New insight into flavivirus maturation from structure/function studies of the yellow fever virus envelope protein complex.

IMPORTANCE: All enveloped viruses enter cells by fusing their envelope with a target cell membrane while avoiding premature fusion with membranes of the producer cell-the latter being particularly important for viruses that bud at internal membranes. Flaviviruses bud in the endoplasmic reticulum, are transported through the TGN to reach the external milieu, and enter other cells via receptor-mediated endocytosis. The trigger for membrane fusion is the acidic environment of early endosomes, which has a similar pH to the TGN of the producer cell. The viral particles therefore become activated to react to mildly acidic pH only after their release into the neutral pH extracellular environment. Our study shows that for yellow fever virus (YFV), the mechanism of activation involves actively knocking out the fusion brake (protein pr) through a localized conformational change of the envelope protein upon exposure to the neutral pH external environment. Our study has important implications for understanding the molecular mechanism of flavivirus fusion activation in general and points to an alternative way of interfering with this process as an antiviral treatment.

Structural effects of monovalent anions on polymorphic lysozyme crystals.

Understanding direct salt effects on protein crystal polymorphism is addressed by comparing different crystal forms (triclinic, monoclinic, tetragonal and orthorhombic) for hen, turkey, bob white quail and human lysozymes. Four new structures of hen egg-white lysozyme are reported: crystals grown in the presence of NapTS diffracted to 1.85 A, of NaI to 1.6 A, of NaNO(3) to 1.45 A and of KSCN to 1.63 A. These new structures are compared with previously published structures in order to draw a mapping of the surface of different lysozymes interacting with monovalent anions, such as nitrate, chloride, iodide, bromide and thiocyanate. An analysis of the structural sites of these anions in the various lysozyme structures is presented. This study shows common anion sites whatever the crystal form and the chemical nature of anions, while others seem specific to a given geometry and a particular charge environment induced by the crystal packing.

Antibody inhibition of the transcriptase activity of the rotavirus DLP: a structural view.

On entering the host cell the rotavirus virion loses its outer shell to become a double-layered particle (DLP). The DLP then transcribes the 11 segments of its dsRNA genome using its own transcriptase complex, and the mature mRNA emerges along the 5-fold axis. In order to better understand the transcription mechanism and the role of VP6 in transcription we have studied three monoclonal antibodies against VP6: RV-238 which inhibits the transcriptase activity of the DLP; and RV-133 and RV-138 which have no effect on transcription. The structures obtained by cryo-electron microscopy of the DLP/Fab complexes and by X-ray crystallography of the VP6 trimer and the VP6/Fab-238 complex have been combined to give pseudo-atomic structures. Steric hindrance between the Fabs results in limited Fab occupancy. In particular, there are on average only three of a possible five Fabs-238 which point towards the 5-fold axis. Thus, Fabs-238 are not in a position to block the exiting mRNA, nor is there any visible conformational change in VP6 on antibody binding at a resolution of 23 A. However, the epitope of the inhibiting antibody involves two VP6 monomers, whereas, those of the non-inhibiting antibodies have an epitope on only one VP6. Thus, the inhibition of transcription may be a result of inhibition of a possible change in the VP6 conformation associated with the transcription of mRNA.

High-resolution structure (1.33 A) of a HEW lysozyme tetragonal crystal grown in the APCF apparatus. Data and structural comparison with a crystal grown under microgravity from SpaceHab-01 mission.

Crystals of tetragonal hen egg-white lysozyme were grown using Advanced Protein Crystallization Facility (APCF) apparatus under a microgravity environment (SpaceHab-01 mission) and ground control conditions. Crystals were grown from NaCl as a crystallizing agent at pH 4.3. The X-ray diffraction patterns of the best diffracting ground- and space-grown crystals were recorded using synchrotron radiation and an image plate on the W32 beamline at LURE. Both ground- and space-grown crystals showed nearly equivalent maximum resolution of 1.3-1.4 A. Refinements were carried out with the program X-PLOR with final R values of 18.45 and 18.27% for structures from ground- and space- grown crystals, respectively. The two structures are nearly identical with the root-mean-square difference on all protein atoms being 0.13 A. Some residues of the two refined structures show multiple alternative conformations. Two ions were localized into the electron-density maps of the two structures: one chloride ion at the interface between two symmetry-related molecules and one sodium ion stabilizing the loop Ser60-Leu75. The sodium ion is surrounded by six ligands which form a bipyramid around it at distances of 2.2-2.6 A.

Crystallization and preliminary X-ray diffraction studies of a protective cloned 28 kDa glutathione S-transferase from Schistosoma mansoni.

