Disrupting domain-domain interactions is indispensable for EngA-ribosome interactions.

EngA consists of two tandem GTPase-domains-GD1 and GD2-followed by a KH-domain. EngA was considered to be a 50S assembly factor since it was shown to bind 50S and its deletion leads to the accumulation of immature 45S ribosomal subunits. Subsequently, we demonstrated an additional ribosome bound state of EngA bound to 50S, 30S, and 70S. While the former (50S binding) is achieved upon GTP binding at both GD1 and GD2, the latter is formed upon GTP hydrolysis at GD1, which is believed to trigger a large conformational change in the protein. The present study brings out two key aspects of EngA regulation: First, that distinctly stabilized GD1-KH interfaces allows EngA to exist in different ribosome bound states, and second is the importance of these states to ribosome assembly. Our analyses suggest that distinct inter-domain (GD-KH) interfaces are stabilized by interactions arising from unique sets of motifs, conserved across EngA homologues, and seem to be mechanistically linked to GTP/GDP binding. By experimentally measuring binding affinities for several interface mutants, we show that disrupting the interface interactions is necessary to realize EngA-ribosome binding. These findings are also supported by a recent cryo-EM structure of EngA bound to 50S, wherein the GD1-KH interface is completely disrupted leading to an 'extended' or 'open state' of the protein. Overall, it appears that the transition of EngA from a 'closed state' with GD1-KH forming a tight interface, to an 'open state' mediates interaction with ribosomal subunits.