Structure of the drug target ClpC1 unfoldase in action provides insights on antibiotic mechanism of action
2022; Elsevier BV; Volume: 298; Issue: 11 Linguagem: Inglês
10.1016/j.jbc.2022.102553
ISSN1083-351X
AutoresKatharina Weinhäupl, Marcos Gragera, M. Teresa Bueno-Carrasco, Rocío Arranz, Olga Krandor, Tatos Akopian, Raquel Soares, Eric J. Rubin, Jan Félix, Hugo Fraga,
Tópico(s)Bacterial Genetics and Biotechnology
ResumoThe unfoldase ClpC1 is one of the most exciting drug targets against tuberculosis. This AAA+ unfoldase works in cooperation with the ClpP1P2 protease and is the target of at least four natural product antibiotics: cyclomarin, ecumicin, lassomycin, and rufomycin. Although these molecules are promising starting points for drug development, their mechanisms of action remain largely unknown. Taking advantage of a middle domain mutant, we determined the first structure of Mycobacterium tuberculosis ClpC1 in its apo, cyclomarin-, and ecumicin-bound states via cryo-EM. The obtained structure displays features observed in other members of the AAA+ family and provides a map for further drug development. While the apo and cyclomarin-bound structures are indistinguishable and have N-terminal domains that are invisible in their respective EM maps, around half of the ecumicin-bound ClpC1 particles display three of their six N-terminal domains in an extended conformation. Our structural observations suggest a mechanism where ecumicin functions by mimicking substrate binding, leading to ATPase activation and changes in protein degradation profile. The unfoldase ClpC1 is one of the most exciting drug targets against tuberculosis. This AAA+ unfoldase works in cooperation with the ClpP1P2 protease and is the target of at least four natural product antibiotics: cyclomarin, ecumicin, lassomycin, and rufomycin. Although these molecules are promising starting points for drug development, their mechanisms of action remain largely unknown. Taking advantage of a middle domain mutant, we determined the first structure of Mycobacterium tuberculosis ClpC1 in its apo, cyclomarin-, and ecumicin-bound states via cryo-EM. The obtained structure displays features observed in other members of the AAA+ family and provides a map for further drug development. While the apo and cyclomarin-bound structures are indistinguishable and have N-terminal domains that are invisible in their respective EM maps, around half of the ecumicin-bound ClpC1 particles display three of their six N-terminal domains in an extended conformation. Our structural observations suggest a mechanism where ecumicin functions by mimicking substrate binding, leading to ATPase activation and changes in protein degradation profile. Clp proteases are composed of two heptameric rings, forming a cylinder with 14 proteolytic sites compartmentalized within its central chamber (1Wang J. Hartling J.A. Flanagan J.M. The structure of ClpP at 2.3 A resolution suggests a model for ATP-dependent proteolysis.Cell. 1997; 91: 447-456Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar). While ClpP alone is able to rapidly hydrolyze peptides, the degradation of large proteins requires the presence of a hexameric AAA+ ATPase complex, such as ClpX or ClpC1. These ATPases activate the Clp proteases but also bind protein substrates, unfold them, and translocate them into the proteolytic compartment. Unlike most bacteria and mitochondria, Mycobacterium tuberculosis (Mtb) contains two clp genes, clpP1 and clpP2, that form an active complex containing one ClpP1 and one ClpP2 ring (unless otherwise stated, ClpP1P2, ClpC1, and ClpX refer to Mtb proteins) (2Akopian T. Kandror O. Raju R.M. Unnikrishnan M. Rubin E.J. Goldberg A.L. The active ClpP protease from M. tuberculosis is a complex composed of a heptameric ClpP1 and a ClpP2 ring.EMBO J. 2012; 31: 1529-1541Crossref PubMed Scopus (88) Google Scholar, 3Raju R.M. Unnikrishnan M. Rubin D.H.F. Krishnamoorthy V. Kandror O. Akopian T.N. et al.Mycobacterium tuberculosis ClpP1 and ClpP2 function together in protein degradation and are required for viability in vitro and during infection.PLoS Pathog. 