Crystal structure of a heterodimer of editosome interaction proteins in complex with two copies of a cross-reacting nanobody

Abstract

The parasite Trypanosoma brucei, the causative agent of sleeping sickness across sub-Saharan Africa, depends on a remarkable U-insertion/deletion RNA editing process in its mitochondrion. A approximately 20 S multi-protein complex, called the editosome, is an essential machinery for editing pre-mRNA molecules encoding the majority of mitochondrial proteins. Editosomes contain a common core of twelve proteins where six OB-fold interaction proteins, called A1–A6, play a crucial role. Here, we report the structure of two single-strand nucleic acid-binding OB-folds from interaction proteins A3 and A6 that surprisingly, form a heterodimer. Crystal growth required the assistance of an anti-A3 nanobody as a crystallization chaperone. Unexpectedly, this anti-A3 nanobody binds to both A3OB and A6, despite only ∼40% amino acid sequence identity between the OB-folds of A3 and A6. The A3OB-A6 heterodimer buries 35% more surface area than the A6 homodimer. This is attributed mainly to the presence of a conserved Pro-rich loop in A3OB. The implications of the A3OB–A6 heterodimer, and of a dimer of heterodimers observed in the crystals, for the architecture of the editosome are profound, resulting in a proposal of a ‘five OB-fold center’ in the core of the editosome.

Structures of P-glycoprotein reveal its conformational flexibility and an epitope on the nucleotide-binding domain

“…The wideness of our P-gp structures is comparable to recently published structures of other ABC transporters having similar ABC B-like protein folds (8,11,12,27). Interestingly, the structures of human ABCB10 and Thermotoga maritima TM287/ 288 (10) also have nucleotide analogs bound to separated NBDs, suggesting a state of these transporters just before the formation of the ABC sandwich that is essential for the structural transition to the outward-facing conformation.…”

The Architecture of Trypanosoma brucei editosomes

“…Biochemical and genetic experiments have predominantly revealed a network of protein-protein interactions that link two heterotrimeric subcomplexes within editosomes (23,29,30,(37)(38)(39)(45)(46)(47). However, the direct interactions of a variety of editosome proteins, including those that act at potentially key points of regulation during editing, were previously unidentified.…”

“…Primary among these are a global understanding of the molecular interactions within editosomes, in addition to interactions between editosomes and other mitochondrial RNA processing complexes. Previous work described a total of 11 direct interactions between 10 common proteins, but these predominantly encompass the insertion and deletion subcomplexes, and the KREPA proteins linking them (37)(38)(39)(45)(46)(47) (SI Appendix, Fig. S1 and Table S2).…”

“…S4 and Table S6) (45)(46)(47)(49)(50)(51) and comparative models (SI Appendix, Figs. S5 and S6 and Table S7, and Dataset S3) of editosome proteins.…”

“…However, cross-links between KREPA2 to other KREPA proteins predominantly involved a region in KREPA2 just N-terminal to the OB-fold. The previously described interactions of the OB-fold-containing proteins, which did not include KREPA5, led to the suggestion of a "five OB-fold center" in the core of the editosome that might perform a critical scaffold function (45)(46)(47). In this model, KREPA6 and the OB-folds of three other KREPA proteins are arranged and interact as a "shifted heterotetramer," whereas another OB-fold interacts with KREPA6 in a different, as yet unknown, manner.…”