Chi-Chun Chen (WPI-SKCM² and Academia Sinica) Evolution of an evolutionarily conserved and topologically knotted SPOUT superfamily

SPOUT (Spou and TrmD) superfamily is a group of Class IV RNA methyltransferases that contain an alpha/beta trefoil knot fold. They use a conserved and topologically knotted structural motif to bind S-adenosyl-L-methionine (SAM) as a methyl donor to achieve the RNA methylation function pertinent to ribosomal RNA (rRNA) and transfer RNA (tRNA) maturation. Because of this important biological function, bacterial TrmD, in particular, has become a validated drug target for antibiotics developments. While many SPOUT family members are multi-domain proteins with different functional module appended before or after the SPOUT domain, all SPOUT members share a deep 3₁ trefoil knot encompassing a long C-terminal alpha helix that thread through a knotting loop to which SAM binds. Importantly, SPOUT proteins are found in all kingdoms of life, underscoring its evolutionary importance. Why would Nature evolve such a complex protein topology to carry out one of the most fundamental biological functions associated with protein biogenesis? By reconstructing the evolutionary trail of the SPOUT superfamily, we aim to reconstruct the sequences at different time points throughout evolution to trace to the last universal common ancestor (LUCA). We aim to test the hypothesis that the LUCA of the SPOUT superfamily is also topologically knotted by combining bioinformatics and experimental structural biology.