Survey of large protein complexes in D. vulgaris reveals great structural diversity

Abstract

An unbiased survey has been made of the stable, most abundant multi-protein complexes in Desulfovibrio vulgaris Hildenborough (DvH) that are larger than Mr approximately 400 k. The quaternary structures for 8 of the 16 complexes purified during this work were determined by single-particle reconstruction of negatively stained specimens, a success rate approximately 10 times greater than that of previous "proteomic" screens. In addition, the subunit compositions and stoichiometries of the remaining complexes were determined by biochemical methods. Our data show that the structures of only two of these large complexes, out of the 13 in this set that have recognizable functions, can be modeled with confidence based on the structures of known homologs. These results indicate that there is significantly greater variability in the way that homologous prokaryotic macromolecular complexes are assembled than has generally been appreciated. As a consequence, we suggest that relying solely on previously determined quaternary structures for homologous proteins may not be sufficient to properly understand their role in another cell of interest.

Fig. 1.

Gallery of three-dimensional reconstructions obtained by single-particle electron microscopy for eight different, large macromolecular complexes isolated from Desulfovibrio vulgaris Hildenborough. Whenever pseudoatomic-resolution models could be created by docking known atomic structures for homologous proteins (or homology models reflecting the DvH sequence), these are shown in color, embedded within the gray isosurfaces for the reconstructed volumes. Homology models were created by using the MODBASE (27) server located at http://modbase.compbio.ucsf.edu/modbase-cgi/index.cgi. In the case of the 70s ribosome, however, united-atom representations of X-ray atomic model structures were used. (A) 70S ribosome, Mr ≈ 3 × 10⁶. The 30S subunit from a X-ray atomic structure (PDB entry 1GIX) is shown in green whereas the 50S subunit (PDB entry 1GIY) is shown in cyan. There is extra density in the EM map at the E site for binding of tRNA, shown in red. (B) Octomeric complex, from (13), of pyruvate:ferredoxin oxidoreductase, Mr ≈ 1 × 10⁶. The top half of the homology model is represented in orchid ribbons and the bottom in turquoise. (C) Icosahedral complex of lumazine synthase (beta subunits of riboflavin synthase), Mr ≈ 1 × 10⁶. One of the pentameric subunits is shown in blue ribbon whereas all others are shown in turquoise. (D) GroEL double ring, Mr ≈ 800 k. Two ribbon diagrams of the pseudoatomic homology model are shown in purple (bottom ring) and magenta (top ring) respectively, whereas all others are shown in pink. (E) Dimer of RNA polymerase, including the transcription elongation factor NusA, Mr ≈ 800 k. Two monomers of the heteromeric core enzyme (PDB entry 2PPB) are shown as pink and green ribbons, respectively. (F) Homo-octomer of putative protein (DVU0671), Mr ≈ 470 k. (G) Homo-octomer of inosine-5′-monophosphate dehydrogenase, Mr ≈ 416 k. The tetramer at the bottom is shown as light green ribbons and the one at the top as light blue ribbons, with a single monomer shown in magenta. (H) Dimer of phosphoenolpyruvate synthase. Mr ≈ 265 k. Although an X-ray crystal structure is available for an homologous protein from Neisseria meningitides (PDB entry 2OLS), the molecular weight of that protein is only ≈2/3 that of the DvH enzyme. As a result, we have not attempted to dock this X-ray structure into the EM map.

Fig. 2.

Comparison of the two types of icosahedral complexes of lumazine synthase (riboflavin synthase beta subunit) formed by the proteins from Aquifex aeolicus (A and C) and from D. vulgaris Hildenborough (B and D), respectively. Note that the vertices of the pentameric subunits of the DvH enzyme meet at the icosahedral threefold axis, thereby resulting in an icosahedral shell with a larger diameter than that produced when the pentamers of the A. aeolicus enzyme interact in a more edge-to-edge fashion. The positions and directions of some of the fivefold axes are indicated with red lines to facilitate the comparison of the two structures. (A and C) Transparent isosurface representations of the X-ray crystal structure of the enzyme from A. aeolicus, computed at the same resolution as that estimated for the structure obtained by electron microscopy for the enzyme from DvH, are shown looking down both the fivefold axis (A) and down the threefold axis (C). A ribbon diagram of the atomic model of the enzyme is shown embedded within the low-resolution isosurface. (B and D) Transparent isosurface representations of the 3D reconstruction of the DvH enzyme obtained by electron microscopy are shown looking down both the fivefold axis (B) and down the threefold axis (D). The ribbon diagram of a homology model of the DvH enzyme shown in this panel was rotated by ≈30° about the icosahedral fivefold axis to produce a good manual fit within the EM density map. The homology model was created by using the MODBASE (27) server located at http://modbase.compbio.ucsf.edu/modbase-cgi/index.cgi.

Fig. 3.

Electron micrograph of a putative carbohydrate phosphorylase complex isolated from DvH and negatively stained with uranyl acetate. The basic ring-shaped nature of this protein complex is quite apparent, thereby establishing that the subunit stoichiometry and quaternary structure of this complex is completely unlike that of any known member of the carbohydrate phosphorylase family. Beyond that, however, many of the rings are broken open or deformed in other ways. It is difficult to know if this structural heterogeneity just reflects a native flexibility and polymorphism of this protein complex or whether the particles were damaged either during purification or during EM sample preparation. In addition, even a subset of the most circular of the intact rings appears to show heterogeneity in particle diameter. These factors, together with the highly preferred orientation adopted by these particles, have made it impractical to obtain a trustworthy 3D reconstruction of this protein complex.