Structural characterisation of PIH-N phospho-binding domains

The R2TP co-chaperone complex plays a critical role in the assembly of multisubunit protein complexes. A number of R2TP targets are recruited via the adaptor protein TEL2 which directly interacts with the R2TP subunit PIH1D1 when phosphorylated by casein kinase 2 (CK2). A recent co-crystal structure of the PIH-N domain of PIH1D1 in complex with a TEL2 phospho-peptide, revealed a novel phospho-binding mechanism. Here, isothermal titration calorimetry (ITC) was utilised to generate a complete thermodynamic description of this binding event. These data show that every residue within a conserved DpSDD motif of TEL2 along with a number of conserved basic PIH1D1 residues are required for the interaction in vitro. Structural studies carried out in parallel resulted in a crystal structure of the unbound PIH-N domain which reveals additional structural features not visible in the bound structure. Subsequent experiments aimed to probe the phospho-binding capabilities of PIH1D1 paralogs. One such paralog, DNAAF2, has been linked to chaperonedependent processes, namely the cytoplasmic assembly of axonemal dynein arms. The structure of a DNAAF2 PIH-N domain was determined revealing similarities with the TEL2 binding site of PIH1D1. ITC experiments confirmed that the human DNAAF2 PIH-N domain is able to bind to phospho-peptides corresponding to the CK2 site of TEL2. This raises the prospect that DNAAF2 recruits clients to chaperone assemblies in a manner analogous to the PIH1D1 subunit of the R2TP complex. Proteomic pull-down experiments then identified a number of putative phospho-dependent DNAAF2 binding partners several of which are linked to dynein-associated flagellar assembly processes