Visualizing Arp2/3 complex activation mediated by binding of ATP and WASp using structural mass spectrometry

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• The actin-related protein (Arp) 2/3 complex is essential for eukaryotic cell motility by nucleating branched actin filaments.
• Activation of the Arp2/3 complex by ATP and WASp-family proteins is critical but mechanistically unclear.

Purpose of the Study:

• To elucidate the structural mechanisms of Arp2/3 complex activation by ATP and WASp.
• To investigate conformational changes induced by nucleotide and protein binding using advanced footprinting techniques.

Main Methods:

• Radiolytic protein footprinting coupled with mass spectrometry was employed in solution.
• This technique required only picomoles of the Arp2/3 complex, enabling analysis of this large assembly.

Main Results:

• ATP binding induces conformational changes in Arp3 (peptides 5-18, 212-225, 318-327) and Arp2 (peptide 300-316), consistent with nucleotide cleft closure.
• WASp binds bivalently to Arp2/3 complex at specific residues and subdomains (Arp3: peptides 162-191, 318-329; Arp2: peptides 66-80, 87-97).
• WASp binding leads to domain rearrangements in Arp2 and Arp3, suggesting a closed, active conformation consistent with an 'actin-dimer' model.

Conclusions:

• ATP binding triggers initial conformational changes in Arp2/3 complex, likely priming it for activation.
• WASp engagement induces significant structural rearrangements, stabilizing an active conformation of the Arp2/3 complex.
• These findings provide a structural basis for understanding Arp2/3 complex regulation in cell motility.