A J-modulated protonless NMR experiment characterizes the conformational ensemble of the intrinsically disordered protein WIP

Abstract

Intrinsically disordered proteins (IDPs) are multi-conformational polypeptides that lack a single stable three-dimensional structure. It has become increasingly clear that the versatile IDPs play key roles in a multitude of biological processes, and, given their flexible nature, NMR is a leading method to investigate IDP behavior on the molecular level. Here we present an IDP-tailored J-modulated experiment designed to monitor changes in the conformational ensemble characteristic of IDPs by accurately measuring backbone one- and two-bond J(N,Cα) couplings. This concept was realized using a unidirectional (H)NCO C-detected experiment suitable for poor spectral dispersion and optimized for maximum coverage of amino acid types. To demonstrate the utility of this approach we applied it to the disordered actin-binding N-terminal domain of WASp interacting protein (WIP), a ubiquitous key modulator of cytoskeletal changes in a range of biological systems. One- and two-bond J(N,Cα) couplings were acquired for WIP residues 2-65 at various temperatures, and in denaturing and crowding environments. Under native conditions fitted J-couplings identified in the WIP conformational ensemble a propensity for extended conformation at residues 16-23 and 45-60, and a helical tendency at residues 28-42. These findings are consistent with a previous study of the based upon chemical shift and RDC data and confirm that the WIP conformational ensemble is biased towards the structure assumed by this fragment in its actin-bound form. The effects of environmental changes upon this ensemble were readily apparent in the J-coupling data, which reflected a significant decrease in structural propensity at higher temperatures, in the presence of 8 M urea, and under the influence of a bacterial cell lysate. The latter suggests that crowding can cause protein unfolding through protein-protein interactions that stabilize the unfolded state. We conclude that J-couplings are a useful measureable in characterizing structural ensembles in IDPs, and that the proposed experiment provides a practical method for accurately performing such measurements, once again emphasizing the power of NMR in studying IDP behavior.

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 ].…”

“…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 ]. Interestingly, a lysate mimicking actin-deficient cellular crowding effects found a decrease in these structural tendencies, presumably due to non-specific protein–protein interactions offering higher stabilization to unfolded conformations of the ABM.…”

“…Notably, a partially pre-formed β-strand similar in significance to residues 45–62 was observed connecting the profilin-binding and amphipathic helix sequences (residues 17–25), a region highly conserved in WIP and its homologs. This may indicate a potential role for this linker in mediating the binding of actin, possibly by interacting with a yet-unknown binding partner [ 73 ].…”