Crystals of the recombinant 28 kDa glutathione S-transferase from Schistosoma mansoni have been obtained by the hanging-drop method of vapor diffusion from ammonium sulfate solutions. The successful crystallization of this enzyme required the presence of a reducing agent and S-hexylglutathione. The crystals belong to the cubic space group P4(1)32 (or P4(3)32), with unit cell dimensions a = 122.6 A and contain one molecule in the asymmetric unit. The crystals diffract to at least 2.8 A resolution and are suitable for X-ray crystallographic structure analysis.

FORME: an interactive package for protein backbone deformation.

The FORME package presented herein is designed for modeling purposes: It allows interactive deformation of the protein backbone. General formalism on transformations is introduced and the operators of stretching inside an "acceptance area" and stretching with end-block invariance (i.e., governed by a translational moving) are described. A discussion is presented on the choice of strategy to achieve an interactive deformation tool. Perspectives about complex transformations are presented.

Symmetry and crystallography: new facilities in the graphic software MANOSK.

The new routine SYMCRY of the graphics program MANOSK is described in this paper. It is designed to analyze interactions between molecular structures related by crystalline symmetry. The symetric objects can be described in the same referential, to be manipulated as an entity, or in a referential of their own, to undergo correlative real-time movements (via the dials), given the symmetry constraints. Crystal packing can be observed, and any command of the main software MANOSK is available for the symmetric objects, including storage of the coordinates of symmetrics in the final orientation.

Crystallization and preliminary X-ray study of porcine trypsin, free and complexed with Ecballium elaterium trypsin inhibitor, a member of the squash inhibitors family.

Porcine trypsin has been crystallized either free or complexed with synthetic Ecballium elaterium trypsin inhibitor II, a 28-residue peptide with three disulfide bridges. The crystals diffract beyond 2.0 A. Crystals are orthorhombic, space group P2(1)2(1)2(1), with cell dimensions a = 77.32 A, b = 53.81 A, c = 46.91 A, for the free trypsin, and a = 62.25 A, b = 62.27 A, c = 84.66 A for the complex with E. elaterium trypsin inhibitor II.

Crystal structure of a cAMP-independent form of catabolite gene activator protein with adenosine substituted in one of two cAMP-binding sites.

Catabolite gene activator protein (CAP) in the presence of cAMP stimulates transcription from several operons in Escherichia coli. A cAMP-independent variant, in which Ala-144 is replaced by Thr (CAP91), is activated by analogues of cAMP, such as adenosine, which do not activate the wild-type CAP. In order to test the effect of adenosine on the structure, a crystal of CAP91 grown as a complex with cAMP was soaked in a solution of 10 mM adenosine, and X-ray diffraction data were measured to 3.5-A resolution. The difference Fourier map calculated with phases from the CAP91 structure showed significant negative density at the position of the phosphate of cAMP bound in one subunit of the CAP91 dimer. Adenosine was preferentially substituted for cAMP in the subunit in the "closed" conformation, while the cAMP-binding site of the "open" subunit was apparently still occupied by cAMP. The structure was refined by restrained least-squares methods to an R factor of 20.2%. Adenosine is not bound in exactly the same position as cAMP; instead, the 5'-OH of adenosine is in a new position that allows formation of two hydrogen bonds with Ser-83, replacing two of the three interactions of the phosphate of cAMP with Arg-82 and Ser-83.

Refinement of the C222(1) crystal form of oxidized uteroglobin at 1.34 A resolution.

The structure of uteroglobin, a progesterone binding protein from rabbit uterine fluid, was determined and refined at 1.34 A resolution to a conventional R-factor of 0.229. The accuracy of the co-ordinates is estimated to be 0.15 A. The isotropic temperature factor of individual atoms was refined and its average value is 11.9 A2 for the 548 non-hydrogen atoms of the protein monomer. A total of 83 water molecules was located in difference electron density maps and refined, first using a constant occupancy factor of 1 and then variable occupancy, the final (Q) being 0.63. The mean temperature factor of the water oxygen atoms is 26.4 A2. Uteroglobin is a dimer and its secondary structure consists of four alpha-helices per monomer that align in an anti-parallel fashion. There is one beta-turn between helix 2 and helix 3 (Lys26 to Glu29); 76% of the residues are part of the alpha-helices. In the core of the dimeric protein molecule, between the two monomers that are held together by two disulfide bridges, we have observed a closed cavity. Its length is 15.6 A and its width is 9 A; 14 water molecules could be positioned inside. In the "bottom" part of the protein, near the C terminus, we have observed a smaller cavity, occupied by two water molecules. The calculation of the molecular surface revealed four surface pockets whose possible functional implications are discussed below.