Abstract
Mitoribosomes are highly specialized to accommodate the translation of mito-specific mRNAs and regulate OXPHOS complex biogenesis. A feature unique to mitoribosomes is the presence of an intrinsic guanine nucleotide binding site, a capability conferred by the mitochondrion-specific GTPase, mS29. Early reconstruction of the human mitoribosome at 3.5 Å attributed GDP binding in mS29 to a highly conserved p-loop region in the protein. Now with an improved resolution at 2.2 Å we observe an ATP where GDP was previously misassigned due to flexibility of the loop. This near atomic resolution map disclosed the presence of a second binding site in mS29. The newly identified moiety corresponds to a GDP and resides in a distinct hydrophobic cavity formed by mS29 where it stabilizes a β-hairpin structure on the top of mS29 in the SSU head, adjacent from sites of intersubunit bridging. Through biochemical and structural analyses, we demonstrate that the GDP supported β-hairpin in mS29 acts as a molecular switch to stabilize the hybrid state during translation, maintaining the only intersubunit communication in the head during rotation. To uncover evolutionary differences between mammalian and fungal mitoribosomes we performed mirror studies in S. cerevisiae. Although structurally similar, we show that fungal and mammalian mS29 possess key features to accommodate species-specific differences in mitochondrial translation. Based on these differences we propose that mammalian mS29 gained GDP binding function as a co-factor solution to structural changes in the peptidyl transferase center during evolution.