Wiskott-Aldrich syndrome protein (WASp) is exclusively expressed in hematopoietic cells and responsible for actin-dependent processes, including cellular activation, migration, and invasiveness. The C-terminal domain of WASp-Interacting Protein (WIP) binds to WASp and regulates its activity by shielding it from degradation in a phosphorylation dependent manner as we previously demonstrated. Mutations in the WAS-encoding gene lead to the primary immunodeficiencies Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT). Here, we shed a first structural light upon this function of WIP using nuclear magnetic resonance (NMR) and in vivo molecular imaging. Coexpression of fragments WASp(20-158) and WIP(442-492) allowed the purification and structural characterization of a natively folded complex, determined to form a characteristic pleckstrin homology domain with a mixed α/β-fold and central two-winged β-sheet. The WIP-derived peptide, unstructured in its free form, wraps around and interacts with WASp through short structural elements. Förster resonance energy transfer (FRET) and biochemical experiments demonstrated that, of these elements, WIP residues 454-456 are the major contributor to WASp affinity, and the previously overlooked residues 449-451 were found to have the largest effect upon WASp ubiquitylation and, presumably, degradation. Results obtained from this complementary combination of technologies link WIP-WASp affinity to protection from degradation. Our findings about the nature of WIP·WASp complex formation are relevant for ongoing efforts to understand hematopoietic cell behavior, paving the way for new therapeutic approaches to WAS and XLT.

intracellular nucleotide binding sites. Fluorescence spectra of ANAP labelled Kir6.2 subunits were acquired after exposure to increasing concentrations of TNP-ATP. Binding of TNP-ATP was measured as quenching of the ANAP fluorescence at 470 nm and could be competed off with addition of unlabelled ATP. Both Kir6.2-I182ANAP and F183ANAP co-expressed with SUR1 bound TNP-ATP in the low mM range in the absence of Mg 2þ , comparable to the apparent affinity for inhibition of wild-type Kir6.2/SUR1 by TNP-ATP. Similar apparent affinities were obtained from C-terminally truncated or GFP-tagged Kir6.2-F183 constructs expressed without SUR1. A mutation (G334D) in the ATP binding site of Kir6.2 that does not affect intrinsic K ATP gating greatly decreased the apparent nucleotide affinity. Similar effects on TNP-ATP binding were observed with the introduction of the C166S gating mutation (which increases open probability), suggesting that conformational changes at the pore of Kir6.2 can influence nucleotide binding.

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