Figure 1.

NMR spectroscopic characterization of PntP2^142–252. (A) Cartoon of the 718-residue Pointed-P2 transcription factor showing the structured PNT domain and DNA-binding ETS domain, along with aligned sequences of PntP2^142–252 and Ets1^29–138. The identified ERK2 phosphoacceptors are in red, and the observed consensus α-helices (or 3₁₀-helix for H2′) in the NMR-derived structural ensembles of Ets1^29–138 (2jv3.pdb) and 2P-Ets1^29–138 (2kmd.pdb) are shaded in light blue.⁴ (B) Superimposed ¹⁵N-HSQC spectra of PntP2^142–252 (red), 2P-PntP2^142–252 (green), and 3P-PntP2^142–252 (blue). The arrows indicate the progressive spectral perturbations upon increased phosphorylation. Amide signals from phosphothreonines are observed selectively in the ¹H–¹⁵N faces of ³¹P-edited HNCA spectra of (C) 2P-PntP2^142–252 and (D) 3P-PntP2^142–252. Although the latter experiment also detects the residue immediately following a phosphothreonine,³¹ only Ala146 is observed as Pro152 lacks an amide ¹H^N and Asn155 has a relatively weak ¹⁵N-HSQC signal. Owing to incomplete separation by anion exchange chromatography, resolved signals from contaminating 2P-PntP2^142–252 were observed in the spectra of 3P-PntP2^142–252 shown in (B and D). The phosphothreonines in (E) 2P-PntP2^142–252 and (F) 3P-PntP2^142–252 were also identified based on a large diagnostic downfield ¹³C^β shift³² relative to the corresponding unmodified amino acid in PntP2^142–252.