The complex formed between Wiskott-Aldrich syndrome protein (WASP) and WASP-Interacting Protein (WIP) is a potent regulator of cytoskeletal changes in hematopoietic cells. Mutations in the WASP N-terminal domain cause the primary immunodeficiencies Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT). Using NMR we determine the structure of the WASP/WIP complex and provide a first molecular view of this key biochemical junction. The central feature of this complex is the extensive binding interface formed by four WIP epitopes that wrap around the canonical EVH1 domain. Phosphoregulation of the WIP chaperone function occurs on two tyrosine residues, and not a distal serine residue as suggested earlier, and involves selective dissociation of the fourth epitope (epiIV), thereby exposing two established WASP ubiquitylation sites. Single-residue WAS-inducing mutations with mild phenotypes all influence the same WASP-epiIV interface, suggesting this is the molecular mechanism behind WAS. This structural viewpoint of WASP/WIP biology creates a much-needed molecular context for understanding hematopoietic cytoskeletal regulation in homeostasis and in WAS/XLT and is expected to be invaluable in the search for new therapeutic approaches to these rare diseases.
Inna Sasson, Saja Baluom, Adi Halle-Bikovski et al.· Journal of Structural Biolog...· 0 citations
Conformational dynamics in toxin inhibitors are an important contributor to ion channel affinity, yet toxin multidimensional energy landscapes remain largely unexplored. In the current work, we combine parallel-bias metadynamics-metainference (PBMetaD) simulations with relaxation dispersion NMR to define, at atomistic resolution, the thermodynamics and kinetics of Hui1, a de novo three disulfide toxin derived from the SAK-I family that targets K+-channels. Using the three χ3 disulfide dihedrals as collective variables, an extensive 48-replica well-tempered PBmetaD simulation (16.2 μs cumulative sampling) resulted in a fully converged three-dimensional (3D) free-energy surface comprising eight Hui1 conformers. These basins account for ∼96% of the bias-weighted ensemble and partition into four low- and four high-energy states separated by 7.5 kJ/mol associated with the (-) and (+)Cys12-Cys28 χ3 states, respectively. Transition-state theory identifies rota-isomerization of Cys3-Cys35 as the slowest, and therefore rate-determining, coordinate, while the analogous motions around Cys12-Cys28 and Cys17-Cys32 are ∼5-fold faster. 15N R1ρ relaxation dispersion NMR measurements confirmed these kinetics, identifying two structurally distinct residue clusters exhibiting intermediate and faster exchange processes. The Key Interaction Finder (KIF) approach reveals that Cys17-Cys32 conformation modifies connectivities within a dense interaction network between Cys17 and residues Gln14, Tyr23, Arg24, and Lys29, and correlates with the accessibility of residues Tyr23 and Arg24 of the helix-kink-helix region for interaction with the channel vestibule. Our work establishes PBMetaD as a powerful framework for mapping coupled disulfide and backbone dynamics in toxins and reveals specific conformers and interactions likely to control K+ channel recognition.
Chen Timsit Shmueli, Miriam Gulman, D. T. Major et al.· Journal of the American Chem...· 0 citations
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