2012; 8: e1002511Crossref PubMed Scopus (130) Google Scholar). In addition to genetic evidence that ClpC1, ClpX, ClpP1, and ClpP2 proteins are essential for viability, the relevance of these targets has been reinforced by the discovery of multiple natural product antibiotics (NPAs) that kill Mtb by targeting this system. The specific potential of ClpC1 as a drug target in Mtb was proven by the discovery of four potent and chemically diverse NPAs acting on this protein (4Gao W. Kim J.Y. Anderson J.R. Akopian T. Hong S. Jin Y.Y. et al.The cyclic peptide ecumicin targeting CLpC1 is active against Mycobacterium tuberculosis in vivo.Antimicrob. Agents Chemother. 2015; 59: 880-889Crossref PubMed Scopus (110) Google Scholar, 5Jung I.P. Ha N.R. Kim A.R. Kim S.H. Yoon M.Y. Mutation analysis of the interactions between Mycobacterium tuberculosis caseinolytic protease C1 (ClpC1) and ecumicin.Int. J. Biol. Macromol. 2017; 101: 348-357Crossref PubMed Scopus (10) Google Scholar, 6Schmitt E.K. Riwanto M. Sambandamurthy V. Roggo S. Miault C. Zwingelstein C. et al.The natural product cyclomarin kills Mycobacterium tuberculosis by targeting the ClpC1 subunit of the caseinolytic protease.Angew. Chem. Int. Ed. Engl. 2011; 50: 5889-5891Crossref PubMed Scopus (124) Google Scholar, 7Weinhäupl K. Brennich M. Kazmaier U. Lelievre J. Ballell L. Goldberg A. et al.The antibiotic cyclomarin blocks arginine-phosphate–induced millisecond dynamics in the N-terminal domain of ClpC1 from Mycobacterium tuberculosis.J. Biol. Chem. 2018; 293: 8379-8393Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 8Choules M.P. Wolf N.M. Lee H. Anderson J.R. Grzelak E.M. Wang Y. et al.Rufomycin targets clpc1 proteolysis in Mycobacterium tuberculosis and M. abscessus.Antimicrob. Agents Chemother. 2019; 63: e02204-e02218Crossref PubMed Scopus (41) Google Scholar, 9Choules M. Yu Y. Cho S.H. Anderson J. Gao W. Klein L. et al.A rufomycin analogue is an anti-tuberculosis drug lead targeting CLPC1 with no cross resistance to ecumicin.Planta Med. 2015; 81: CL2Crossref Google Scholar, 10Gavrish E. Sit C.S. Cao S. Kandror O. Spoering A. Peoples A. et al.Lassomycin, a ribosomally synthesized cyclic peptide, kills Mycobacterium tuberculosis by targeting the ATP-dependent protease ClpC1P1P2.Chem. Biol. 2014; 21: 509-518Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). Indeed, the cyclic peptides ecumicin, cyclomarin, rufomycin, and lassomycin, all binding ClpC1, are among the most powerful anti-tuberculosis (TB) molecules to emerge recently. Ecumicin, for example, displays potent selective anti-TB activity with an MIC value 50 times lower than that of rifampicin or isoniazid, the first line drugs for the treatment of TB (4Gao W. Kim J.Y. Anderson J.R. Akopian T. Hong S. Jin Y.Y. et al.The cyclic peptide ecumicin targeting CLpC1 is active against Mycobacterium tuberculosis in vivo.Antimicrob. Agents Chemother. 2015; 59: 880-889Crossref PubMed Scopus (110) Google Scholar, 5Jung I.P. Ha N.R. Kim A.R. Kim S.H. Yoon M.Y. Mutation analysis of the interactions between Mycobacterium tuberculosis caseinolytic protease C1 (ClpC1) and ecumicin.Int. J. Biol. Macromol. 2017; 101: 348-357Crossref PubMed Scopus (10) Google Scholar). ClpC1 is a member of the class II AAA+ family of proteins, which contains an N-terminal domain (NTD) and two distinct ATP-binding modules, D1 and D2. While no full-length structure of MtbClpC1 is currently available, considerable structural work has been performed on the easy-to-handle NTD domain (7Weinhäupl K. Brennich M. Kazmaier U. Lelievre J. Ballell L. Goldberg A. et al.The antibiotic cyclomarin blocks arginine-phosphate–induced millisecond dynamics in the N-terminal domain of ClpC1 from Mycobacterium tuberculosis.J. Biol. Chem. 2018; 293: 8379-8393Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 11Vasudevan D. Rao S.P.S. Noble C.G. Structural basis of mycobacterial inhibition by cyclomarin A.J. Biol. Chem. 2013; 288: 30883-30891Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 12Wolf N.M. Lee H. Choules M.P. Pauli G.F. Phansalkar R. Anderson J.R. et al.High-resolution structure of ClpC1-rufomycin and ligand binding studies provide a framework to design and optimize anti-tuberculosis leads.ACS Infect. Dis. 2019; 5: 829-840Crossref PubMed Scopus (23) Google Scholar). Curiously, despite representing only a small portion of the full protein, all the NPAs have been shown to bind to the ClpC1-NTD domain, and high-resolution