The FHA Domain Is a Modular Phosphopeptide Recognition Motif Daniel Durocher, Julia Henckel, Alan R Fersht, Stephen P Jackson  Molecular Cell  Volume 4, Issue 3, Pages 387-394 (September 1999) DOI: 10.1016/S1097-2765(00)80340-8

Figure 1 The FHA1 Domain of Rad53p Binds to Phosphorylated Rad9p Extracts (1 mg protein) from cultures treated with MMS or 4-NQO as indicated were incubated in the presence of equal amounts (500 ng) of immobilized GST–FHA1, GST–FHA2, GST–FHA1(R70A), GST–FHA1(H88A), GST–FHA2, or GST–FHA2(R605A), as indicated. The fusion protein complexes were separated by SDS-PAGE and subjected to immunoblotting with an anti-Rad9p polyclonal antiserum. Molecular Cell 1999 4, 387-394DOI: (10.1016/S1097-2765(00)80340-8)

Figure 2 The FHA1 Domain Binds to Phosphorylated Rad9p by Recognizing a Phosphoepitope (A) List of the peptides used in this study. Note that peptides, except for the H2A.X-derived peptides, are biotinylated and contain the linker Ser-Gly-Ser-Gly between the biotin group and the N terminus of the peptide sequence given. Thr-18 of p53 has been shown to be targeted by DNA-PK, at least in vitro (N. Lakin, personal communication). (B, C, and D) Competition of the FHA1–Rad9 interaction using phosphorylated peptides. Immobilized GST–FHA1 (B and C) or GST–FHA2 (D) was incubated in the presence of 1 mg of protein extracts prepared from MMS-treated cells, in the presence or absence of the indicated peptides. The presence of Rad9p in the FHA–protein complexes was detected by Western blotting. In (C), the Rad9p–FHA1 interaction was competed by increasing amounts of the ST(P) peptide. The relative amount of Rad9p bound was quantitated using the NIH image software, the R square of the logarithmic regression is 0.88. The ST peptide does not compete significantly the FHA1–Rad9p interaction at 500 μM (data not shown). Molecular Cell 1999 4, 387-394DOI: (10.1016/S1097-2765(00)80340-8)

Figure 3 The FHA1 Domain of Rad53p Binds Directly to Specific Phosphopeptides (A and B) Biotinylated peptide [5 ng; ST, S(P)T, ST(P), as indicated] was incubated with 50 ng of the indicated GST fusion protein. Protein–peptide complexes were retrieved using streptavidin paramagnetic beads and were separated by SDS-PAGE. Fusion proteins were detected by Western blotting using an anti-GST monoclonal antibody. Note that the multiple bands observed in the input lanes correspond to GST–FHA fusion degradation products. One degraded form of GST–FHA1 can still bind the ST(P) peptide, and this form corresponds to a C-terminal truncation of GST–FHA1 that still harbors an intact FHA domain (data not shown). (C and D) The binding of FHA1 to peptides requires a phosphothreonine and the presence of an aspartic acid at position +3. GST–FHA1 (50 ng) was incubated with 5 ng of biotinylated peptides. Peptide–protein complexes were retrieved and analyzed as described in (A). (E) The FHA domain protects the phosphate group from phosphatase activity. λ protein phosphatase was added to binding reactions containing the ST(P) peptide and GST–FHA1 (FHA1) or GST–FHA1(H88A) fusion proteins, and aliquots were taken after 30 and 60 min. Reactions were then stopped with EDTA and were spotted on nitrocellulose. The ST(P) peptide was detected by Western blotting using a phospho-specific p53 Thr18 antibody and was quantitated using the NIH image software. Each point of the graph represents the mean of a duplicate. Molecular Cell 1999 4, 387-394DOI: (10.1016/S1097-2765(00)80340-8)

Figure 4 The FHA1 Domain Binds Specific Phosphopeptides with High Affinity (A) Binding of wild-type FHA1 to the ST(P) peptide using ITC. The titrations and the calculations leading to the KD, enthalpy (ΔH) as well as the stoichiometry per FHA unit (N) were performed as described in the Experimental Procedures. (B) Binding of wild-type FHA1 to the peptides ST, ST(P)+3A, ST(P)5A, ST(P), SS(P), and S(P)T measured by SPR. Binding experiments were performed as described in the Experimental Procedures. (C) Comparison of wild-type FHA1 and mutants (R70A, H88A, or E117A) binding to phosphorylated peptide ST(P). Binding experiments were performed and analyzed as described in (B). Molecular Cell 1999 4, 387-394DOI: (10.1016/S1097-2765(00)80340-8)

Figure 5 The FHA Domain Is a Modular Phosphopeptide Recognition Motif GST fusion proteins corresponding to FHA1 and FHA1(H88A) (B); FHA2 and FHA2(R605A) (C); KAPP (accession number 1709235) (D); KIAA0170 (accession number 1136400) (E); Yhr115cp (accession number S48957) (F); and MtuCY1A11.16C (accession number 2496539) (G) were tested for their ability to bind to biotinylated degenerate peptide libraries, described in (A), immobilized on streptavidin-coated chips. The binding activity was measured by SPR and expressed as corrected resonance units (RU). Molecular Cell 1999 4, 387-394DOI: (10.1016/S1097-2765(00)80340-8)