New Structural Insights into Formation of the Key Actin Regulating WIP-WASp Complex Determined by NMR and Molecular Imaging

Abstract

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.

WIP, YAP/TAZ and Actin Connections Orchestrate Development and Transformation in the Central Nervous System

“…Binding of the N-terminal domain of WASP by the WIP C-terminal domain was demonstrated in initial descriptions of the complex (Ramesh et al, 1997), and its contribution to shield WASP proteasome-mediated degradation was identified soon after (Sasahara et al, 2002). Advanced nuclear magnetic resonance structures of purified co-expressed fragments from and have confirmed these results and unraveled WIP's wrapping around WASP (Halle-Bikovski et al, 2018). WIP residues a.a. 454-456 are the major contributor to WASP affinity, while residues a.a. 449-451 have the greatest effect on WASP phosphorylation, and likely its degradation.…”

The Actin Regulators Involved in the Function and Related Diseases of Lymphocytes

“…Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency disease characterized by thrombocytopenia, eczema, episodes of fever, bloody diarrhea, recurrent bacterial infections, innate and adaptive immune deficiency, and a high rate of autoimmunity and malignancies ( 60 , 61 ). The disease is due to mutations of the gene which encodes the WAS protein (WASp) ( 62 ).…”

The Disordered Cellular Multi-Tasker WIP and Its Protein–Protein Interactions: A Structural View

“…More importantly, the analysis also identified a previously undetected fourth segment (residues EDEWES, 447–452) with a strong helical tendency and high conservation level (DDFE, residues 417–420 in CR16, or 394–397 of WICH), suggesting a potential involvement in EVH1 binding ( Figure 5 C,D) [ 106 ]. Indeed, an NMR investigation of a complex of the T cell WASp EVH1 domain bound to an extended WIP polypeptide including this additional epitope (residues 442–492) showed the DEWE segment to adopt a turn conformation and interact with a helical segment (ENQRLFE, WASp residues 31–37) preceding the β-sandwich and overlooked in earlier structural studies [ 107 ]. Homologs of this additional helix appear in N-WASp (ENESLFT, residues 23–29) and in related pleckstrin homology (PH) domains [ 108 , 109 ], suggesting it should be included in the functional EVH1 domain.…”

“…While WIP mutated at the FYF (454–456) epitope lost the ability to bind WASp, loss of the polyproline and DEWE (448–451) epitopes incurred equally significant reductions in affinity to WASp. In addition, of all epitopes, the DEWE was shown to have the greatest effect upon ubiquitylation levels, indicating that this additional binding interface was important for protecting WASp from proteasomal degradation [ 107 ]. The mechanism by which this occurs in yet unclear, since confirmed WASp ubiquitylation sites K76 and K81 [ 111 ] are distant from DEWE interaction surface, and it is possible that this interaction interferes with another component of the ubiquitylation machinery.…”

“…Structures of single-chain tethered complexes (in which the WIP polypeptide was connected to the N-WASp N-terminal) were either missing the relevant residues [ 34 , 104 ] or unable to observe changes induced by phospho-mimicking mutants S488D/S488E [ 113 ]. The later NMR-based analysis of the WIP/WASp complex did not find secondary structure differences between free and bound WIP for residues 483–492, and WIP dissociation-inducing mutations (as shown in cells) caused little (if any) change in chemical shifts within this region [ 107 ]. Concomitantly, introduction of the phospho-mimicking S488E mutation had no effect on WASp resonance frequencies (Halle-Bikovski A, Baluom S, Chill JH, unpublished results).…”