X-ray structures of the binding sites are available for cyclomarin, ecumicin, and rufomycin (7Weinhäupl K. Brennich M. Kazmaier U. Lelievre J. Ballell L. Goldberg A. et al.The antibiotic cyclomarin blocks arginine-phosphate–induced millisecond dynamics in the N-terminal domain of ClpC1 from Mycobacterium tuberculosis.J. Biol. Chem. 2018; 293: 8379-8393Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 11Vasudevan D. Rao S.P.S. Noble C.G. Structural basis of mycobacterial inhibition by cyclomarin A.J. Biol. Chem. 2013; 288: 30883-30891Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 12Wolf N.M. Lee H. Choules M.P. Pauli G.F. Phansalkar R. Anderson J.R. et al.High-resolution structure of ClpC1-rufomycin and ligand binding studies provide a framework to design and optimize anti-tuberculosis leads.ACS Infect. Dis. 2019; 5: 829-840Crossref PubMed Scopus (23) Google Scholar). While this allows a proper mapping of the NPA-binding pockets, it is still not clear how binding to the NTD can translate into functional impairment of the remaining protein. The NTD lacks ATPase activity, which is present in the D1 and D2 domains, and is connected to these domains by a long, disordered loop. Somehow, binding to the NTD must induce changes in the remaining domains. Curiously, despite binding to similar parts of the NTD, the different NPAs lead to distinct effects. Using small-angle X-ray scattering, we have recently shown that ClpC1 exists in an equilibrium between a resting state and the active hexameric state, but how this equilibrium is modulated in Mtb is still difficult to understand (7Weinhäupl K. Brennich M. Kazmaier U. Lelievre J. Ballell L. Goldberg A. et al.The antibiotic cyclomarin blocks arginine-phosphate–induced millisecond dynamics in the N-terminal domain of ClpC1 from Mycobacterium tuberculosis.J. Biol. Chem. 2018; 293: 8379-8393Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). MecA, an adapter that modulates ClpC in other species, does not exist in the Mtb genome, but a homolog of the ClpS adaptor protein has been shown to bind to ClpC1 (13Marsee J.D. Ridings A. Yu T. Miller J.M. Mycobacterium tuberculosis ClpC1 N-terminal domain is dispensable for adaptor protein-dependent allosteric regulation.Int. J. Mol. Sci. 2018; 19: 3651Crossref Scopus (11) Google Scholar). Another possibility is that substrate binding, as proposed by others, can shift the ClpC1 equilibrium towards a hexamer (14Carroni M. Franke K.B. Maurer M. Jäger J. Hantke I. Gloge F. et al.Regulatory coiled-coil domains promote head-to-head assemblies of AAA+ chaperones essential for tunable activity control.Elife. 2017; 6: 724-741Crossref Scopus (24) Google Scholar). It is assumed, based on the extensive AAA+ literature and obtained structural data, that the hexameric state is the ClpC1 functional form. Indeed, only the hexameric form permits the formation of a surface for the interaction with ClpP as well as a pore linked with loops that can couple ATP hydrolysis to mechanical substrate pulling. It is rather unlikely that the structure observed for the resting state, despite the low resolution, can interact with ClpP. Taking advantage of a single mutation, we were able to stabilize the hexameric state of this important drug target and obtain structures in the presence of cyclomarin and ecumicin. Interestingly, half of the ecumicin-bound particles display three of their six NTDs in an extended conformation, suggesting a potential mechanism of how ecumicin modulates ClpC1 function. Although the ClpC1-NTD has been extensively studied by X-ray crystallography and solution NMR (7Weinhäupl K. Brennich M. Kazmaier U. Lelievre J. Ballell L. Goldberg A. et al.The antibiotic cyclomarin blocks arginine-phosphate–induced millisecond dynamics in the N-terminal domain of ClpC1 from Mycobacterium tuberculosis.J. Biol. Chem. 2018; 293: 8379-8393Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 11Vasudevan D. Rao S.P.S. Noble C.G. Structural basis of mycobacterial inhibition by cyclomarin A.J. Biol. Chem. 2013; 288: 30883-30891Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 12Wolf N.M. Lee H. Choules M.P. Pauli G.F. Phansalkar R. Anderson J.R. et al.High-resolution structure of ClpC1-rufomycin and ligand binding studies provide a framework to design and optimize