Deciphering divergent trypanosomatid nuclear complexes by analyzing interactomic datasets with AlphaFold2 and genetic approaches

Abstract

AbstractAcetylation signaling pathways in trypanosomatids, a group of early branching organisms, are poorly understood due to highly divergent protein sequences. To overcome this challenge, we used interactomic datasets and AlphaFold2-multimer to predict direct interactions and validated them using yeast two and three-hybrid assays. We focused on MRG domain-containing proteins and their interactions, typically found in histone acetyltransferase/deacetylase complexes. The results identified a structurally conserved complex,TcTINTIN, which is orthologous to human and yeast TINTIN complexes; and another trimeric complex involving an MRG domain, only seen in trypanosomatids. The identification of a key component ofTcTINTIN,TcMRGBP, would not have been possible through traditional homology-based methods. We also conducted molecular dynamics simulations, revealing a conformational change that potentially affects its affinity forTcBDF6. The study also revealed a novel way in which an MRG domain participates in simultaneous interactions with two MRG binding proteins binding two different surfaces, a phenomenon not previously reported. Overall, this study demonstrates the potential of using AlphaFold2-processed interactomic datasets to identify protein complexes in deeply branched eukaryotes, which can be challenging to study based on sequence similarity. The findings provide new insights into the acetylation signaling pathways in trypanosomatids, specifically highlighting the importance of MRG domain-containing proteins in forming complexes, which may have important implications for understanding the biology of these organisms and developing new therapeutics. On the other hand, our validation of AlphaFold2 models for the determination of multiprotein complexes illuminates the power of using such artificial intelligence-derived tools in the future development of biology.Abstract Figure