Acetylation 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 (AF2)-multimer to predict direct interactions
and validated them using yeast two and three-hybrid assays. We focused
on MORF4 related gene (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 trimer independent
of NuA4 for transcription interaction (TINTIN) complexes; and another
trimeric complex involving an MRG domain, only seen in trypanosomatids.
The identification of a key component of TcTINTIN, 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
for TcBDF6. 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 AF2-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 AF2 models for the determination of multiprotein
complexes illuminates the power of using such artificial intelligence-derived
tools in the future development of biology.