Ebola virus (EBOV) and Marburg virus are members of the family Filoviridae. Both are highly pathogenic and cause hemorrhagic fever, lethal in 90% of infected people. There is fear that the viruses can be used as bioterrorism agents. There are no approved vaccines, and intense effort is underway to discover drugs targeting these viruses. The EBOV genome encodes seven proteins, two of which have no known structures: RNA polymerase (L) and nucleoprotein (NP). NP is essential for packaging viral genomic RNA into the nucleocapsid. Other viruses also contain nucleoproteins, but only the Ebola and Marburg NP proteins contain two distinct domains. The C-terminal domain (Ct; ~100 residues) has no homologues; it acts as a hub for protein-protein interactions important for the assembly of the nucleocapsid and for the interaction with the VP40 matrix protein, embedded in the viral membrane. We obtained three distinct crystal forms of the Ct domain of NP from EBOV, and solved the structures using anomalous scattering from Se, and Molecular Replacement. High-quality NMR data were also collected. The models were refined at 1.6-2.0 Å resolution to R factors ~20%. The protein has a novel fold, with topology distantly related to the β-grasp fold. In spite of its small size, the Ct domain shows high melting temperature of ~60°C. Our efforts focus on the identification of how the C-terminal domain of NP binds to its partners. As part of an effort towards anti-filovirus drug discovery, proteins NP, VP24, VP35 and VP40 are being targeted for small molecule inhibition using a yeast-based phenotypic assay. Each protein, when expressed in budding yeast, produces a slow-growth phenotype. Chemical suppressors of the slow-growth phenotype will be identified and used in viral growth assays to confirm their antiviral activity. The structure of NP will be used to complement small molecule screening methods.

This study examined the role of guanine exchange factors (GEFs) and their specific domains involved in mediating Ca2+ sensitization. Here, we identify two GEFs, LARG and PDZ-RhoGEF (PRG) in rabbit pulmonary artery smooth muscle (PA), which have been shown to interact with Gα12/13 family members via their regulators of G protein signaling (RGSL) domain. We determined whether these RhoGEFs and which specific domains were involved in Ca2+ sensitizing actions of U46619, a thromboxane A2 analogue. U-46619 was a strong mediator of Ca2+ sensitization in PA muscle strips. We found that the DH/PH domains of PRG and LARG significantly increased Ca2+ sensitization (1). The RGSL domains of PRG and LARG, implicated in GEF/RhoA regulation, function as DN inhibitors of this pathway, but surprisingly, no significant effect on U-46619-induced sensitization occurred. However, PDZ domains from LARG and PRG caused significant inhibition of U-46619-induced sensitization. In control experiments, two PDZ domain fragments of Syntenin, a non-RhoGEF PDZ domain containing protein showed no inhibition of sensitization. Preliminary studies show co-localization and interaction of the TP and the PDZ domain of PRG and endogenous PRG in cultured cells. Together, these data suggest that LARG and PRG are involved in signaling via stimulation of GPCR leading to RhoA-dependent activation of Ca2+ sensitization in SM. These interactions suggest a mechanistic role for the TP/GEF/RhoA pathway leading to Ca2+ sensitization in smooth muscle. Supported by NIH P01 HL48807.

The crystal structure of the common house mite (Dermatophagoides sp.) Der p 2 allergen was solved at 2.15Å resolution using the MAD phasing technique, and refined to an R-factor of 0.209. The refined atomic model, which reveals an immunoglobulin-like tertiary fold, differs in important ways from the previously described NMR structure, because the two β-sheets are significantly further apart and create an internal cavity, which is occupied by a hydrophobic ligand. This interaction is structurally reminiscent of the binding of a prenyl group by a regulatory protein, the Rho guanine nucleotide exchange inhibitor. The crystal structure suggests that binding of non-polar molecules may be essential to the physiological function of the Der p 2 protein.

