A structural mechanism for phosphorylation-dependent inactivation of the AP2 complex

Area of Science:

• Cell biology
• Molecular and structural biology

Background:

• Endocytosis relies on the AP2 clathrin adaptor complex to internalize transmembrane proteins.
• AP2 transitions from an inactive, closed state in the cytosol to an active, open conformation on the plasma membrane.
• Phosphorylation of membrane-activated AP2 is a known modification, but its functional significance remains unclear.

Purpose of the Study:

• To elucidate the regulatory mechanism of AP2 by NECAP, focusing on the role of phosphorylation.
• To determine the structural basis of NECAP-AP2 interaction using high-resolution cryo-electron microscopy (cryo-EM).
• To investigate how NECAP binding affects AP2 conformation and activity.

Main Methods:

• High-resolution cryo-electron microscopy (cryo-EM) to visualize NECAP-AP2 complexes.
• Utilizing membrane mimetics to generate conformationally open AP2 states.
• In vitro and in vivo biochemical and cellular assays to assess AP2 activity and NECAP regulation.

Main Results:

• Cryo-EM structures reveal NECAP binding to phosphorylated AP2, with key interactions involving the NECAP PHear domain.
• NECAP's second domain binds to conformationally open AP2 complexes.
• Both NECAP domains cooperate to bind and inactivate AP2 in both in vitro and in vivo settings.
• Genetic screens in *C. elegans* predicted the residues involved in NECAP-AP2 phosphorylation site coordination.

Conclusions:

• NECAP acts as a negative regulator of AP2, binding to its open and phosphorylated forms.
• The study proposes that phosphorylation serves as a signal for AP2 inactivation by NECAP.
• These findings provide structural insights into the regulation of endocytosis by adaptor protein complexes.