NMR determines transient structure and dynamics in the disordered C-terminal domain of WASp interacting protein

WASp-interacting protein (WIP) is a 503-residue proline-rich polypeptide expressed in human T cells. The WIP C-terminal domain binds to Wiskott-Aldrich syndrome protein (WASp) and regulates its activation and degradation, and the WIP-WASp interaction has been shown to be critical for actin polymerization and implicated in the onset of WAS and X-linked thrombocytopenia. WIP is predicted to be an intrinsically disordered protein, a class of polypeptides that are of great interest because they violate the traditional structure-function paradigm. In this first (to our knowledge) study of WIP in its unbound state, we used NMR to investigate the biophysical behavior of WIP^C, a C-terminal domain fragment of WIP that includes residues 407-503 and contains the WASp-binding site. In light of the poor spectral dispersion exhibited by WIP^C and the high occurrence (25%) of proline residues, we employed 5D-NMR ¹³C-detected NMR experiments with nonuniform sampling to accomplish full resonance assignment. Secondary chemical-shift analysis, ¹⁵N relaxation rates, and protection from solvent exchange all concurred in detecting transient structure located in motifs that span the WASp-binding site. Residues 446-456 exhibited a propensity for helical conformation, and an extended conformation followed by a short, capped helix was observed for residues 468-478. The ¹³C-detected approach allows chemical-shift assignment in the WIP^C polyproline stretches and thus sheds light on their conformation and dynamics. The effects of temperature on chemical shifts referenced to a denatured sample of the polypeptide demonstrate that heating reduces the structural character of WIP^C. Thus, we conclude that the disordered WIP^C fragment is comprised of regions with latent structure connected by flexible loops, an architecture with implications for binding affinity and function.