Ribosome‐associated factor Y adopts a fold resembling a double‐stranded RNA binding domain scaffold

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Abstract
Escherichia coli protein Y (pY) binds to the small ribosomal subunit and stabilizes ribosomes against dissociation when bacteria experience environmental stress. pY inhibits translation in vitro, most probably by interfering with the binding of the aminoacyl‐tRNA to the ribosomal A site. Such a translational arrest may mediate overall adaptation of cells to environmental conditions. We have determined the 3D solution structure of a 112‐residue pY and have studied its backbone dynamic by NMR spectroscopy. The structure has a βαβββα topology and represents a compact two‐layered sandwich of two nearly parallel α helices packed against the same side of a four‐stranded β sheet. The 23 C‐terminal residues of the protein are disordered. Long‐range angular constraints provided by residual dipolar coupling data proved critical for precisely defining the position of helix 1. Our data establish that the C‐terminal region of helix 1 and the loop linking this helix with strand β2 show significant conformational exchange in the ms–µs time scale, which may have relevance to the interaction of pY with ribosomal subunits. Distribution of the conserved residues on the protein surface highlights a positively charged region towards the C‐terminal segments of both α helices, which most probably constitutes an RNA binding site. The observed βαβββα topology of pY resembles the αβββα topology of double‐stranded RNA‐binding domains, despite limited sequence similarity. It appears probable that functional properties of pY are not identical to those of dsRBDs, as the postulated RNA‐binding site in pY does not coincide with the RNA‐binding surface of the dsRBDs.