The I domain of the AAA+ HslUV protease coordinates substrate binding, ATP hydrolysis, and protein degradation

In the AAA+ HslUV protease, substrates are bound and unfolded by a ring hexamer of HslU, before translocation through an axial pore and into the HslV degradation chamber. Here, we show that the N‐terminal residues of an Arc substrate initially bind in the HslU axial pore, with key contacts mediated by a pore loop that is highly conserved in all AAA+ unfoldases. Disordered loops from the six intermediate domains of the HslU hexamer project into a funnel‐shaped cavity above the pore and are positioned to contact protein substrates. Mutations in these I‐domain loops increase K_M and decrease V_max for degradation, increase the mobility of bound substrates, and prevent substrate stimulation of ATP hydrolysis. HslU‐ΔI has negligible ATPase activity. Thus, the I domain plays an active role in coordinating substrate binding, ATP hydrolysis, and protein degradation by the HslUV proteolytic machine.