of the crystals in the beam was increased from one exposure per crystal volume to between four and eight exposures per crystal volume. More than 500 images were collected in two trips, allowing the extension of the resolution of the data set and the structure determination to 3.5 A resolution. Factors in the increased resolution may include X-ray flux, beamline geometry and low background scatter. These results suggest that other crystals with large unit cells and pronounced intensity falloff with resolution may benefit from the use of this or similar beamlines.

Maturation dynamics of a viral capsid: visualization of transitional intermediate states.

Typical of DNA bacteriophages and herpesviruses, HK97 assembles in two stages: polymerization and maturation. First, capsid protein polymerizes into closed shells; then, these precursors mature into larger, stabler particles. Maturation is initiated by proteolysis, producing a metastable particle primed for expansion-the major structural transition. We induced expansion in vitro by acidic pH and monitored the resulting changes by time-resolved X-ray diffraction and cryo-electron microscopy. The transition, which is not synchronized over the population, proceeds in a series of stochastically triggered subtransitions. Three distinct intermediates were identified, which are comparable to transitional states in protein folding. The intermediates' structures reveal the molecular events occurring during expansion. Integrated into a movie (see Dynamic Visualization below), they show capsid maturation as a dynamic process.

Virus assembly: Imaging a molecular machine.

A recent structural study of a double-stranded DNA bacteriophage has provided remarkable new insights into the assembly of a complex virus particle and ushers in a new era in the imaging of non-icosahedral viruses.

Crystallographic analysis of the dsDNA bacteriophage HK97 mature empty capsid.

HK97 is a member of the Siphovirus family of dsDNA bacteriophages. It is similar in architecture to bacteriophage lambda, the type member of this family, with an icosahedral capsid of triangulation number T = 7. No high-resolution structural information is available for the dsDNA phages, and HK97 is the only dsDNA bacteriophage capsid to produce crystals which diffract X-rays. At 650 A in diameter, the large size of the particle and resultant large unit cell create crystallographic challenges. The empty Head II (mature) particles were expressed in Escherichia coli and assembled in vitro, but they have the same morphology as the mature HK97 capsid. Previously reported Head II crystals diffracting to 3.5 A resolution are examined here in detail. Although the cell dimensions suggest an orthorhombic lattice, further analysis demonstrated that the space group was monoclinic. This has been confirmed by the present study. Images were recorded on the F1 beamline at CHESS and they were processed and scaled, resulting in a data set with a cumulative completeness of 65% and a scaling R factor of 7.7% to 7 A. The cell dimensions after post-refinement were a = 580, b = 626, c = 788 A, beta = 90.0 degrees. From the particle dimensions determined by cryo-electron microscopy (cryo-EM), there were determined to be two particles per unit cell. Systematic absences of even reflections along the 0k0 lattice line indicate that the space group is P21. The rotation function was used to determine the orientation of the particles in the unit cell and to confirm the space group. An icosahedral twofold axis is approximately, but not exactly, aligned with the crystallographic screw (b) axis. An icosahedral twofold axis orthogonal to the one approximately parallel to the b axis, is rotated 18 degrees away from the a axis. The centers of the two particles must be positioned close to the minimum-energy packing arrangement for spheres, which places one particle at ((1/4), 0, (1/4)) and the other particle at ((3/4), (1/2), (3/4)). The particle position and orientation were confirmed by calculating a Patterson function. The particles interact closely along icosahedral threefold axes, which occurs both along the crystallographic a axis and along the b axis. The particle dimensions derived from this packing arrangement agree well with those determined by cryo-EM and image reconstruction. The cryo-EM reconstruction will be used as a model to initiate phase determination; structure determination at 7 A is under way.

Crystallization and preliminary X-ray analysis of the dsDNA bacteriophage HK97 mature empty capsid.

