Figure 9.

Quantitative HDX analysis and NHERF1 conformation. A, differential deuterium uptake for WT–NHERF1 and S290A–NHERF1 and at 10 s (black), 100 s (blue), 1000 s (green), 10000 s (pink), and 100,000 s (gray). Peptides exhibiting significant differential deuterium uptake greater than 50% are indicated; others were omitted for clarity. Of note, there are large peaks of differential deuterium uptake surrounding Ser²⁹⁰ from Ala²⁸⁹ to Phe³²³ in the S290A phosphoresistant construct. The (39–58)-peptide, where Glu⁴³ is adjacent to the PDZ1 ²³GYGF²⁶ core-binding motif as shown in Fig. 8A, displays high differential deuterium uptake. B, representative isotope profile of the (39–58)-peptide at the indicated times for WT (black) and S290A–NHERF1 (red). Nondeuterated controls for WT and S290A peptides are indistinguishable. A bimodal distribution at 10, 100, and 1000 s (panels ii–iv) reflects conformational change between phosphorylated and unphosphorylated conditions. C, dynamic exchange profiles for the indicated peptides from WT (black) and S290A–NHERF1 (red). Peptides with unique, time-dependent HDX behavior are shown. C-terminal residues in S290A–NHERF1 display greater deuterium uptake. D, model showing residues exhibiting differential deuterium uptake between WT and S290A–NHERF1 at 100 s mapped on the structure of PDZ1 (PDB code 1I92). The critical residues His²⁷, Glu⁴³, and the GYGF core-binding motif for PDZ ligand binding are colored in purple. Residues undergoing increased deuteration of 30% or more are shown. E, differential deuterium uptake between WT and S290A–NHERF1 at 100 s mapped on PDZ2 (PDB code 4Q3H). Critical binding residues based on the same criteria as in D are colored purple. The differential deuteration levels are color-coded as in D. The high HDX rate at Ile²¹⁹, Arg²²⁰, Glu²²⁵, Thr²²⁶, and Lys²²⁷ suggests that these residues undergo significant structural movement in the opposite direction from what occurs in PDZ1.