anti-tuberculosis leads.ACS Infect. Dis. 2019; 5: 829-840Crossref PubMed Scopus (23) Google Scholar, 15Wolf N.M. Lee H. Zagal D. Nam J.W. Oh D.C. Lee H. et al.Structure of the N-terminal domain of ClpC1 in complex with the antituberculosis natural product ecumicin reveals unique binding interactions.Acta Crystallogr. D Struct. Biol. 2020; 76: 458-471Crossref PubMed Scopus (13) Google Scholar), no structural information on the full-length ClpC1 has so far been reported. This can be rationalized by several key factors. Firstly, we have shown that ClpC1 exists in an equilibrium between a resting state and the active hexameric state (7Weinhäupl K. Brennich M. Kazmaier U. Lelievre J. Ballell L. Goldberg A. et al.The antibiotic cyclomarin blocks arginine-phosphate–induced millisecond dynamics in the N-terminal domain of ClpC1 from Mycobacterium tuberculosis.J. Biol. Chem. 2018; 293: 8379-8393Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Second, the active hexamer is only formed in the presence of ATP, which is rapidly converted into ADP. Finally, the ClpC1P1P2 Mtb system is, compared to homologs from Staphylococcus aureus and Bacillus subtilis, more insoluble and therefore harder to work with in vitro (7Weinhäupl K. Brennich M. Kazmaier U. Lelievre J. Ballell L. Goldberg A. et al.The antibiotic cyclomarin blocks arginine-phosphate–induced millisecond dynamics in the N-terminal domain of ClpC1 from Mycobacterium tuberculosis.J. Biol. Chem. 2018; 293: 8379-8393Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 16Maurer M. Linder D. Franke K.B. Jäger J. Taylor G. Gloge F. et al.Toxic activation of an AAA+ protease by the antibacterial drug cyclomarin A.Cell Chem. Biol. 2019; 26: 1169-1179.e4Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Indeed, these limitations and the fact that so far all the NPAs targeting ClpC1 bind to the ClpC1 NTD have led others to employ chimeras of ClpC1 and the D1D2 domains of S. aureus to study the function and mode of action of cyclomarin (16Maurer M. Linder D. Franke K.B. Jäger J. Taylor G. Gloge F. et al.Toxic activation of an AAA+ protease by the antibacterial drug cyclomarin A.Cell Chem. Biol. 2019; 26: 1169-1179.e4Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). While this is an interesting approach, it has the limitation of inferring results from nonphysiological proteins and therefore not providing any direct and useful structural data for drug development. This is particularly evident, considering the existing mechanistic differences between the S. aureus and Mtb proteins. For example, whereas SaClpC depends on MecA to catalyze the degradation of GFPssra by ClpP, ClpC1 is fully capable of doing so, independently of the adapter (2Akopian T. Kandror O. Raju R.M. Unnikrishnan M. Rubin E.J. Goldberg A.L. The active ClpP protease from M. tuberculosis is a complex composed of a heptameric ClpP1 and a ClpP2 ring.EMBO J. 2012; 31: 1529-1541Crossref PubMed Scopus (88) Google Scholar, 17Li M. Kandror O. Akopian T. Dharkar P. Wlodawer A. Maurizi M.R. et al.Structure and functional properties of the active form of the proteolytic complex, ClpP1P2, from Mycobacterium tuberculosis.J. Biol. Chem. 2016; 291: 7465-7476Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 18Fraga H. Rodriguez B. Bardera A. Cid C. Akopian T. Kandror O. et al.Development of high throughput screening methods for inhibitors of ClpC1P1P2 from Mycobacteria tuberculosis.Anal. Biochem. 2019; 567: 30-37Crossref PubMed Scopus (8) Google Scholar, 19Akopian T. Kandror O. Tsu C. Lai J.H. Wu W. Liu Y. et al.Cleavage specificity of Mycobacterium tuberculosis ClpP1P2 protease and identification of novel peptide substrates and boronate inhibitors with anti-bacterial activity.J. Biol. Chem. 2015; 290: 11008-11020Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). In fact, MecA does not exist in the Mtb genome. It is therefore fundamental to obtain structural information on ClpC1 in its functional state. Given the presence of an equilibrium between a decameric resting state and the active hexameric ClpC1 states in solution, we rationalized that the stabilization of the ClpC1 hexamer could allow us to obtain structural information on this important drug target. Previously, stabilization was achieved by removing intrinsically disordered loops, allowing the elucidation of the only ClpC X-ray structure described to date (20Wang F. Mei Z. Qi Y. Yan C. Hu Q. Wang J. et al.Structure and mechanism of the hexameric MecA-ClpC molecular machine.Nature. 