Engineered FHA domains can bind to a variety of Phosphothreonine-containing peptides

Abstract

Antibodies play a crucial role in monitoring post-translational modifications, like phosphorylation, which regulates protein activity and location; however, commercial polyclonal and monoclonal antibodies have limitations in renewability and engineering compared to recombinant affinity reagents. A scaffold based on the Forkhead-associated domain (FHA) has potential as a selective affinity reagent for this post-translational modification. Engineered FHA domains, termed phosphothreonine-binding domains (pTBDs), with limited cross-reactivity were isolated from an M13 bacteriophage display library by affinity selection with phosphopeptides corresponding to human mTOR, Chk2, 53BP1, and Akt1 proteins. To determine the specificity of the representative pTBDs, we focused on binders to the pT543 phosphopeptide (536-IDEDGENpTQIEDTEP-551) of the DNA repair protein 53BP1. ELISA and western blot experiments have demonstrated the pTBDs are specific to phosphothreonine, demonstrating the potential utility of pTBDs for monitoring the phosphorylation of specific threonine residues in clinically relevant human proteins.

Keywords: 53BP1; Akt1; Chk2; FHA domain; affinity selection; library; mTOR; phosphoepitope; phosphopeptide; phosphoserine; phosphothreonine; sortase.

Graphical Abstract

Figure 1

The Forkhead-associated (FHA) domain as a phosphothreonine-binding affinity reagent. The yeast Rad53 protein contains two FHA domains, and a three-dimensional structure (PDB: 1j4q) is shown complexed with the Rad9 phosphopeptide, SLEVpTEAD. We have used the FHA1 domain as a scaffold for protein engineering experiments and generated a library of 10⁹ variants in which two loops of the Rad53 FHA1 domain were randomized with NNK codons. The library was affinity selected with four different phosphopeptides to yield binders characterized in this report. Once validated, binders can be used in ELISAs, western blot, pull-down, and in vivo expression experiments

Figure 2

Phage ELISA to examine the specificity of phage clones. Biotinylated peptides containing the mTOR, Chk2, 53BP1, and Akt1 sequences were synthesized with phosphothreonine (pT), threonine (T), or phosphoserine (pS) and immobilized by NeutrAvidin. A biotinylated ATR peptide was also synthesized with pT and immobilized by NeutrAvidin. From top to bottom, the binding of anti-mTOR C9, anti-Chk2 A4, anti-53BP1 H6, and anti-Akt1 G5 clones was detected with α-M13 HRP. Error bars represent standard error of triplicate measurements

Figure 3

a) Comparison of the binding profile of A2 virions and a commercial polyclonal antibody to a panel of peptides. Biotinylated 53BP1 peptides with phosphothreonine (pT), threonine (T) and phosphoserine (pS), and other unrelated phosphopeptides were captured on NeutrAvidin-coated microtiter plate wells. Binding of A2 virions and the commercial rabbit polyclonal antibody (pAb) to the peptides was detected with α-M13 HRP and α-rabbit IgG HRP, respectively. Error bars represent standard error of triplicate measurements. b) Binding of A2 virions to the 53BP1 phosphopeptide in a complex mixture. Biotinylated peptides, corresponding to the 53BP1 sequence with phosphothreonine (pT) or threonine (T) at position 543, were captured on NeutrAvidin-coated microtiter plate wells. Binding of virions, in the presence (+) or absence (−) of 10% human serum, was detected with α-M13 HRP. Error bars represent standard error of triplicate measurements

Figure 4

Recombinant proteins. a) SDS-PAGE analysis. Bio-Rad’s Precision Plus Protein Unstained Standards ladder (Lane 1), cellulose binding domain (CBD, Lane 2), CBD-A2 (Lane 3), and CBD-G12 (Lane 4) proteins were resolved in a Bio-Rad Mini-PROTEAN Stain-Free Gel (8–16%). The calculated molecular weights of CBD, CBD-A2, and CBD-G12 proteins are listed (kilodaltons, kDa). b) ELISA with HRP conjugated CBD-FHA domain fusions. Biotinylated phosphopeptides were captured on microtiter plate wells coated with NeutrAvidin, and binding of CBD-A2-HRP and CBD-G12-HRP was detected enzymatically. Error bars represent standard error of triplicate measurements

Figure 5

Sortase tagging ELISA. a) An illustration showing the conjugation of a phosphopeptide to the Cellulose Binding Domain (CBD). When sortase recognizes the pentapeptide sequence, LPETG, of the CBD-LPETG-H₆ fusion protein, it forms a peptide bond with threonine and releases the G-H₆ peptide. The N-terminal glycine of the phosphopeptide acts as a nucleophile and attacks the intermediate to form the CBD-LPETG- phosphopeptide conjugate. b) ELISA showing the detection of the CBD-phosphopeptide ligation product. Microtiter plate wells are coated with various sortase reaction mixtures containing CBD (+/−), 53BP1 pT543 phosphopeptide (+/−), unrelated mTOR phosphopeptide (+/−), and sortase (+/), and then probed with virions displaying the A2 clone, which binds the 53BP1 pT543 phosphopeptide, followed by anti-M13 Ab-HRP. The optical density (OD) of wells is measured at 475 nm wavelength (OD₄₇₅). Error bars represent standard error of triplicate measurements

Figure 6

Western blot analysis of sortase ligation products with two anti-53BP1 phosphopeptide pTBDs. a) SDS-PAGE analysis of CBD and phosphopeptide ligation reactions. Twenty microliters of the Sortase reaction mixtures with CBD (lane 1), CBD-mTOR phosphopeptide (lane 2), and CBD-53BP1 phosphopeptide (lane 3) conjugates were resolved in a Bio-Rad Mini-PROTEAN Stain-Free Gel (8–16%). The calculated molecular weights of CBD, CBD-mTOR, and CBD-53BP1 sortase ligation products are listed. b) and c) Western blots of sortase ligation products with CBD-A2-HRP and CBD-G12-HRP, respectively, as probes. Five microliters of the Sortase reactions with only CBD (lane 1), CBD and mTOR phosphopeptide (lane 2), and CBD and 53BP1 phosphopeptide (lane 3) were resolved in an SDS-gel and transferred to a PVDF membrane. The membrane was probed with either CBD-A2-HRP or CBD-G12-HRP, incubated with SuperSignal West Pico Chemiluminescent Substrate (ThermoFisher Scientific), and chemiluminescence detected with a Bio-Rad ChemiDoc MP imaging system