The Heat‐Shock Protein HslVU from Escherichia Coli is a Protein‐Activated ATPase as well as an ATP‐Dependent Proteinase

Abstract

HslVU in Escherichia coli a new two-component ATP-dependent protease composed of two heatshock proteins, the HslU ATPase and the HslV peptidase which is related to proteasome p-type subunits. Here we show that the reconstituted HslVU enzyme degrades not only certain hydrophobic peptides but also various polypeptides, including insulin B-chain, casein, and carboxymethylated lactalbumin. Maximal proteolytic activity was obtained with a 1 : 2 molar ratio of HslV (a 250-kDa complex) to HslU (a 450-kDa complex). By itself, HslV could slowly hydrolyze these polypeptides, but its activity was stimulated 20-fold by HslU in the presence of ATP. The ATPase activity of HslU was stimulated up to 50% by the protein substrates, but not by nonhydrolyzed proteins, and this stimulation further increased 2-3-fold in the presence of HslV. Concentrations of insulin B-chain that maximally stimulated the ATPase allowed maximal rates of the B-chain hydrolysis. Furthermore, addition of increasing amounts of ADP or N-ethylmaleimide reduced ATP and protein or peptide hydrolysis in parallel. Thus, HslVU is a proteinactivated ATPase as well as an ATP-dependent proteinase, and these processes appear linked. Surprisingly, the protein and peptide substrates do not compete with each other for hydrolysis. Lactacystin strongly inhibits protein degradation, but has little effect on peptide hydrolysis, while the peptide aldehydes are potent inhibitors of hydrolysis of small peptides, but have little effect on proteins. Thus, the functional requirements for ATP-dependent hydrolysis of peptides and proteins appear different.

The C-terminal Tails of HslU ATPase Act as a Molecular Switch for Activation of HslV Peptidase

“…After incubation, the samples were subjected to SDS-PAGE followed by staining with Coomassie Blue R-250. As previously reported (44), HslV efficiently degraded ␣-casein in the presence of HslU but much less efficiently in its absence (Fig. 5A).…”

Binding of MG132 or Deletion of the Thr Active Sites in HslV Subunits Increases the Affinity of HslV Protease for HslU ATPase and Makes This Interaction Nucleotide-independent

“…ATP hydrolysis by HslU is known to be stimulated by protein substrates (16,37), thus acting in favor of providing mechanical energy for substrate unfolding. Conversely, the increased ATP hydrolysis also leads to the generation of ADP, which causes the reverse movement of the HslU C-terminal tails back to the HslU-HslU subunit interfaces and thereby weakening the interaction between HslV and HslU.…”

“…Protein Expression and Purification-HslU and HslV were purified as described previously (2,16). pETDuet-1 vectors (Novagen) were used for co-expression of HslU and His-tagged HslV proteins.…”

“…Hexamerization of HslU itself is largely favored by the nucleotide binding to the ATPase (17). Moreover, HslV that by itself is a weak peptidase can be activated 1-2 orders of magnitude by ATP-bound HslU (15,16). ATP␥S, 4 a nonhydrolyzable ATP analog, also supports HslV-mediated hydrolysis of small peptides but not that of native protein substrates, such as SulA, suggesting the role of ATP hydrolysis by HslU in unfolding of protein substrates for their access to and subsequent degradation at the inner proteolytic chamber of dodecameric HslV (18).…”

“…Biochemical studies have shown that ATP binding and its subsequent hydrolysis by HslU play essential roles in controlling the proteolytic function of HslV and the interaction between HslU and HslV (10,12,(15)(16)(17). Hexamerization of HslU itself is largely favored by the nucleotide binding to the ATPase (17).…”