2011; 471: 331-337Crossref PubMed Scopus (106) Google Scholar), but at the expense of enzymatic activity and therefore mechanistic relevance. Carroni et al. (14Carroni M. Franke K.B. Maurer M. Jäger J. Hantke I. Gloge F. et al.Regulatory coiled-coil domains promote head-to-head assemblies of AAA+ chaperones essential for tunable activity control.Elife. 2017; 6: 724-741Crossref Scopus (24) Google Scholar) have demonstrated that point mutations in the middle domain (MD) of SaClpC shift the equilibrium towards the hexameric state. We hypothesized that similar modifications could also stabilize the active hexameric state of ClpC1, allowing subsequent structural characterization. In particular, residue F436 in SaClpC (F444 in Mtb) has been shown to be important for the resting state–hexamer equilibrium (Figs. 1, A and B and S2A). By means of site-directed mutagenesis, we mutated the homologous residue in ClpC1 to an alanine (F444A), to test if it could shift the ClpC1 equilibrium. Using a Superose 6 10/300 GL column, we observed that, as previously shown (7Weinhäupl K. Brennich M. Kazmaier U. Lelievre J. Ballell L. Goldberg A. et al.The antibiotic cyclomarin blocks arginine-phosphate–induced millisecond dynamics in the N-terminal domain of ClpC1 from Mycobacterium tuberculosis.J. Biol. Chem. 2018; 293: 8379-8393Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar), WT ClpC1 (ClpC1WT) migrates as a species larger than the canonical hexamer. By contrast, in the presence of ATP, the F444A main species (ClpC1F444A) eluted in a volume consistent with the size expected for a ClpC1 hexamer (Fig. 1C). Furthermore, the presence of hexameric rings in the mutant was also confirmed by negative stain EM (Fig. 1D). After successful stabilization of the hexameric state, we sought to verify that biological activity was maintained in mutant ClpC1F444A. ClpC1F444A ATPase activity was checked using an assay monitoring the fluorescence decrease at 340 nm, associated with NADH to NAD+ conversion as the ADP formed by ClpC1 ATPase activity is reconverted into ATP by pyruvate kinase and phosphoenolpyruvate dehydrogenase (18Fraga H. Rodriguez B. Bardera A. Cid C. Akopian T. Kandror O. et al.Development of high throughput screening methods for inhibitors of ClpC1P1P2 from Mycobacteria tuberculosis.Anal. Biochem. 2019; 567: 30-37Crossref PubMed Scopus (8) Google Scholar). As shown in Figure 2A, ClpC1F444A displayed an increase in the ATPase activity in comparison to ClpC1WT. An increase in ATPase activity was also observed for the SaClpC1F436A mutant, which was explained by a shift in the resting state–active hexamer equilibrium towards the latter, which is consistent with the described size-exclusion chromatography experiments (14Carroni M. Franke K.B. Maurer M. Jäger J. Hantke I. Gloge F. et al.Regulatory coiled-coil domains promote head-to-head assemblies of AAA+ chaperones essential for tunable activity control.Elife. 2017; 6: 724-741Crossref Scopus (24) Google Scholar). Although cyclomarin and ecumicin binding sites are located at the ClpC1 NTD and are therefore distant from the F444A mutation, we tested the functional consequences of cyclomarin and ecumicin binding on the mutant. Using saturating concentrations (10 μM), ecumicin binding to the WT results in a strong increase in ClpC1 ATPase activity, while cyclomarin does not. Curiously, a similar increase in ATPase activity is observed using the F444A mutant, showing that ecumicin effects do not exclusively result from the modulation of the ClpC1 resting state–hexamer equilibrium (Fig. 2A). An important in vivo function of ClpC1, if not the most relevant, is to associate with ClpP1P2 and target proteins for degradation by the protease. The roles of ClpC1 in this process are multiple, as it is responsible for substrate recognition, unfolding, and translocation but also for association with ClpP1P2 and its concomitant allosteric activation (2Akopian T. Kandror O. Raju R.M. Unnikrishnan M. Rubin E.J. Goldberg A.L. The active ClpP protease from M. tuberculosis is a complex composed of a heptameric ClpP1 and a ClpP2 ring.EMBO J. 2012; 31: 1529-1541Crossref PubMed Scopus (88) Google Scholar, 18Fraga H. Rodriguez B. Bardera A. Cid C. Akopian T. Kandror O. et al.Development of high throughput screening methods for inhibitors of ClpC1P1P2 from Mycobacteria tuberculosis.Anal. Biochem. 