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

ACS Chem Biol. 2018 Jan 19;13(1):100-109. doi: 10.1021/acschembio.7b00486. Epub 2017 Dec 7.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actins / metabolism
  • Binding Sites
  • Cytoskeletal Proteins / chemistry*
  • Cytoskeletal Proteins / genetics
  • Cytoskeletal Proteins / immunology
  • Cytoskeletal Proteins / metabolism*
  • Epitopes
  • Fluorescence Resonance Energy Transfer
  • Humans
  • Intracellular Signaling Peptides and Proteins / chemistry*
  • Intracellular Signaling Peptides and Proteins / genetics
  • Intracellular Signaling Peptides and Proteins / immunology
  • Intracellular Signaling Peptides and Proteins / metabolism*
  • Jurkat Cells
  • Magnetic Resonance Spectroscopy
  • Molecular Chaperones / chemistry
  • Molecular Chaperones / metabolism
  • Molecular Imaging / methods
  • Multiprotein Complexes
  • Mutation
  • Protein Domains
  • Protein Folding
  • Ubiquitination
  • Wiskott-Aldrich Syndrome Protein / chemistry*
  • Wiskott-Aldrich Syndrome Protein / genetics
  • Wiskott-Aldrich Syndrome Protein / metabolism*

Substances

  • Actins
  • Cytoskeletal Proteins
  • Epitopes
  • Intracellular Signaling Peptides and Proteins
  • Molecular Chaperones
  • Multiprotein Complexes
  • WAS protein, human
  • WIPF1 protein, human
  • Wiskott-Aldrich Syndrome Protein