NMR-Based Molecular view of the Biology and Biophysics of WIP, An Intrinsically Disordered Protein

Abstract

Intrinsically disordered proteins (IDPs) are multi-conformational polypeptides that lack a stable three-dimensional structure, forcing structural biology to reconsider the structure-function paradigm. The versatile IDPs play key roles in a multitude of biological processes, and, given their inherently flexible nature, nuclear magnetic resonance (NMR) is a leading method for investigating IDP behavior on the molecular level. Our research has focused on the intrinsically disordered WASp Interacting Protein (WIP) from human T cells, whose C-terminal binds Wiskott-Aldrich syndrome protein (WASp), and N-terminal interacts with actin, both crucial factors in mediating the cytoskeletal changes accompanying cell activation.A range of NMR experiments tailored for IDP study was employed to discover transient structural motifs in the two terminal WIP domains. Chemical shifts, relaxation rates, residual dipolar couplings and solvent exposure all concurred in identifying WIP segments exhibiting a secondary structure bias in their conformational ensemble. Remarkably, these transient structural elements echo the conformation of WIP bound to actin or WASp. In addition, in each domain we identified a previously unrecognized binding epitope, and later confirmed these with binding-induced effects observed by NMR.IDPs are essentially an ensemble of multiple fast-interchanging conformations, and we have utilized WIP also as a model to monitor this ensemble behavior under various conditions. Temperature effects were consistent with thermal unfolding and exhibited a strong correlation with structural propensities. Similarly, the effects of denaturing or crowding conditions could be evaluated with this approach. Finally, we obtain insight into how proteins fold and interact with peptidic ligands by following the effects of sub-stochiometric actin concentrations on the WIP conformational ensemble. Our findings highlight the potential impact of high-resolution NMR upon the field of IDPs, which can be enhanced by complementary biophysical and computational approaches.