Abstract
HslVU is an ATP-dependent proteolytic complex present in certain bacteria and in the mitochondrion of some primordial eukaryotes, including deadly parasites such as Leishmania. It is formed by the dodecameric protease HslV and the hexameric ATPase HslU, which binds via the C-terminal end of its subunits to HslV and activates it by a yet unclear allosteric mechanism. We undertook the characterization of HslV from Leishmania major (LmHslV), a trypanosomatid that expresses two isoforms for HslU, LmHslU1 and LmHslU2. Using a novel and sensitive peptide substrate, we found that LmHslV can be activated by peptides derived from the C-termini of both LmHslU1 and LmHslU2. Truncations, Ala- and D-scans of the C-terminal dodecapeptide of LmHslU2 (LmC12-U2) showed that five out of the six C-terminal residues of LmHslU2 are essential for binding to and activating HslV. Peptide cyclisation with a lactam bridge allowed shortening of the peptide without loss of potency. Finally, we found that dodecapeptides derived from HslU of other parasites and bacteria are able to activate LmHslV with similar or even higher efficiency. Importantly, using electron microscopy approaches, we observed that the activation of LmHslV was accompanied by a large conformational remodeling, which represents a yet unidentified layer of control of HslV activation.
Highlights
ATP-dependent proteases are present in all living organisms [1] and play major roles in intracellular proteolysis, and in cell homeostasis
As the protease is normally expressed with a N-terminal mitochondrial signal peptide that is removed upon translocation to the mitochondrion, we produced a modified form of the protein, in which the signal peptide has been replaced by a N-terminal methionine that precedes the ‘TTI’ motif required for activity (Figure S1A)
This configuration is that observed in E. coli HslV, for which it is known that the N-terminal methionine is cleaved upon expression, exposing a N-terminal threonine (Thr1) that is the catalytic residue
Summary
ATP-dependent proteases are present in all living organisms [1] and play major roles in intracellular proteolysis, and in cell homeostasis. The major ATP-dependent protease is the 26S proteasome responsible for the degradation of poly-ubiquitylated proteins This multi-enzymatic complex is composed of the 20S catalytic core that is capped at one or both ends by its regulatory subunit, the 19S complex or PA700 [2,3]. HslVU, called ClpYQ, is a proteolytic complex initially described in Escherichia coli (E. coli) [4,5,6] It is a self-compartmentalized protease constituted of two HslV hexameric rings that enclose a proteolytic chamber containing 12 active sites whose catalytic residues are the N-terminal Thr (Thr1) of each subunit. Substrate recognition is most likely performed by the HslU regulator that unfolds and translocates the captured protein into the internal proteolytic chamber of HslV, by an ATP-dependent mechanism [6,8,9,10]
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