לוח שנה משולב

WASP-Interacting Protein (WIP) is a multifunctional key participant in mediating actin-related cytoskeletal changes in human T cells. WIP is also an intrinsically disordered protein (IDP), lacking any significant secondary or tertiary structure across its 503 residues, and thus defies the ordinarily reliable structure-function paradigm. Our research focuses on how interactions between this ‘hub’ multi-tasker and its various structured binding partners delicately control T cell destiny, in particular the role played by disorder-to-order transitions. Three such critical protein-protein contacts involve the WIP N-terminal domain (with actin), a proline-rich central segment (with cortactin) and the C-terminal domain (with Wiskott-Aldrich syndrome protein, WASP). The first two are of intermediate binding energy (KD ~ 50-3000 nM) and transiently modulate WIP interactions with the actin polymerization machinery. In contrast, the latter forms a tight complex with WASP and inhibits both its activity and eventual degradation in a phosphorylation-dependent manner, explaining why the hereditary Wiskott-Aldrich syndrome immunodeficiency results from WASP mutants unable to bind WIP.
As an IDP, WIP ‘structure’ is essentially an ensemble of multiple conformations contributing to function, and this complexity gives solution NMR – armed with new IDP-optimized methodologies – unrivaled insight into how IDPs exert their biological influence. We established that transient structure in free WIPN and WIPC echoes their bound conformations, uncovering novel binding epitopes in the process. We also observed subtle ensemble shifts induced by environmental factors, such as temperature, denaturant or crowding agents, revealing the biophysics governing WIP behavior in the cellular environment. We further investigated the largest conformational change, experienced by WIPC upon binding to WASP, by determining the contribution of various WIP epitopes to complex affinity, and eventually the structure of the WIP-WASP complex. Finally, we offer an unexpected structural explanation for phosphorylation-induced dissociation of this complex that may explain how this phospho-switch controls WASP degradation. Taken together our results provide a comprehensive map of WIP structure and dynamics and how these affect its interaction with T cell binding partners, and highlight the great impact of high-resolution NMR studies upon the field of biologically active unstructured proteins.