New insights into the role of the disordered WIP N‐terminal domain revealed by NMR structural characterization

Abstract

WASp-interacting protein (WIP) is an intrinsically disordered 503-residue polypeptide with a key role in actin polymerization in activated T cells. Its interaction with actin is mediated by a pair of conserved actin binding motifs (ABMs) at the WIP N-terminus, a domain that has not been investigated in its unbound form. Here we use NMR to investigate the biophysical behavior of the N-terminal ABM in WIP using protonless 13 C 0 -detected spectroscopy. Secondary chemical shifts, residual dipolar couplings and temperature effects identify residual structure throughout the ABM, which exhibits transient helical and b-strand character for residues 30-42 and 44-62, respectively. These observed structural propensities echo the structure observed in the actin-bound state of the ABM. Furthermore, residues preceding the canonical ABM (17-25) and conserved among WIP-related proteins exhibit transient b-strand character, suggesting that the WIP N interaction epitope extends towards the N-terminal polyproline motif. This suggests a possible role for this region in mediating the WIP interaction with polyproline binders such as profilin. In revealing these features of the WIP ABM this study demonstrates the unique ability of NMR in characterizing unstructured domains and provides necessary information for further investigation of WIP-mediated protein-protein interactions.

13C Direct Detected NMR for Challenging Systems

“…Other motifs in which prolines are flanked by specific amino acids are also emerging, as described above for the interaction of α-synuclein with calcium ions through the DPD or EPE motifs. Similar motifs were also identified in different proteins, such as viral proteins ,− as well as in WASP-interacting protein (WIP), an intrinsically disordered polypeptide with a key role in actin polymerization in activated T cells. , Another interesting example in this context is provided by aromatic-proline pairs as recently monitored in different proteins ,, and illustrated below through the example of quail osteopontin . The 13 C detected experiments used to complete resonance assignment revealed in addition to the major form, also a subset of peaks with lower intensity that could be clearly identified in the clean region of CON spectra reporting the correlation of proline nitrogen (Figure ).…”

The Disordered Cellular Multi-Tasker WIP and Its Protein–Protein Interactions: A Structural View

“…Secondary backbone chemical shifts, temperature-induced chemical shift effects, backbone heteronuclear coupling constants, and analysis of residual dipolar couplings for this segment all concurred in identifying a helical propensity for residues 30–42 and partial extended β-strand character for residues 45–62. These propensities echo the ABM actin-bound structure, suggesting this pre-formed conformation may contribute to the actin binding mode [ 72 , 73 ]. As shown by changes in backbone J-couplings, this structural bias in the WIP conformational ensemble was obviated by exposure to denaturing conditions [ 73 ].…”