Abstract
A critical yet challenging step in protein complex assembly is the formation of a dimeric intermediate that serves as a seed for incorporating additional subunits. We hypothesized that this step could be facilitated by "bi-handed" chaperones that recognize two different subunits through distinct domains (hands). However, whether such chaperones exist remained unknown. Here, we identify AAGAB as a bona fide bi-handed chaperone. AAGAB uses its C-terminal domain (CTD) to bind the α subunit and its GTPase-like domain (GD) to bind the σ2 subunit of the AP2 adaptor complex, a central player in membrane trafficking. AAGAB first recruits α via its CTD; σ2 then joins through interaction with α, forming a conformationally immature α:σ2 hemicomplex at the CTD. This hemicomplex is subsequently transferred to the GD via a GD:σ2 binding interface, accompanied by conformational maturation. These findings establish AAGAB as the founding member of a bi-handed chaperone family and reveal an intramolecular handover mechanism that underlies their mode of action.