Generating and Characterizing Phosphospecific Affinity Reagents Using the Forkhead-Associated 1 Domain

Phosphorylation is an important post-translational event that has a wide array of functional consequences. With advances in the ability of various technologies in revealing and mapping new phosphosites in proteins, it is equally important to develop affinity reagents that can monitor such posttranslational modifications in eukaryotic cells. While monoclonal and polyclonal antibodies have been shown to be useful in assessing the phosphoproteome, we have expanded our efforts to exploit the Forkhead-associated 1 (FHA1) domain as scaffold for generating recombinant affinity reagents that recognize phosphothreonine-containing peptides. A phage display library of FHA1 variants was screened by affinity selection with 15 phosphothreonine-containing peptides corresponding to various human transcription factors and kinases. The library yielded binding variants against 10 targets (66% success rate); success was largely determined by what residue occurred at the +3 position (C-terminal) to the pThr moiety (i.e., pT+3). Three FHA domains binding transcription factor,c-Myc (Myc), calmodulin-dependent protein kinase II (CaMKII), and extracellular-signal regulated kinases 1 and 2 (ERK1/2) were characterized and compared against commercially available antibodies. All FHA domains were shown to be phosphorylation-dependent and phosphothreonine-specific in their binding, unlike several commercial monoclonal and polyclonal antibodies. The crystal structure of the engineered FHA Myc-pThr-binding domain (Myc-pTBD) was solved in complex with its cognate ligand. The Myc-pTBD was observed to be structurally similar to the yeast Rad9 FHA1 domain, except that its β4-β5 and β10-β11 loops form a hydrophobic pocket to facilitate the interaction between the domain and the peptide ligand. Both the pThr and the residue at the pT+3 position were major factors in defining the specificity of the FHA domains. To enhance apparent affinity of the pTBDs, the Myc-pTBD, ERK1/2-pTBD, and CaMKII-pTBD were converted to homodimers by either fusing the domains to a leucine zipper (LZ) dimerizing sequence (to generate pTBD LZs) or linking them in tandem (to generate pTBD-Tandem repeats, TR). To estimate the affinity of the various constructs, the concentration of each that gives the half maximal response (EC50) was measured in an ELISA. Monomers were observed to have an average EC50 value of 10-8 M, whereas pTBD-LZs and pTBD-TRs had EC50 values of 10-9 M and 10-10 M, respectively. Interestingly, there was no apparent loss of specificity for either dimeric form of the three pTBDs tested. The utility of Myc-pTBD LZ was further evaluated by probing western blots of lysates of HEK 293 cells engineered to overexpress Myc-pT58. Curiously, while the Myc-pTBD LZ failed to detect Myc-pT58, it bound to two unrelated, phosphorylated protein species. Future engineering experiments on the FHA scaffold may contribute to enhanced detection of cell signaling events and biomarkers of disease.