Interaction of elongation factor eEF-2 with ribosomal P proteins

Area of Science:

• Molecular Biology
• Ribosome biogenesis and function
• Protein-protein interactions

Background:

• Eukaryotic P1 and P2 ribosomal proteins form part of the 60S subunit's lateral stalk.
• These proteins differ from prokaryotic equivalents and are subject to phosphorylation, with unclear functional significance.
• Their interaction with elongation factor eEF-2 is crucial for protein synthesis.

Purpose of the Study:

• To investigate the interaction between eukaryotic ribosomal proteins P1 and P2 and elongation factor eEF-2.
• To determine the role of P1 and P2, including phosphorylated P2, in eEF-2's GTPase activity.
• To analyze the effect of P1 and P2 binding on eEF-2 conformation.

Main Methods:

• Overproduction of rat liver P1 and P2 proteins in Escherichia coli.
• Assay of ribosome-dependent GTPase activity of eEF-2.
• Surface plasmon resonance (SPR) to measure binding affinities.
• Proteolytic accessibility assay to detect conformational changes in eEF-2.

Main Results:

• Both P1 and P2 are required for eEF-2's GTPase activity, particularly phosphorylated P2.
• SPR analysis showed specific binding of P1 and P2 to eEF-2, with higher affinity for P1.
• Phosphorylation moderately increased the binding affinities of P1 and P2 to eEF-2.
• Binding of P1 and P2 induced a conformational change in eEF-2, altering Glu554 accessibility, independent of GTP/GDP binding.

Conclusions:

• Eukaryotic ribosomal proteins P1 and P2 interact with eEF-2, modulating its activity and conformation.
• Phosphorylation plays a role in regulating the interaction and function of P2.
• These findings highlight the distinct evolutionary properties acquired by P1 and P2 in eukaryotes.