| Mitochondrial mRNA editing in trypanosomatid parasites involves several multiprotein assemblies including three very similar complexes which contain the key enzymatic editing activities and sediment at ∼20S on glycerol gradients. These ∼20S editosomes have a common set of twelve proteins, including enzymes for uridylyl (U) removal and addition, two RNA ligases, two proteins with RNase III-like domains, and six proteins with predicted oligonucleotide binding (OB) folds. In addition, each of the three distinct ∼20S editosomes contains a different RNase III-type endonuclease, one of three related proteins and, in one case, an additional exonuclease.;A protein-protein interaction map was obtained through a combination of yeast two-hybrid analysis and subcomplex reconstitution with recombinant protein. This map interlinks ten of the proteins and in several cases localizes the protein region mediating the interaction, which often includes the predicted OB-fold domain. The results indicate that the OB-fold proteins form an extensive protein-protein interaction network that connects the two trimeric subcomplexes that catalyze U removal or addition and RNA ligation.;KREPA6 performs a central role in maintaining the integrity of the editosome core and also binds to ssRNA. The use of llama VHH domains accelerated crystal growth of KREPA6 from Trypanosoma brucei dramatically. All three resultant structures are heterotetramers with a KREPA6 dimer in the center, and one VHH domain bound to each KREPA6 subunit.;Two of the resultant heterotetramers use primarily CDR2 and framework 3 residues to interact with a KREPA6 subunit to form a parallel pair of beta strands---a mode of interaction not seen before in VHH domain-antigen complexes. The third type of VHH domain binds in a totally different manner to KREPA6.;Intriguingly, while KREPA6 forms tetramers in solution adding either one of the three VHH domains results in the formation of a heterotetramer in solution, in perfect agreement with the crystal structures. Biochemical solution studies indicate that the C-terminal tail of KREPA6 is involved in the dimerization of KREPA6 dimers to form tetramers. The implications of these crystallographic and solution studies for possible modes of interaction of KREPA6's many binding partners with KREPA6 in the center of the editosome are discussed. |
