antibiotics ͉ functional flexibility ͉ ribosome crystallography ͉ peptidyl transferase center ͉ retapamulin

Trigger factor (TF), the first chaperone in eubacteria to encounter the emerging nascent chain, binds to the large ribosomal subunit in the vicinity of the protein exit tunnel opening and forms a sheltered folding space. Here, we present the 3.5-Å crystal structure of the physiological complex of the large ribosomal subunit from the eubacterium Deinococcus radiodurans with the N-terminal domain of TF (TFa) from the same organism. For anchoring, TFa exploits a small ribosomal surface area in the vicinity of proteins L23 and L29, by using its ''signature motif'' as well as additional structural elements. The molecular details of TFa interactions reveal that L23 is essential for the association of TF with the ribosome and may serve as a channel of communication with the nascent chain progressing in the tunnel. L29 appears to induce a conformational change in TFa, which results in the exposure of TFa hydrophobic patches to the opening of the ribosomal exit tunnel, thus increasing its affinity for hydrophobic segments of the emerging nascent polypeptide. This observation implies that, in addition to creating a protected folding space for the emerging nascent chain, TF association with the ribosome prevents aggregation by providing a competing hydrophobic environment and may be critical for attaining the functional conformation necessary for chaperone activity.protein folding ͉ nascent chain ͉ ribosomal exit tunnel ͉ ribosomal crystallography ͉ Deinococcus radiodurans

Bacterioferritins, also known as cytochrome b 1 , are oligomeric iron-storage proteins consisting of 24 identical amino acid chains, which form spherical particles consisting of 24 subunits and exhibiting 432 point-group symmetry. They contain one haem b molecule at the interface between two subunits and a di-nuclear metal binding center. The X-ray structure of bacterioferritin from Mycobacterium smegmatis (Ms-Bfr) was determined to a resolution of 2.7 Å in the monoclinic space group C2. The asymmetric unit of the crystals contains 12 protein molecules: five dimers and two half-dimers located along the crystallographic twofold axis. Unexpectedly, the di-nuclear metal binding center contains zinc ions instead of the typically observed iron ions in other bacterioferritins.

The crystallization of ribosomal particles is associated with extraordinary challenging demands. This originates mainly from the ribosome's natural tendency to deteriorate and from its multi-conformational heterogeneity, both of which stem from its functional flexibility. To increase the level of homogeneity of ribosomal preparations, systematic searches for conditions yielding populations of fully defined chemical compositions were employed and the variables essential for high functional activity were analyzed and optimized. These include temperature, cell-growth duration and media, the cell-harvesting stage, ribosomal purification and storage. The functional state that is most suitable to yield quality crystals was identified as that of the polysome and it was found that this fraction reproducibly yielded crystals of superior properties.

In comparison to existing structural, biochemical, and therapeutical data, the crystal structures of large ribosomal subunit from the eubacterial pathogen model Deinococcus radiodurans in complex with the 14-membered macrolides erythromycylamine, RU69874, and the 16-membered macrolide josamycin, highlighted the similarities and differences in macrolides binding to the ribosomal tunnel. The three compounds occupy the macrolide binding pocket with their desosamine or mycaminose aminosugar, the C4-C7 edge of the macrolactone ring and the cladinose sugar sharing similar positions and orientations, although the latter, known to be unnecessary for antibiotic activity, displays fewer contacts. The macrolactone ring displays altogether few contacts with the ribosome and can, therefore, tilt in order to optimize its interaction with the 23S rRNA. In addition to their contacts with nucleotides of domain V of the 23S RNA, erythromycylamine and RU69874 interact with domain II nucleotide U790, and RU69874 also reaches van der Waals distance from A752, in a fashion similar to that observed for the ketolides telithromycin and cethromycin. The variability in the sequences and consequently the diversity of the conformations of macrolide binding pockets in various bacterial species can explain the drug's altered level of effectiveness on different organisms and is thus an important factor in structure-based drug design.