, Methods Cloning of LmHslV: LmHslV gene was amplified with its mitochondrial signal sequence using DNA from Leishmania major Friedlin strain and subcloned into PQE60 vector to obtain the C-terminally 6xHis-tagged LmHslV protein. For expression in E. coli, LmHslV-6His was subcloned without its mitochondrial targeting signal into the vector pRSET-B. Protein expression: The proteins of interest (L. major and E. coli HslV, both with a C-terminal 6xHis tag), were expressed overnight at 20 ? C in E. coli BL21 (LB medium + ampicilline 100 µg/mL), by adding 1 mM IPTG in a culture with an OD between 0.5 and 0.8. Protein purification by Ni 2+ -NTA chromatography: All purification steps were performed at 4 ? C. Bacteria cultures (usually 500 mL) were centrifuged at 4000 RPM for 15 min, the pellet was collected, washed with PBS, then resuspended in 45 mL lysis buffer (PBS 2X, 10 mM Tris-HCl pH 8.0, 1 mM EDTA). Bacteria were then lysed with a high-pressure homogeneizer (Emulsiflex-C3, Avestin, Ottawa, Canada). 5 mL of 10% Triton X-100 (1% final) was added to the homogenate, which was then clarified by centrifugation (1,2000× g, 20 min). The supernatant was collected and incubated overnight with 500 µL of Ni-NTA beads (Qiagen, les Ulis, France) pre-equilibrated with PBS 2X, All peptides (Table 1 and Table S1) were assembled on a 2-chloro-trityl resin following either manual or microwave-assisted solid phase synthetic protocols with Fmoc as the N?-protecting group and HBTU or HATU/DIEA as coupling agents. Their synthesis as well that of the substrate JMV4482 (Z-EVNL-AMC) routinely used for activity tests are described in detail in the Supplementary Materials. Suc-LLVY-AMC and Z-GGL-AMC were purchased from Bachem. pET-Duet-1 plasmid with inducible E. coli C-terminally 6His-tagged HslV were kindly given by Dr Chin Ha

, As the initial iterative alignments of negatively stained LmHslV particles revealed common structural features with other already characterized HslV, particles were then consequently aligned iteratively against the envelop of bacterial HslV (pdb 1NED) filtered at 40 Å using the multi-reference alignment program, Briefly, the phase-contrast-transfer function was corrected by phase flipping using defocus parameters obtained using Gctf, vol.47

, Sequence alignments: All sequence alignments were performed using the ClustalW software

, Supplementary Materials: Supplementary Materials can be

O. C. , J. H. , M. P. , P. B. Patrick-bastien, ). et al., Realization of experiments, Author Contributions: Conceptualization

R. T. Sauer and T. A. Baker, AAA+ proteases: ATP-fueled machines of protein destruction, Annu. Rev. Biochem, vol.80, pp.587-612, 2011.

D. Finley, X. Chen, K. J. Walters, and . Gates, Channels, and Switches: Elements of the Proteasome Machine, Trends Biochem. Sci, vol.41, pp.77-93, 2015.

G. A. Collins and A. L. Goldberg, The Logic of the 26S Proteasome, vol.169, pp.792-806, 2017.

D. Missiakas, F. Schwager, J. M. Betton, C. Georgopoulos, and S. Raina, Identification and characterization of HsIV HsIU (ClpQ ClpY) proteins involved in overall proteolysis of misfolded proteins in Escherichia coli, EMBO J, vol.15, pp.6899-6909, 1996.

M. Rohrwild, O. Coux, H. C. Huang, R. P. Moerschell, S. J. Yoo et al., HslV-HslU: A novel ATP-dependent protease complex in Escherichia coli related to the eukaryotic proteasome, Proc. Natl. Acad. Sci, vol.93, pp.5808-5813, 1996.

S. J. Yoo, J. H. Seol, D. H. Shin, M. Rohrwild, M. S. Kang et al., Purification and characterization of the heat shock proteins HslV and HslU that form a new ATP-dependent protease in Escherichia coli, J. Biol. Chem, vol.271, pp.14035-14040, 1996.

A. F. Neuwald, L. Aravind, J. L. Spouge, and E. V. Koonin, AAA+: A class of chaperone-like ATPases associated with the assembly, operation, and disassembly of protein complexes, Genome Res, vol.9, pp.27-43, 1999.

M. C. Sousa, C. B. Trame, H. Tsuruta, S. M. Wilbanks, V. S. Reddy et al., Crystal and solution structures of an HslUV protease-chaperone complex, Cell, vol.103, pp.633-643, 2000.

J. Wang, J. J. Song, I. S. Seong, M. C. Franklin, S. Kamtekar et al., Nucleotide-dependent conformational changes in a protease-associated ATPase HsIU, Structure, vol.9, pp.1107-1116, 2001.

