Interaction of elongation factor eEF-2 with ribosomal P proteins.
The eukaryotic P1 and P2 ribosomal proteins which constitute, with P0, a pentamer forming the lateral stalk of the 60 S ribosomal subunit, exhibit several differences from their prokaryotic equivalents L7 and L12; in particular, 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 coli cells and their interaction with elongation factor eEF-2 was studied. Both recombinant proteins were found to be required for the ribosome-dependent GTPase activity of eEF-2, with P2 in the phosphorylated form. The surface plasmon resonance technique revealed that, in vitro, both proteins interact specifically with eEF-2, with a higher affinity for P1 (Kd = 3.8 x 10-8 m) than for P2 (Kd = 2.2 x 10-6 m). Phosphorylation resulted in a moderate increase (two- to four-fold) in these affinities. The interaction of both P1 and P2 (phosphorylated or not) with eEF-2 resulted in a conformational change in the factor, revealed 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, which themselves produced marked opposite effects on the conformation of eEF-2. Our results suggest that the two proteins P1 and P2 both interact with eEF-2 inducing a conformational transition of the factor, but have acquired some specific properties during evolution.
Bargis-Surgey
P
P
Laboratoire de Biochimie Médicale, Institut de Biologie et Chimie des Protéines, CNRS, Lyon, France.
Lavergne
J P
JP
Gonzalo
P
P
Vard
C
C
Filhol-Cochet
O
O
Reboud
J P
JP
D001483:Base Sequence; D017931:DNA Primers; D006868:Hydrolysis; D007700:Kinetics; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D012269:Ribosomal Proteins
D017931:DNA Primers; D020652:Peptide Elongation Factor 2; D010445:Peptide Elongation Factors; D010750:Phosphoproteins; D012269:Ribosomal Proteins; C031937:phosphoprotein P2, ribosomal; C035690:ribosomal phosphoprotein P1