The structures of HslU and the ATP-dependent protease HslU–HslV

The degradation of cytoplasmic proteins is an ATP-dependent process1. Substrates are targeted to a single soluble protease, the 26S proteasome2,3, in eukaryotes and to a number of unrelated proteases in prokaryotes4,5. A surprising link emerged with the discovery of the ATP-dependent protease HslVU (heat shock locus VU)6,7,8 in Escherichia coli. Its protease component HslV shares ∼20% sequence similarity6 and a conserved fold9 with 20S proteasome β-subunits. HslU is a member of the Hsp100 (Clp) family of ATPases. Here we report the crystal structures of free HslU and an 820,000 relative molecular mass complex of HslU and HslV–the first structure of a complete set of components of an ATP-dependent protease. HslV and HslU display sixfold symmetry, ruling out mechanisms of protease activation that require a symmetry mismatch between the two components. Instead, there is conformational flexibility and domain motion in HslU and a localized order–disorder transition in HslV. Individual subunits of HslU contain two globular domains in relative orientations that correlate with nucleotide bound and unbound states. They are surprisingly similar to their counterparts in N-ethylmaleimide-sensitive fusion protein10,11, the prototype of an AAA-ATPase. A third, mostly α-helical domain in HslU mediates the contact with HslV and may be the structural equivalent of the amino-terminal domains in proteasomal AAA-ATPases.