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.

中文翻译：

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通过核磁共振和分子成像确定的关键肌动蛋白调控WIP-WASp复合物形成的新结构见解

Wiskott–Aldrich综合征蛋白（WASp）仅在造血细胞中表达，并负责肌动蛋白依赖性过程，包括细胞激活，迁移和侵袭性。如前所述，WASp相互作用蛋白（WIP）的C末端结构域与WASp结合并通过保护其免受磷酸化依赖性方式的降解来调节其活性。WAS编码基因的突变导致原发性免疫缺陷Wiskott-Aldrich综合征（WAS）和X连锁性血小板减少症（XLT）。在这里，我们通过核磁共振（NMR）和体内实验对WIP的这一功能进行了初步的结构分析分子成像。片段WASp（20–158）和WIP（442–492）的共表达可对天然折叠的复合物进行纯化和结构表征，确定其形成具有α/β折叠和中央双翼β混合的特征性pleckstrin同源结构域-床单。WIP衍生肽，以其自由形式未结构化，通过短的结构元件环绕WASp并与WASp相互作用。Förster共振能量转移（FRET）和生化实验表明，在这些元素中，WIP残基454-456是WASp亲和力的主要贡献者，发现先前被忽视的449-451残基对WASp泛素化的影响最大，并且大概是退化。从这种互补的技术组合中获得的结果将WIP-WASp亲和力与防止降解相关联。