Combining natural sequence variation with high throughput mutational data to reveal protein interaction sites

Area of Science:

• Molecular Biology
• Evolutionary Biology
• Protein Interactions

Background:

• Protein interactions are often conserved across species, even with sequence divergence.
• Inter-species complementation experiments use homologous sequences to identify functional sites.
• Previous methods were limited by small-scale analyses of sequence differences.

Purpose of the Study:

• To analyze interaction sites within the RRM2 domain of Saccharomyces cerevisiae poly(A)-binding protein (Pab1) using high-throughput mutational data.
• To investigate the impact of evolutionary sequence differences on Pab1 function and protein interactions.

Main Methods:

• Utilized existing high-throughput mutational data on Pab1 RRM2 domain in vivo function.
• Performed inter-species complementation by substituting homologous sequences into yeast Pab1.
• Conducted large-scale mutational analysis using a two-hybrid assay for eIF4G1 binding.
• Created triple mutants with human orthologue substitutions to assess binding specificity switch.

Main Results:

• 17 out of 197 single amino acid differences in 52 Pab1 homologues reduced yeast Pab1 function.
• Most deleterious mutations affected binding of the RRM2 domain to translation initiation factors eIF4G1 and eIF4G2.
• Three substitutions corresponding to human Pab1 residues eliminated binding to yeast eIF4G isoforms.
• A triple mutant demonstrated a switch in RRM2 binding specificity from yeast to human eIF4G.

Conclusions:

• Large-scale mutational data combined with evolutionary conservation effectively characterizes protein interaction sites at single amino acid resolution.
• Identified specific residues critical for Pab1 interaction with translation factors and demonstrated the potential to engineer binding specificity.
• Mapped other deleterious substitutions to inter-domain and protein-RNA interaction sites within Pab1.