Interaction of elongation factor eEF-2 with ribosomal P proteins

The eukaryotic P1 and P2 ribosomal proteins which constitute, with PO, a pe ntamer forming the lateral stalk of the 60 S ribosomal subunit, exhibit sev eral differences from their prokaryotic equivalents L7 and L12; in particul ar, P1 does not have the same primary structure as P2 and both of them are phosphorylated, the significance of the latter remaining unclear. Rat liver P1 and P2 were overproduced in Escherichia coil cells and their interactio n with elongation factor eEF-2 was studied. Both recombinant proteins were found to be required for the ribsome-dependent GTPase activity of eEF-2, wi th P2 in the phosphorylated form. The surface plasmon resonance technique r evealed that, in vitro, both proteins interact specifically with eEF-2, wit h a higher affinity for P1 (K-d = 3.8 x 10(-8) M) than for P2 (K-d = 7.2 x 10(-6) M). Phosphorylation resulted in a moderate increase (two- to four-fo ld) in these affinities. The interaction of both P1 and P2 (phosphorylated or not) with eEF-2 resulted in a conformational change in the factor, revea led by an increase in the accessibility of Glu554 to proteinase Glu-C. This increase was observed in both the presence and absence of GTP and GDP, whi ch themselves produced marked opposite effects on the conformation of eEF-2 . Our results suggest that the two proteins P1 and P2 both interact with eE F-2 inducing a conformational transition of the factor, but have acquired s ome specific properties during evolution.