HslVU

Abstract: The crystal structure of the 20S proteasome from the yeast Saccharomyces cerevisiae shows that its 28 protein subunits are arranged as an (α1...α7, β1...β7)2 complex in four stacked rings and occupy unique locations. The interior of the particle, which harbours the active sites, is only accessible by some very narrow side entrances. The β-type subunits are synthesized as proproteins before being proteolytically processed for assembly into the particle. The proforms of three of the seven different β-type subunits, (β1/PRE3, β2/PUP1 and β5/PRE2, are cleaved between the threonine at position 1 and the last glycine of the pro-sequence, with release of the active-site residue Thr 1. These three β-type subunits have inhibitor-binding sites, indicating that PRE2 has a chymotrypsin-like and a trypsin-like activity and that PRE3 has peptidylglutamyl peptide hydrolytic specificity. Other β-type subunits are processed to an intermediate form, indicating that an additional nonspecific endopeptidase activity may exist which is important for peptide hydrolysis and for the generation of ligands for class I molecules of the major histocompatibility complex.

Abstract: The catalytic mechanism of the 20S proteasome from the archaebacterium Thermoplasma acidophilum has been analyzed by site-directed mutagenesis of the beta subunit and by inhibitor studies. Deletion of the amino-terminal threonine or its mutation to alanine led to inactivation of the enzyme. Mutation of the residue to serine led to a fully active enzyme, which was over ten times more sensitive to the serine protease inhibitor 3,4-dichloroisocoumarin. In combination with the crystal structure of a proteasome-inhibitor complex, the data show that the nucleophilic attack is mediated by the amino-terminal threonine of processed beta subunits. The conservation pattern of this residue in eukaryotic sequences suggests that at least three of the seven eukaryotic beta-type subunit branches should be proteolytically inactive.

Abstract: Escherichia coli FtsH is an essential integral membrane protein that has an AAA-type ATPase domain at its C-terminal cytoplasmic part, which is homologous to at least three ATPase subunits of the eukaryotic 26S proteasome. We report here that FtsH is involved in degradation of the heat-shock transcription factor sigma 32, a key element in the regulation of the E. coli heat-shock response. In the temperature-sensitive ftsH1 mutant, the amount of sigma 32 at a non-permissive temperature was higher than in the wild-type under certain conditions due to a reduced rate of degradation. In an in vitro system with purified components, FtsH catalyzed ATP-dependent degradation of biologically active histidine-tagged sigma 32. FtsH has a zinc-binding motif similar to the active site of zinc-metalloproteases. Protease activity of FtsH for histidine-tagged sigma 32 was stimulated by Zn2+ and strongly inhibited by the heavy metal chelating agent o-phenanthroline. We conclude that FtsH is a novel membrane-bound, ATP-dependent metalloprotease with activity for sigma 32. These findings indicate a new mechanism of gene regulation in E. coli.

Abstract: 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.