Fig 2: Isothermal titration calorimetry of PNK binding to XRCC1 phospho-peptides. (a) ITC-binding curves for injection of XRCC1 peptides phosphorylated on Thr523 (left), Thr519 (middle), and on both Thr519 and Thr523 (right), into a microcalorimeter cell containing PNK–FHA (see Methods section). The mono-phosphorylated peptides show weak binding, while the doubly phosphorylated peptide binds with sub-micromolar affinity. Both peptides containing pThr518 show binding stoichiometry, (corresponding to the inflexion point of the curve) consistent with a 2:1 PNK–FHA:XRCC1 peptide complex. (b) As (a) but for peptide phosphorylated on pSer518 and pThr519 (left), and on pSer518, pThr519 and pThr523 (right). Affinities are >8-fold and >50-fold tighter, respectively, than the mono-phosphorylated pThr519 peptide. Binding stoichiometry in both cases indicates a 2:1 PNK–FHA:XRCC1 peptide complex. (c) Binding curves for injection of a pSer518 and pThr519 peptide (left) or a pSer518, pThr519, pThr523 and pSer525 peptide (right), into a cell containing full length human PNK. Affinities compare with those for the isolated FHA domain, and the binding stoichiometry remains consistent with a 2:1 PNK:XRCC1 peptide complex. (d) Inverted experiment, where PNK–FHA protein is injected into a cell containing an XRCC1 pSer518, pThr519 peptide. In this orientation of the experiment the binding stoichiometry is again consistent with formation of a 2:1 PNK:XRCC1 peptide complex, confirming that the observation is not a result of the experimental set-up. (e) Binding curves for injection of an XRRC1 peptide, phosphorylated on Ser518 and on Thr519, but with Thr523 mutated to valine. Removal of the threonine residue ablates the second binding site, so that only a single FHA domain now binds.

Fig 3: PNK–FHA protects CK2-phosphorylated XRCC1 from dephosphorylation. (a) Schematic representation of the domain structure of human XRCC1. The predicted amino-acid boundaries for each domain are indicated, with a dotted box delineating the extent of the XRCC1?N expression construct used in this study. The positions of the known phosphorylation sites within the linker region connecting the two C-terminal BRCT domains are also shown. (b) XRCC1?N expressed in insect cells (Sf9) is phosphorylated, as detected by a phospho-specific polyclonal antibody (Bethyl Laboratories, A300-059A). Incubation with ?-phosphatase readily removes the detected sites of phosphorylation. In contrast, XRCC1?N expressed in E.coli is not phosphorylated. One hundred micrograms of purified XRCC1?N protein was incubated with 50 µg of ?-phosphatase for a period of 1 h at room temperature, before being analysed by SDS–PAGE. (c) Incubation of the E.coli expressed XRCC1?N with recombinant maize CKII reproduces the phosphorylation observed in Sf9 cells. Sixty micrograms of purified XRCC1?N was incubated with increasing amounts of CKII (ranging from 0 to 95 µg), for a period of 2 h at room temperature. (d) Cell lysates from insect cells expressing His6-tagged XRCC1?N and E.coli expressing GST-tagged PNK–FHA were mixed, then applied to two sequential affinity columns (Talon/IMAC and Glutathione Sepharose). The SDS–PAGE analyses of the eluates from each step are shown. (e) The presence of the PNK–FHA domain protects, at least, the three phosphorylation sites detected by the polyclonal antibody in XRCC1 from the enzymatic action of ?-phosphatase. SDS–PAGE gels were stained with SimplyBlue SafeStain (Invitrogen, Paisley, UK).

Fig 4: Different modes of bis-phosphopeptide-binding in PNK and Dun1. The binding mode to PNK–FHA of the XRCC1 bis-phosphopeptide (a) with adjacent pSer-pThr residues is markedly different to that recently observed for a yeast Rad53 phosphopeptide containing two non-adjacent pThr residues, to the FHA of the DNA damage checkpoint kinase Dun1 (PDB code 2JQL) (b). While the C-terminal phosphorylated residue is in a similar topological position on the FHA domain, the N-terminal pThr in Dun1 binds in the -4 subsite, which is occupied by a tyrosine in PNK–FHA complexes with XRCC1 and XRCC4 peptides.

Fig 5: Comparison of PNK–FHA interactions with XRCC1 and XRCC4. PNK–FHA interacts in a similar way with the XRCC1 phosphopeptide (a), as with the XRCC4 peptide (b) apart from the additional interactions with pSer518 and Asn522 in the XRCC1 complex. That Ser518 was not phosphorylated in the XRCC4 complex is probably responsible for the different conformation observed for Arg44 in the two complexes. In both cases, the residues at -2 and -3 relative to the pThr, make no side-chain contacts with the protein that could mediate selectivity for these positions, and the specificity of FHA for the XRCC1 and XRCC4 sequences resides in the polar and electrostatic interactions with phosphorylated and acidic residues at 0, -1, +1 and +2, the hydrophobic interaction with Tyr at -4, and (for XRCC1) with the amide-side chain of the Asn at +3. In the schematics on the right, residues with a red (strictly) or orange (highly) background are conserved across FHA domains. Those with a green background are involved in hydrophobic contacts with the bound XRCC1/XRCC4 peptide, whilst those with a grey background are involved in making specific hydrogen bonds (as indicated by a line).