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A Quantitative Dot Blot Assay for AAV Titration and Its Use for Functional Assessment of the Adeno-associated Virus Assembly-activating Proteins
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Identification and structural basis for a novel interaction between Vav2 and Arap3.

Bo Wu1, Fengsong Wang, Jiahai Zhang

  • 1Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, PR China.

Journal of Structural Biology
|July 4, 2012
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Summary

Vav2, a guanine nucleotide exchange factor, interacts with Arap3, a GTPase-activating protein. This study reveals the structural basis for Vav2

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Area of Science:

  • Molecular and Cellular Biology
  • Protein Interactions and Structural Biology

Background:

  • Vav2 is a guanine nucleotide exchange factor (GEF) crucial for Rac1 GTPase activity.
  • Vav2 regulates fundamental cellular processes like migration, neuronal development, and phagocytosis.
  • Identifying novel Vav2 interaction partners is key to understanding its diverse functions.

Purpose of the Study:

  • To identify and characterize novel interaction partners of Vav2.
  • To elucidate the structural basis of the interaction between Vav2 and its novel partner, Arap3.
  • To determine the specificity of the Vav2 SH2 domain for phosphotyrosine recognition.

Main Methods:

  • In vitro and in vivo interaction assays to identify Vav2-Arap3 binding.
  • Isothermal Titration Calorimetry (ITC) and NMR chemical shift perturbation to quantify binding affinity.
  • X-ray crystallography and NMR spectroscopy to determine the solution structures of Vav2 SH2 domain.

Main Results:

  • Arap3, a dual RhoA/Arf6 GTPase-activating protein (GAP), was identified as a novel Vav2 interaction partner.
  • Vav2 SH2 domain binds directly to phosphorylated Y1403 and Y1408 on Arap3 with high affinity (Kd ≈ 0.27 μM and 1.40 μM).
  • Structural analysis revealed a specific pY +3 pocket in the Vav2 SH2 domain, mediated by a Phe residue, dictating recognition specificity.

Conclusions:

  • Arap3 is a novel interaction partner of Vav2, suggesting a role in regulating Rac1 signaling pathways.
  • The Vav2 SH2 domain exhibits specific recognition for phosphotyrosine residues at the pY +3 position.
  • The determined structures provide insights into the molecular mechanisms underlying Vav2-Arap3 interaction and specificity.