J. A. Yakamavich, T. A. Baker, and R. T. Sauer, Asymmetric nucleotide transactions of the HslUV protease, J. Mol. Biol, vol.380, pp.946-957, 2008.

J. Wang, J. J. Song, M. C. Franklin, S. Kamtekar, Y. J. Im et al., Crystal structures of the HslVU peptidase-ATPase complex reveal an ATP-dependent proteolysis mechanism, Structure, vol.9, pp.177-184, 2001.

E. Park, J. W. Lee, H. M. Yoo, B. H. Ha, J. Y. An et al., Structural Alteration in the Pore Motif of the Bacterial 20S Proteasome Homolog HslV Leads to Uncontrolled Protein Degradation, J. Mol. Biol, vol.425, pp.2940-2954, 2013.

M. Bochtler, C. Hartmann, H. K. Song, G. P. Bourenkov, H. D. Bartunik et al., The structures of HsIU and the ATP-dependent protease HsIU-HsIV, Nature, vol.403, pp.800-805, 2000.

M. C. Sousa, B. M. Kessler, H. S. Overkleeft, and D. B. Mckay, Crystal structure of HslUV complexed with a vinyl sulfone inhibitor: corroboration of a proposed mechanism of allosteric activation of HslV by HslU, J. Mol. Biol, vol.318, pp.779-785, 2002.

R. Ramachandran, C. Hartmann, H. K. Song, R. Huber, and M. Bochtler, Functional interactions of HslV (ClpQ) with the ATPase HslU (ClpY), Proc. Natl. Acad. Sci, vol.99, pp.7396-7401, 2002.

S. E. Chuang, V. Burland, G. Plunkett, D. L. Daniels, and F. R. Blattner, Sequence analysis of four new heat-shock genes constituting the hslTS/ibpAB and hslVU operons in Escherichia coli, Gene, vol.134, pp.1-6, 1993.

M. Bochtler, L. Ditzel, M. Groll, and R. Huber, Crystal structure of heat shock locus V (HslV) from Escherichia coli, Proc. Natl. Acad. Sci, vol.94, pp.6070-6074, 1997.

B. Couvreur, R. Wattiez, A. Bollen, P. Falmagne, D. Le-ray et al., Eubacterial HslV and HslU subunits homologs in primordial eukaryotes, Mol. Biol. Evol, vol.19, pp.2110-2117, 2002.

C. Gille, A. Goede, C. Schlöetelburg, R. Preissner, P. M. Kloetzel et al., A comprehensive view on proteasomal sequences: implications for the evolution of the proteasome, J. Mol. Biol, vol.326, pp.1437-1448, 2003.

M. X. Ruiz-gonzález and I. Marín, Proteasome-related HslU and HslV genes typical of eubacteria are widespread in eukaryotes, J. Mol. Evol, vol.63, pp.504-512, 2006.

M. Groll, M. Bochtler, H. Brandstetter, T. Clausen, and R. Huber, Molecular machines for protein degradation, Chembiochem, vol.6, pp.222-256, 2005.

Z. Li, M. E. Lindsay, S. A. Motyka, P. T. Englund, and C. C. Wang, Identification of a bacterial-like HslVU protease in the mitochondria of Trypanosoma brucei and its role in mitochondrial DNA replication, PLoS Pathog, 2008.

S. Rathore, S. Jain, D. Sinha, M. Gupta, M. Asad et al., Disruption of a mitochondrial protease machinery in Plasmodium falciparum is an intrinsic signal for parasite cell death, Cell Death Dis, 2011.

M. Chrobak, S. Förster, S. Meisel, R. Pfefferkorn, F. Förster et al., Leishmania donovani HslV does not interact stably with HslU proteins, Int. J. Parasitol, vol.42, pp.329-339, 2012.

S. Jain, S. Rathore, M. Asad, M. E. Hossain, D. Sinha et al., Mohmmed, A. The prokaryotic ClpQ protease plays a key role in growth and development of mitochondria in Plasmodium falciparum, Cell. Microbiol, vol.15, pp.1660-1673, 2013.

D. Mbang-benet, Y. Sterkers, C. Morelle, N. Kebe, L. Crobu et al., The bacterial-like HslVU protease complex subunits are involved in the control of different cell cycle events in trypanosomatids, Acta Trop, vol.131, pp.22-31, 2014.
URL : https://hal.archives-ouvertes.fr/hal-02518501

G. De-bettignies and O. Coux, Proteasome inhibitors: Dozens of molecules and still counting, Biochimie, vol.92, pp.1530-1545, 2010.

G. Lin, D. Li, L. P. De-carvalho, H. Deng, H. Tao et al., Inhibitors selective for mycobacterial versus human proteasomes, Nature, vol.461, pp.624-626, 2009.