HK97 is a temperate dsDNA bacteriophage of Escherichia coli that is structurally similar to phage lambda, with an icosahedral head of triangulation (7) number 7. Although the capsids of several large dsDNA phages have been studied extensively using a variety of biophysical approaches, no high-resolution structure is available. We have grown crystals of mature but empty bacteriophage HK97 capsids that diffract to at least 3.5 A using synchrotron radiation. The HK97 Head II crystals are the first capsid crystals from a dsDNA bacteriophage that diffract X-rays to high resolution. A capsid precursor (prohead) was made in vivo by expressing capsid proteins in E. coli. This prohead was purified, converted to Head II in vitro, and used to grow crystals. The empty Head II has the same form as the mature HK97 capsid, but without DNA. The crystals were grown in a mixture of ammonium sulfate and PEG 8000, directly in an X-ray capillary to minimize crystal handling. The unit cell is monoclinic, with dimensions a = 580 A, b = 625 A, c = 790 A, beta = 90.0 degrees and two particles per unit cell.

Macromolecular assembly: chainmail stabilization of a viral capsid.

The subunits that make up the capsid of a double-stranded DNA phage have been found to be arranged as covalently bonded, interlinked pentamer and hexamer rings. This remarkable 'chainmail' arrangement raises interesting new questions about macromolecular assembly.

Imaging RNA and dynamic protein segments with low-resolution virus crystallography: experimental design, data processing and implications of electron density maps.

Single crystal diffraction data were collected from virus crystals in the resolution range of 270 to 14 A using a synchrotron X-ray source and a small-angle scattering instrument adapted for single crystal measurements. Reflections were measured from single crystals of the capsid of the double-stranded DNA bacteriophage HK97 and synthetic Flock House virus-like particles (sFHV). The quality of the low-resolution measurements was confirmed by excellent scaling statistics for both data sets. The sFHV amplitudes between 270 and 90 A resolution were closely similar to independently measured solution scattering data, and to data calculated from the Fourier transform of a uniform density sphere of 315 A diameter. A rotation function computed with the sFHV data between 70 and 20 A resolution was readily interpretable. A uniform density sphere model was used to compute phases for measured amplitudes between 270 and 68 A resolution. The calculated phases were refined and extended to 14 A resolution with real space averaging employing an external mask shape defined by the high-resolution structure. The resulting electron density map displayed regions interpretable as loosely ordered RNA that connected ordered RNA segments seen in a published 3.0 A resolution map. The published high-resolution electron density map lacked data inside 15 A resolution and the interior of the particle in that map appeared hollow. Difference electron density maps corresponding to bulk RNA were computed by subtracting the contribution of the protein shell, based on the available high-resolution atomic model, from either the cryo-electron microscopy density or the low-resolution X-ray density. Features of the RNA were closely similar in the cryo-electron microscopy and X-ray maps, demonstrating the consistency of the two imaging methods. Electron density maps computed at 14 and 6 A resolution with the X-ray amplitudes showed that RNA contributed little to the scattering beyond 14 A resolution.

The structure of cucumber mosaic virus: cryoelectron microscopy, X-ray crystallography, and sequence analysis.

The three-dimensional structure of cucumber mosaic virus (CMV) was analyzed at 23 A resolution by cryoelectron microscopy and image reconstruction, demonstrating structural similarity to cowpea chlorotic mottle virus (CCMV), another member of the Bromoviridae family. The CMV structure was determined at 8 A resolution by X-ray crystallography with phases determined by single isomorphous replacement and refined by fivefold noncrystallographic symmetry averaging. The X-ray structure agreed with the electron microscopy reconstruction; the electron density is consistent with beta-barrel subunits arranged with T = 3 quasi-symmetry in an orientation similar to that observed in CCMV. Strong density surrounding the icosahedral threefold axes (quasi sixfold axes in the T = 3 particle) between 80 and 100 A from the particle center formed a cylinder of radius 11 A, similar to the density observed in the same region of CCMV. This density corresponds to the beta-annulus of CCMV, which differentiates hexamers from pentamers and determines the formation of the T = 3 particles. The CMV and CCMV amino acid sequences were aligned, providing information (based on the CCMV atomic model) about the probable distribution of residues in the three-dimensional structure of CMV.

Structure determination of minute virus of mice.