2019; 567: 30-37Crossref PubMed Scopus (8) Google Scholar). Because these processes are very hard to study independently, ClpC1 function is often evaluated indirectly by measuring protein target degradation, that is, the rate at which a protein is degraded by ClpP1P2 in association with ClpC1 (2Akopian T. Kandror O. Raju R.M. Unnikrishnan M. Rubin E.J. Goldberg A.L. The active ClpP protease from M. tuberculosis is a complex composed of a heptameric ClpP1 and a ClpP2 ring.EMBO J. 2012; 31: 1529-1541Crossref PubMed Scopus (88) Google Scholar). Two substrates that are commonly used are FITC-casein (casein with a fluorescein fluorophore attached to lysine residues) and GFP, representing two classes of proteins. FITC-Casein, which lacks a well-defined tertiary structure, is usually used as a model for unfolded substrates, while the stable, beta-barrel containing GFP is used as a model for a structured protein. While in the case of FITC-casein, protein degradation results in a net fluorescence increase as fluorescein quenching is reduced with degradation; in the case of GFPssra, degradation of the protein fluorophore results in a decrease in fluorescence (18Fraga H. Rodriguez B. Bardera A. Cid C. Akopian T. Kandror O. et al.Development of high throughput screening methods for inhibitors of ClpC1P1P2 from Mycobacteria tuberculosis.Anal. Biochem. 2019; 567: 30-37Crossref PubMed Scopus (8) Google Scholar). As can be seen in Figure 2, B and C, the F444A mutant is fully functional to catalyze the degradation of both FITC-casein and GFPssra by ClpP1P2, showing that this mutation does not impair ClpC1 enzymatic activity. Reflecting different modes of action, the effects of cyclomarin and ecumicin in protein degradation by the ClpC1P1P2 complex are distinct. At the concentration used, cyclomarin is a moderate activator of FITC-casein degradation and a weak inhibitor of GFPssra degradation, whereas for ecumicin, we observe a mild activation of FITC-casein degradation and a strong inhibition of GFPssra degradation (Fig. 2, B and C). The different effects of these two NPAs are also clear when they are used together. Indeed, cyclomarin is able to prevent both ATPase activation (Fig .2D) as well as GFP degradation inhibition induced by ecumicin (Fig. S1), showing that they are competing for a similar pocket but they induce distinct biochemical effects. The distinct structural consequences resulting from cyclomarin and ecumicin binding are also clear from other biophysical data. Differential scanning fluorimetry is a very useful method to monitor ligand binding to a given target. We have previously shown that arginine phosphate (ArgP) is able to stabilize the NTD, and we aimed to test the effects of cyclomarin and ecumicin on the NTD (7Weinhäupl K. Brennich M. Kazmaier U. Lelievre J. Ballell L. Goldberg A. et al.The antibiotic cyclomarin blocks arginine-phosphate–induced millisecond dynamics in the N-terminal domain of ClpC1 from Mycobacterium tuberculosis.J. Biol. Chem. 2018; 293: 8379-8393Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Using Sypro orange as a fluorescence reporter, we obtained a Tm of 77.7 (±0.3) °C and 83.6 (±0.3) °C, for the apo and ArgP-bound NTD. These values are higher than the ones we previously reported using intrinsic tryptophan fluorescence as a reporter that were 69 °C and 79 °C for the WT and ArgP-bound NTD (7Weinhäupl K. Brennich M. Kazmaier U. Lelievre J. Ballell L. Goldberg A. et al.The antibiotic cyclomarin blocks arginine-phosphate–induced millisecond dynamics in the N-terminal domain of ClpC1 from Mycobacterium tuberculosis.J. Biol. Chem. 2018; 293: 8379-8393Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Quite striking was the difference observed between cyclomarin and ecumicin. While cyclomarin binding resulted in a very strong stabilization of NTD, with a calculated Tm of 91.5 (±0.9) °C, ecumicin, on the contrary, did not stabilize the domain and in fact lead to a decrease in the Tm (Fig. S2B). Having shown that the