H. Li, A. J. O'donoghue, W. A. Van-der-linden, S. C. Xie, E. Yoo et al., Structure-and function-based design of Plasmodium-selective proteasome inhibitors, Nature, vol.530, pp.233-236, 2016.

S. Khare, A. S. Nagle, A. Biggart, Y. H. Lai, F. Liang et al., Proteasome inhibition for treatment of leishmaniasis, Chagas disease and sleeping sickness, Nature, vol.537, pp.229-233, 2016.

Y. Rashid, M. Kamran-azim, Z. S. Saify, K. M. Khan, and R. Khan, Small molecule activators of proteasome-related HslV peptidase, Bioorg. Med. Chem. Lett, vol.22, pp.6089-6094, 2012.

K. H. Sung, S. Y. Lee, and H. Song, Structural and Biochemical Analyses of the Eukaryotic Heat Shock Locus V (HslV) from Trypanosoma brucei, J. Biol. Chem, vol.288, pp.23234-23243, 2013.

K. H. Sung and H. K. Song, Insights into the molecular evolution of HslU ATPase through biochemical and mutational analyses, PLoS ONE, vol.9, 2014.

G. Ramasamy, D. Gupta, A. Mohmmed, and V. S. Chauhan, Characterization and localization of Plasmodium falciparum homolog of prokaryotic ClpQ/HslV protease, Mol. Biochem. Parasitol, vol.152, pp.139-148, 2007.

I. S. Seong, M. S. Kang, M. K. Choi, J. W. Lee, O. J. Koh et al., The C-terminal tails of HslU ATPase act as a molecular switch for activation of HslV peptidase, J. Biol. Chem, vol.277, pp.25976-25982, 2002.

J. H. Seol, S. J. Yoo, D. H. Shin, Y. K. Shim, M. S. Kang et al., The heat-shock protein HslVU from Escherichia coli is a protein-activated ATPase as well as an ATP-dependent proteinase, Eur. J. Biochem, vol.247, pp.1143-1150, 1997.

M. C. Manning, M. Illangasekare, and R. W. Woody, Circular dichroism studies of distorted alpha-helices, twisted beta-sheets, and beta turns, Biophys. Chem, vol.31, pp.77-86, 1988.

A. Miranda, S. C. Koerber, J. Gulyas, S. L. Lahrichi, A. G. Craig et al., Conformationally restricted competitive antagonists of human/rat corticotropin-releasing factor, J. Med. Chem, vol.37, pp.1450-1459, 1994.

N. Greenfield and G. D. Fasman, Computed circular dichroism spectra for the evaluation of protein conformation, Biochemistry, vol.8, pp.4108-4116, 1969.

A. V. Terskikh, J. M. Le-doussal, R. Crameri, I. Fisch, J. P. Mach et al., Peptabody": A new type of high avidity binding protein, Proc. Natl. Acad. Sci, vol.94, pp.1663-1668, 1997.

J. Wang, A corrected quaternary arrangement of the peptidase HslV and atpase HslU in a cocrystal structure, J. Struct. Biol, vol.134, pp.15-24, 2001.

H. K. Song, M. Bochtler, M. K. Azim, C. Hartmann, R. Huber et al., Isolation and characterization of the prokaryotic proteasome homolog HslVU (ClpQY) from Thermotoga maritima and the crystal structure of HslV, Biophys. Chem, vol.100, pp.437-452, 2003.

L. Shi and L. E. Kay, Tracing an allosteric pathway regulating the activity of the HslV protease, vol.111, pp.2140-2145, 2014.

G. Tang, L. Peng, P. R. Baldwin, D. S. Mann, W. Jiang et al., EMAN2: an extensible image processing suite for electron microscopy, J. Struct. Biol, vol.157, pp.38-46, 2007.

M. Van-heel, G. Harauz, E. V. Orlova, R. Schmidt, and M. Schatz, A new generation of the IMAGIC image processing system, J. Struct. Biol, vol.116, pp.17-24, 1996.

P. Bron, E. Giudice, J. Rolland, R. M. Buey, P. Barbier et al., Apo-Hsp90 coexists in two open conformational states in solution, Biol. Cell, vol.100, pp.413-425, 2008.
URL : https://hal.archives-ouvertes.fr/hal-00493797

K. Zhang and . Gctf, Real-time CTF determination and correction, J. Struct. Biol, vol.193, pp.1-12, 2016.

M. Van-heel, B. Gowen, R. Matadeen, E. V. Orlova, R. Finn et al., Single-particle electron cryo-microscopy: towards atomic resolution, Q. Rev. Biophys, vol.33, pp.307-369, 2000.

E. F. Pettersen, T. D. Goddard, C. C. Huang, G. S. Couch, D. M. Greenblatt et al., UCSF Chimera-a visualization system for exploratory research and analysis, J. Comput. Chem, vol.25, pp.1605-1612, 2004.