Abstract
HslVU is a bacterial ATP-dependent protease distantly related to eukaryotic proteasomes consisting of hexameric HslU ATPase and dodecameric HslV protease. As a homolog of the 20 S proteasome beta-subunits, HslV also uses the N-terminal threonine as the active site residue. However, unlike the proteasome that has only 6 active sites among the 14 beta-subunits, HslV has 12 active sites that could potentially contribute to proteolytic activity. Here, by using a series of HslV dodecamers containing different numbers of active sites, we demonstrate that like the proteasome, HslV with only approximately 6 active sites is sufficient to support full catalytic activity. However, a further reduction of the number of active sites leads to a proportional decrease in activity. Using proteasome inhibitors, we also demonstrate that substrate-mediated stabilization of the HslV-HslU interaction remains unchanged until the number of the active sites is decreased to approximately 6 but is gradually compromised upon further reduction. These results with a mathematical model suggest HslVU utilizes no more than 6 active sites at any given time, presumably because of the action of HslU. These results also suggest that each ATP-bound HslU subunit activates one HslV subunit and that substrate bound to the HslV active site stimulates the HslU ATPase activity by stabilizing the HslV-HslU interaction. We propose this mechanism plays an important role in supporting complete degradation of substrates while preventing wasteful ATP hydrolysis in the resting state by controlling the interaction between HslV and HslU through the catalytic engagement of the proteolytic active sites.
Highlights
ATP-dependent proteases are cellular machines that play essential roles in the controlled turnover of regulatory proteins and the clearance of damaged proteins
A further increase in the number of T1A-His subunits led to a proportional decrease in the rate of peptide hydrolysis, as revealed by plotting the observed rates of peptide hydrolysis against the average number of HslV subunits per dodecamer (Fig. 3B). These results suggest that approximately half of the 12 Thr1 active sites of HslV are sufficient for exhibiting its full peptidase activity, but a further reduction in the number of Thr1 residues leads to a decrease in peptide hydrolysis, presumably because of a lack of the active sites
ATP, ϳ6 of 12 active sites of HslV are sufficient for its full proteolytic activity
Summary
Z-GGL-AMC, carbobenzoxy-Gly-Gly-Leu-7-amido4-methyl coumarin; ATP␥S, adenosine 5Ј-O-(thiotriphosphate); NLVS, 4-hydroxy-5-iodo-3-nitrophenylacetyl-Leu-Leu-Leu-vinylsulfone; NTA, nitrilotriacetic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. Deletion of the Thr residues (T1⌬) causes a dramatic increase in the HslV-HslU interaction even in the absence of ATP [17]. We demonstrate that among the 12 Thr residues in HslV, only ϳ6 of them participate in the hydrolysis of substrates as well as in the stable interaction between HslV and HslU This conclusion is based on our findings that in the presence of HslU and ATP, increasing the inactive T1A subunits up to ϳ6 in a dodecamer causes little or no effect on the proteolytic activity of HslV toward all tested substrates and on proteasome inhibitor-mediated stabilization of the HslV-HslU interaction. Our data suggest that each ATP-bound HslU subunit conformationally communicates with one HslV subunit, and catalytic engagement of the HslV active sites stabilizes the HslV-HslU interaction to support efficient degradation of substrates
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