Directed Evolution of the Forkhead-Associated Domain to Generate Anti-Phosphospecific Reagents by Phage Display

Abstract

While affinity reagents are valuable tools for monitoring protein phosphorylation and studying signaling events in cells, generating them through immunization of animals with phosphopeptides is expensive, laborious and time consuming. An attractive alternative is to use protein evolution techniques and isolate new anti-phosphopeptide binding specificities from a library of variants of a phosphopeptide-binding domain. To explore this strategy, we attempted to display on the surface of bacteriophage M13, the N-terminal Forkhead-associated domain (FHA1) of yeast Rad53p, which is a naturally occurring phosphothreonine (pT)-binding domain, and found it to be non-functional due to misfolding in the bacterial periplasm. To overcome this limitation, a library of FHA1 variants was constructed by mutagenic PCR and functional variants were isolated after three rounds of affinity selection with its pT peptide ligand. A hydrophobic residue at position 34 in the β1-strand was discovered to be essential for phage-display of a functional FHA1 domain. Additionally, by heating the phage library to 50°C prior to affinity selection with its cognate pT peptide, we identified a variant (G2) that was ~8°C more thermal stable than the wild-type domain. Using G2 as a scaffold, we constructed phage-displayed libraries of FHA1 variants and affinity selected for variants that bound selectively to five pT peptides. These reagents are renewable and have high protein yields (~20–25 mg/L), when expressed in Escherichia coli. Thus, we have changed the specificity of the FHA1 domain and demonstrated that engineering phosphopeptide-binding domains is an attractive avenue for generating new anti-phosphopeptide binding specificities in vitro by phage-display.

A comparison of phosphospecific affinity reagents reveals the utility of recombinant Forkhead-associated domains in recognizing phosphothreonine-containing peptides

“…Our findings confirm that the pT+3 position is critical for binding for this FHA domain variant. This is consistent with the previous finding of Pershad et al (22), which demonstrated the importance of the pT+3 in the peptide ligand for the FHA domain that binds MAPK3. However, it is likely that other positions in the peptide likely contribute somewhat to binding, as the peptide AAApTAALA does not bind to the same level as the target sequence, LLPpTPPLS.…”

“…To generate recombinant affinity reagents that are phosphothreonine-specific, a phage display library was constructed by randomizing residues in the β4-β5 and β10-β11 loop regions of a thermostable variant (FHA1G2) of the FHA1 domain of the yeast Rad53 protein (22, 28) (Fig. 1A).…”

“…Italicized residues are in the +3 position, where the pT is assigned as the “0” position, and residues N-terminal and C-terminal to the pT are denoted as “-” and “+,” respectively. * Previously reported in (22). …”

“…One strategy to overcome these limitations is to use recombinant affinity reagents, as they eliminate the need for animals, there is more control in epitope recognition, they are sequenced and renewable reagents and they are amenable to protein engineering (17, 18). To this extent, several engineered phosphate-binding domains, such as the Src Homology 2 (SH2) domain (19), a recombinant phosphospecific antibody fragment (20), the 10 th fibronectin type III domain (10FnIII) (21), and the Forkhead-associated 1 (FHA) domain (22), have all been used successfully for generating recombinant affinity reagents to phosphopeptides.…”

“…Within the FHA domain, there is a pocket that interacts with the γ-methyl group and phosphate of pThr, which allows the domain to discriminate between phosphoserine (pSer) and pThr (24). Utilizing the domain's natural ability to discriminate between pSer and pThr, the specificity of one particular FHA domain, the FHA1 domain of yeast Rad53 protein, was reengineered through phage display (22). In this report, we demonstrate that the engineered FHA domains are exquisitely selective in binding pThr-, and not pSer- or phosphotyrosine (pTyr)-containing peptides, unlike several polyclonal and monoclonal antibodies tested.…”