MD mutant (ClpC1F444A) is active, we attempted unsuccessfully to crystallize the resulting hexamer. We therefore set out to obtain structural information on ClpC1 via cryo-EM. To further stabilize the sample, considering that ATP is being continuously degraded even at low temperatures and that some AAA+ proteins have been shown to hydrolyze ATP analogs, we introduced two additional mutations in the double walker B motif (E288A and E626A). We hypothesized that these mutations allow nucleotide binding but prevent hydrolysis and therefore further stabilize the hexameric assembly. After initial cryo-EM grid screening, a dataset was collected of the apo form of MtbClpC1E288A/F444A/E626A (see Experimental procedures). Processing of the data resulted in a structure for apo ClpC1 determined to a resolution of 3.6 Å (see Experimental procedures, Figs. S3 and S4, and Table S1). As expected, the overall structure of apo MtbClpC1E288A/F444A/E626A consists of a hexamer composed of six ClpC1 subunits (A–F), with the D1 and D2 domains arranged in a ring that together forms a pore through which the substrate can be actively translocated. In line with recent structures, including the recent cryo-EM structure of B. subtilis ClpC (21Morreale F.E. Kleine S. Leodolter J. Junker S. Hoi D.M. Ovchinnikov S. et al.BacPROTACs mediate targeted protein degradation in bacteria.Cell. 2022; 185: 2338-2353.e18Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar), the observed ClpC1 hexamer is not symmetric, as observed originally in the crystal structure of ClpC (20Wang F. Mei Z. Qi Y. Yan C. Hu Q. Wang J. et al.Structure and mechanism of the hexameric MecA-ClpC molecular machine.Nature. 2011; 471: 331-337Crossref PubMed Scopus (106) Google Scholar), and instead adopts an asymmetric spiral structure (Fig. 3A). An important characteristic of our structure is the observation of a 23-residue long peptide visible in its central pore. The presence of substrates in the pore formed by D1 and D2 has been previously reported for other members of the family, but as no substrate has been added to our cryo-EM sample preparation, it was probably taken up and trapped inside of the inactive protein during purification (Fig. 3, A and B). This unexpected finding is nevertheless useful as it provides important details on the ClpC1 mechanism of action. Indeed, the substrate is bound by each of the two pore loops from subunits A to E in both D1 and D2, with the exception of pore loop 2 in D1 subunit E, forming a spiral along the substrate. Subunit E is positioned at the highest point and subunit A at the lowest. Subunit F is detached from the substrate in both D1 and D2, and the pore loops are not visible. Furthermore, all nucleotide-binding sites in D1 are occupied by ADP, while for D2, four sites are occupied by ADP (subunits B, C, D, and E), while two are in their apo state (subunits A and F) (Fig. 3C). This organization is in line with the asymmetric disposition we referred to above and has been reported for other members of the family, particularly for structures obtained using cryo-EM (21Morreale F.E. Kleine S. Leodolter J. Junker S. Hoi D.M. Ovchinnikov S. et al.BacPROTACs mediate targeted protein degradation in bacteria.Cell. 2022; 185: 2338-2353.e18Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar, 22Lopez K.E. Rizo A.N. Tse E. Lin J.B. Scull N.W. Thwin A.C. et al.Conformational plasticity of the ClpAP AAA+ protease couples protein unfolding and proteolysis.Nat. Struct. Mol. Biol. 2020; 27: 406-416Crossref PubMed Scopus (34) Google Scholar, 23Lee S. Roh S.H. Lee J. Sung N. Liu J. Tsai F.T.F. Cryo-EM structures of the Hsp104 protein disaggregase captured in the ATP conformation.Cell Rep. 2019; 26: 29-36.e3Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Unfortunately, several structural features are not visible in the ClpC1 apo structure, presumably due to flexibility or intrinsic disorder. One of these missing features are the LGF loops which, in line with other AAA+ ATPase structures, will likely only become structured upon interaction with the ClpP protease (22Lopez K.E. Rizo A.N. Tse E. Lin J.B. Scull N.W. Thwin A.C. et al.Conformational plasticity of the ClpAP AAA+ protease couples protein unfolding and p
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