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

Abstract

In the AAA+ HsIUV protease, substrates are bound and unfolded by a ring hexamer of HsIU, before translocation through an axial pore and into the HsIV degradation chamber. Here, we show that the N-terminal residues of an Arc substrate initially bind in the HsIU 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 HsIU 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. HsIU-ΔI has negligible ATPase activity. Thus, the I domain plays an active role in coordinating substrate binding, ATP hydrolysis, and protein degradation by the HsIUV proteolytic machine. Published by Wiley-Blackwell. © 2011 The Protein Society.