Structures of Ebola and Reston Virus VP35 Oligomerization Domains and Comparative Biophysical Characterization in All Ebolavirus Species.

The multifunctional virion protein 35 (VP35) of ebolaviruses is a critical determinant of virulence and pathogenesis indispensable for viral replication and host innate immune evasion. Essential for VP35 function is homo-oligomerization via a coiled-coil motif. Here we report crystal structures of VP35 oligomerization domains from the prototypic Ebola virus (EBOV) and the non-pathogenic Reston virus (RESTV), together with a comparative biophysical characterization of the domains from all known species of the Ebolavirus genus. EBOV and RESTV VP35 oligomerization domains form bipartite parallel helix bundles with a canonical coiled coil in the N-terminal half and increased plasticity in the highly conserved C-terminal half. The domain assembles into trimers and tetramers in EBOV, whereas it exclusively forms tetramers in all other ebolavirus species. Substitution of coiled-coil leucine residues critical for immune antagonism leads to aberrant oligomerization. A conserved arginine involved in inter-chain salt bridges stabilizes the VP35 oligomerization domain and modulates between coiled-coil oligomeric states.

Lipoprotein-like particles in a prokaryote: quinone droplets of Thermoplasma acidophilum.

Cytosolic, globular droplets with an average diameter of 50 nm were observed in vitrified Thermoplasma acidophilum cells by means of cryo-electron tomography. These droplets were isolated by column chromatography and immunoprecipitation protein purification methods. Subsequent chemical and biochemical analyses identified lipid and protein components, respectively. Two major lipid components, comigrating menaquinones at the solvent front and the slower migrating Thermoplasma polar lipid U4, were detected by TLC experiments. The major protein component was identified as the 153 amino acid long Ta0547 vitellogenin-N domain protein. This domain has been found so far exclusively in large lipid transport proteins of vertebrates and non-vertebrates. Blast protein database homology searches with Ta0547 did not return any eukaryal hits; homologous sequences were found only in thermo-acidophilic archaeons. However, a profile-sequence domain search performed with the vitellogenin-N domain (PF01347) hmm-profile against the T. acidophilum proteome returned Ta0547 as hit. Electron microscopy appearance of isolated droplets resembled to lipoprotein particles. However, no (tetraether) lipid layer could be detected on the droplets surface, rather hydrophobic compounds of the electron dense lumen were surrounded by a denser discontinuous protein boundary. Based on described features, these particles qualify for a novel lipoprotein particle category, what we nominated Thermoplasma Quinone Droplet.

Peptidyl prolyl cis/trans-isomerases: comparative reactivities of cyclophilins, FK506-binding proteins, and parvulins with fluorinated oligopeptide and protein substrates.

Peptidyl prolyl cis/trans-isomerases catalyze the cis-trans isomerization of prolyl bonds in oligopeptides and various folding states of proteins. The proline residue in PPIase substrates at the P1' subsite, which follows the isomerizing peptide bond, appears to be the common recognition element for all subfamilies of this enzyme class. The molecular principles that govern substrate specificity at the P1' subsite were analyzed using 4-fluoroproline-containing tetrapeptide 4-nitroanilides and barstar Cys40Ala/Cys82Ala/Pro27Ala/Pro48-->4-fluoroproline quadruple variants. Generally, PPIase catalysis demonstrated stereospecificity for monofluoro substitutions at the 4-position of the pyrrolidine ring. However, the replacement of hydrogens with fluoro atoms did not impair productive interactions for the majority of PPIase-substrate complexes. Comparison of specificity constants for oligopeptide and protein substrates revealed striking differences in the 4-fluoroproline substituent effects between members of the PPIase families. Introduction of 4(R)-fluoroproline resulted in an oligopeptide substrate completely resistant to catalytic effects of FKBP-like PPIases. By contrast, the 4(R)-fluoroproline barstar variant demonstrated only slightly reduced or even better catalytic susceptibility when compared to the parent barstar Cys40Ala/Cys82Ala/Pro27Ala/Pro48 substrate. On the other hand, Suc-Ala-Ser-4(S)-FPro-Phe-pNA exhibits a discriminating specificity toward the prototypic parvulin, the Escherichia coli Par10. The E. coli trigger factor, in the extreme, catalyzes Cys40Ala/Cys82Ala/Pro27Ala/4-F(2)Pro48 with a more than 20-fold higher efficiency when compared to the proline-containing congener. These findings support the combined subsite concept for PPIase catalysis in which the positioning of a substrate in the active cleft must activate a still unknown number of remote subsites in the transition state of the reaction. The number of critical subsites was shown to vary between the PPIase families.