The three-dimensional crystal structure of the single-stranded DNA-containing ('full') parvovirus, minute virus of mice (MVM), has been determined to 3.5 A resolution. Both full and empty particles of MVM were crystallized in the monoclinic space group C2 with cell dimensions of a = 448.7, b = 416.7, c = 305.3 A and beta = 95.8 degrees. Diffraction data were collected at the Cornell High Energy Synchrotron Source using an oscillation camera. The crystals have a pseudo higher R32 space group in which the particles are situated at two special positions with 32 point symmetry, separated by (1/2)c in the hexagonal setting. The self-rotation function showed that the particles are rotated with respect to each other by 60 degrees around the pseudo threefold axis. Subsequently, a more detailed analysis of the structure amplitudes demonstrated that the correct space-group symmetry is C2 as given above. Only one of the three twofold axes perpendicular to the threefold axis in the pseudo R32 space group is a 'true' crystallographic twofold axis; the other two are only 'local' non-crystallographic symmetry axes. The known canine parvovirus (CPV) structure was used as a phasing model to initiate real-space electron-density averaging phase improvement. The electron density was easily interpretable and clearly showed the amino-acid differences between MVM and CPV, although the final overall correlation coefficient was only 0.63. The structure of MVM has a large amount of icosahedrally ordered DNA, amounting to 22% of the viral genome, which is significantly more than that found in CPV.

The structure of a neutralized virus: canine parvovirus complexed with neutralizing antibody fragment.

BACKGROUND: Members of the Parvovirus genus cause a variety of diseases in mammals, including humans. One of the major defences against viral infection is the presence of neutralizing antibodies that prevent virus particles from infecting target cells. The mechanism of neutralization is not well understood. We therefore studied the structure of canine parvovirus (CPV) complexed with the Fab fragment of a neutralizing antibody, A3B10, using image reconstruction of electron micrographs of vitrified samples, together with the already known structure of CPV from X-ray crystallographic data. RESULTS: The structure of the complex of CPV with Fab A3B10 has been determined to 23 A resolution. The known CPV atomic structure was subtracted from the electron density of the complex, and the difference map was used to fit the atomic coordinates of a known Fab fragment, HyHEL-5. The long axis of each Fab molecule is oriented in a near radial direction, inclined away from the two-fold axes. The viral epitope consists of 14 amino acid residues found in loops 1, 2 and 3 on the capsid surface, which include previously identified escape mutations. CONCLUSIONS: The mode of Fab binding suggests that the A3B10 neutralizing antibody cannot bind bivalently to the capsid across the two-fold axes, consistent with the observation that whole A3B10 antibody readily precipitates CPV. Since Fab A3B10 can also neutralize the virus, mechanisms of neutralization such as interference with cell attachment, cell entry, or uncoating, must be operative.

Substrate properties of C1 inhibitor Ma (alanine 434----glutamic acid). Genetic and structural evidence suggesting that the P12-region contains critical determinants of serine protease inhibitor/substrate status.

The serine protease inhibitor (serpin) C1 inhibitor inactivates enzymes involved in the regulation of vascular permeability. A patient from the Ma family with the genetic disorder hereditary angioedema inherited a dysfunctional C1 inhibitor allele. Relative to normal plasma, the patients's plasma contained an additional C1 inhibitor immunoreactive band, which comigrated with normal C1 inhibitor cleaved by plasma kallikrein, C1s, or factor XIIa. C1 inhibitor Ma did not react with a monoclonal antibody to a neoepitope that is present in complexed and cleaved normal C1 inhibitor, suggesting conformational differences between cleaved normal C1- inhibitor and cleaved C1 inhibitor Ma. Molecular cloning and sequencing of exon 8 of the C1 inhibitor Ma allele revealed a single C to A mutation, changing alanine 434 to glutamic acid. Ala 434 of C1 inhibitor aligns with the P12 residue of the prototypical serpin alpha 1-antitrypsin. The P12 amino acid of all inhibitory serpins is alanine, and it is present in a highly conserved region on the amino-terminal side of the serpin-reactive center loop. Whereas normal C1 inhibitor expressed by transfected COS-1 cells formed complexes with and was cleaved by kallikrein, fXIIa, and C1s, COS-1-expressed Ala434---Glu C1 inhibitor was cleaved by these enzymes but did not form complexes with them. These results, together with evidence from other studies, suggest that serpin protease inhibitor activity is the result of protein conformational change that occurs when the P12 region of a serpin moves from a surface location, on the reactive site loop of the native molecule, to an internal location within sheet A of the complexed inhibitor.