We describe the construction of expression vectors based on three of the most frequently used gene fusion affinity tags [glutathione S-transferase (GST), maltose binding protein (MBP), and the His(6) peptide]. The polylinkers of pGEX4T1, pMal-c2, and a pET vector were replaced with the polylinker isolated from the baculovirus expression plasmid pFastBac. Once appropriate restriction sites have been introduced into a gene, it can be fused to all three affinity tags with little effort, allowing expression-screening experiments to be performed efficiently. We discuss the development and use of these vectors with respect to overcoming purification problems encountered for the RhoA GDP/GTP nucleotide dissociation inhibitor (RhoGDI) and their advantages over commercially available expression vectors, (C) 1999 Academic Press.

The assembly of the insulin hexamer brings the six B13 glutamate side-chains at the centre into close proximity. Their mutual repulsion is unfavourable and zinc co-ordination to B10 histidine is necessary to stabilize the well known zinc-containing hexamers. Since B13 is always a carboxylic acid in all known sequences of hexamer forming insulins, it is likely to be important in the hormone's biology. The mutation of B13 Glu → Gln leads to a stable zinc-free hexamer with somewhat reduced potency. The structures of the zinc-free B13 Gln hexamer and the 2Zn B13 insulin hexamer have been determined by X-ray analysis and refined with 2.5 Å and 2.0 Å diffraction data, respectively.

A crystal structure of a totally inactive insulin molecule has been determined. For this insulin molecule, the first without detectable activity to be characterized, the A and B-chains are linked by a peptide bond between A1 Gly and B29 Lys. The molecule has retained all its normal self-association properties and it can also accommodate the two different conformations designated T and R, as seen in 4Zn native pig insulin crystals. The hexamers of the crosslinked insulin molecule were crystallized using the 4Zn insulin recipe of Schlichtkrull. The structure has been crystallographically refined with data extending to 2 Å using restrained least-square methods. Comparison of the B29-A1 peptide crosslink insulin and the 4Zn native insulin reveals close structural similarities with the native dimer. The analysis of the structure confirms the earlier hypothesis that insulin structures in crystals are not in an active conformation and that a separation of N-terminal A-chain and C-terminal B-chain is required for interaction with the insulin receptor.

We have obtained well-ordered single crystals of the flavoenzyme trypanothione reductase from Crithidia fasciculata. The crystals are tetragonal rods with unit cell dimensions a = 128·6 Å, c = 92·5 Å. The diffraction pattern corresponds to a primitive lattice, Laue class 4/m. Diffraction to better than 2·4 Å has been recorded at the Daresbury Synchrotron. The accurate elucidation of the three-dimensional structure of this enzyme is required to support the rational design of compounds active against a variety of tropical diseases caused by trypanosomal parasites.

X-ray diffraction analysis (at 2.1-A resolution) of an acid alpha-amylase from Aspergillus niger allowed a detailed description of the stereochemistry of the calcium-binding sites. The primary site (which is essential in maintaining proper folding around the active site) contains a tightly bound Ca2+ with an unusually high number of eight ligands (O delta 1 and O delta 2 of Asp175, O delta of Asn121, main-chain carbonyl oxygens of Glu162 and Glu210, and three water molecules). A secondary binding site was identified at the bottom of the substrate binding cleft; it involves the residues presumed to play a catalytic role (Asp206 and Glu230). This explains the inhibitory effect of calcium observed at higher concentrations. Neutral Aspergillus oryzae (TAKA) alpha-amylase was also refined in a new crystal at 2.1-A resolution. The structure of this homologous (over 80%) enzyme and additional kinetic studies support all the structural conclusions regarding both calcium-binding sites.

The structures of carbonmonoxyhaemoglobins A and Cowtown (His146 beta----Leu) have been refined at 2.2 A (1 A = 0.1 nm) and 2.3 A resolution, respectively. The least squares fit to the Fe-C-O line makes an angle to the haem normal of about 6 degrees. The Fe-C-O group is bent from linearity by about 7 degrees. The porphyrins in the CO liganded haemoglobins are ruffled. This deformation of the haem and the distortion of the Fe-C-O group may explain the low CO affinity of haemoglobin. The electron density for the C-terminal residues is low but sufficient to distinguish the histidyl and leucyl residues clearly. The similarity between these two structures, apart from 146 beta, means that the reduced alkaline Bohr effect is due solely to the replacement of histidine by a leucine.