Phosphorylation-dependent PIH1D1 interactions define substrate specificity of the R2TP cochaperone complex

Area of Science:

• Molecular Biology
• Protein Complexes
• Biochemistry

Background:

• The R2TP cochaperone complex is vital for assembling key cellular machines like RNA polymerase II.
• The precise mechanism by which R2TP recognizes its substrates is not well understood.

Purpose of the Study:

• To elucidate the molecular basis of substrate recognition by the R2TP cochaperone complex.
• To identify the specific domain and motif responsible for binding phosphorylated substrates.

Main Methods:

• Co-crystallization of the PIH1D1 subunit's PIH-N domain with a TEL2 phosphopeptide.
• Proteomic analysis to identify PIH1D1 interactors and substrates.
• Structural analysis of the protein-peptide complex.

Main Results:

• A phosphopeptide binding domain (PIH-N) in PIH1D1 was identified, showing preferential binding to acidic phosphorylated proteins.
• Structural data revealed specific recognition involving Lys57 and Lys64 in PIH1D1 and a DpSDD motif in TEL2.
• Proteomic analysis confirmed R2TP substrates are recruited by PIH-N in a phosphorylation-dependent manner.

Conclusions:

• The PIH1D1 subunit's PIH-N domain mediates substrate recognition through specific phosphopeptide binding.
• This mechanism, involving phosphorylation of a specific motif, is proposed as a common way R2TP assembles protein complexes.