Structure of the drug target ClpC1 unfoldase in action provides insights on antibiotic mechanism of action

Abstract

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. 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 cyclomarin in the N-terminal domain of ClpC1 from Mycobacterium Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Anderson J.R. et targets in Mycobacterium tuberculosis and M. Agents Chemother. PubMed Scopus Google Scholar, M. S.H. Anderson J. W. L. et is an drug targeting with to 2015; Google Scholar, S. Kandror O. A. A. et a cyclic kills Mycobacterium tuberculosis by targeting the ATP-dependent protease Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). the cyclic ecumicin, cyclomarin, and lassomycin, ClpC1, are the most molecules to for displays potent with an that of or the first for the of (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 of the AAA+ family of contains an N-terminal domain and two and While structure of is structural has been on the domain K. Brennich M. Kazmaier U. Lelievre J. Ballell L. Goldberg A. et al.The cyclomarin in the N-terminal domain of ClpC1 from Mycobacterium Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, of by cyclomarin Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Anderson J.R. et structure of and a to and PubMed Scopus Google Scholar). a of the the NPAs have been to bind to the and structures of the sites are for cyclomarin, ecumicin, and K. Brennich M. Kazmaier U. Lelievre J. Ballell L. Goldberg A. et al.The cyclomarin in the N-terminal domain of ClpC1 from Mycobacterium Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, of by cyclomarin Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Anderson J.R. et structure of and a to and PubMed Scopus Google Scholar). While this a of the is to the into of the The ATPase is in the and and is to these domains by a to the changes in the to of the the NPAs to we have that ClpC1 in an between a and the active hexameric but this is in Mtb is to K. Brennich M. Kazmaier U. Lelievre J. Ballell L. Goldberg A. et al.The cyclomarin in the N-terminal domain of ClpC1 from Mycobacterium Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). an that in other in the Mtb but a of the protein has been to bind to ClpC1 A. T. J.M. Mycobacterium tuberculosis ClpC1 N-terminal domain is for J. Scopus Google Scholar). is that substrate binding, as by the ClpC1 a M. M. J. et domains of AAA+ essential for 2017; Scopus Google Scholar). is on the AAA+ and obtained structural that the hexameric is the ClpC1 the hexameric form the of a for the with ClpP as as a with that to substrate is that the structure observed for the the with Taking advantage of a we able to the hexameric of this drug target and structures in the presence of cyclomarin and half of the ecumicin-bound particles display three of their six in an extended a potential mechanism of ecumicin ClpC1 Although the has been by and K. Brennich M. Kazmaier U. Lelievre J. Ballell L. Goldberg A. et al.The cyclomarin in the N-terminal domain of ClpC1 from Mycobacterium Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, of by cyclomarin Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Anderson J.R. et structure of and a to and PubMed Scopus Google Scholar, et of the N-terminal domain of ClpC1 in complex with the natural product ecumicin Biol. PubMed Scopus Google structural on the ClpC1 has been This by we have that ClpC1 in an between a and the active hexameric K. Brennich M. Kazmaier U. Lelievre J. Ballell L. Goldberg A. et al.The cyclomarin in the N-terminal domain of ClpC1 from Mycobacterium Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the active is in the presence of is rapidly into the Mtb to from and and to with in vitro K. Brennich M. Kazmaier U. Lelievre J. Ballell L. Goldberg A. et al.The cyclomarin in the N-terminal domain of ClpC1 from Mycobacterium Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M. J. et activation of an AAA+ protease by the drug cyclomarin Chem. Biol. 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Kandror O. et of for of from PubMed Scopus Google Scholar, T. Kandror O. C. W. et of Mycobacterium tuberculosis ClpP1P2 protease and of peptide and with Biol. Chem. 2015; Full Text Full Text PDF PubMed Scopus Google Scholar). In in the Mtb is to structural on ClpC1 in its the presence of an between a and the active hexameric ClpC1 states in we that the of the ClpC1 to structural on this drug was by the of the structure to C. J. et and mechanism of the hexameric 2011; PubMed Scopus Google but at the of and et M. M. J. et domains of AAA+ essential for 2017; Scopus Google have that in the middle domain of the the hexameric that also the active hexameric of ClpC1, structural In in in has been to for the A and and of we the in ClpC1 to an to the ClpC1 a we observed as K. Brennich M. Kazmaier U. Lelievre J. Ballell L. Goldberg A. et al.The cyclomarin in the N-terminal domain of ClpC1 from Mycobacterium Biol. Chem. Full Text Full Text PDF PubMed Scopus Google ClpC1 as a the in the presence of the in a with the for a ClpC1 the presence of hexameric in the was also by EM of the hexameric we to that was in ATPase was an the at with to as the by ClpC1 ATPase is into by and A. C. Akopian T. Kandror O. et of for of from PubMed Scopus Google Scholar). in an in the ATPase in to in ATPase was also observed for the mutant, was by a in the the is with the M. M. J. et domains of AAA+ essential for 2017; Scopus Google Scholar). Although cyclomarin and ecumicin sites are at the ClpC1 and are from the we the of cyclomarin and ecumicin on the ecumicin to the in a in ClpC1 ATPase cyclomarin a in ATPase is observed the mutant, that ecumicin from the of the ClpC1 in function of ClpC1, the most is to with ClpP1P2 and target proteins for degradation by the The of ClpC1 in this are as is for substrate and but also for with ClpP1P2 and its 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, A. C. Akopian T. Kandror O. et of for of from PubMed Scopus Google Scholar). these are to ClpC1 function is by protein target that the at a protein is by ClpP1P2 in 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). that are are with a to and two of a is as a model for substrates, the containing is as a model for a While in the of protein degradation in a as is with in the of degradation of the protein in a in A. C. Akopian T. Kandror O. et of for of from PubMed Scopus Google Scholar). in and the is to the degradation of and by ClpP1P2, that this ClpC1 of the of cyclomarin and ecumicin in protein degradation by the complex are the cyclomarin is a of degradation and a of for ecumicin, we a activation of degradation and a of degradation and The of these two NPAs are also are cyclomarin is able to ATPase activation as as degradation by ecumicin that are for a but The structural from cyclomarin and ecumicin are also from other is a to to a have that is able to the and we to the of cyclomarin and ecumicin on the K. Brennich M. Kazmaier U. Lelievre J. Ballell L. Goldberg A. et al.The cyclomarin in the N-terminal domain of ClpC1 from Mycobacterium Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). as a we obtained a of and for the apo and These are the we as a that and for the and K. Brennich M. Kazmaier U. Lelievre J. Ballell L. 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J. et and mechanism of the hexameric 2011; PubMed Scopus Google and an structure of structure is the of a peptide in its central The presence of in the by and has been for other members of the but as substrate has been to was and of the protein during A and This is as provides on the ClpC1 mechanism of the substrate is by of the two from A to in and with the of in subunit forming a the is at the and subunit A at the is from the substrate in and and the are sites in are by for four sites are by and two are in their apo A and This is in with the we to and has been for other members of the for structures obtained S. J. S. S. et protein degradation in Full Text Full Text PDF PubMed Scopus Google Scholar, et of the AAA+ protease protein and Biol. PubMed Scopus Google Scholar, S. S.H. J. 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In this we advantage of a to the ClpC1 hexameric This was on obtained for and that the of an between a and a hexameric form K. Brennich M. Kazmaier U. Lelievre J. Ballell L. Goldberg A. et al.The cyclomarin in the N-terminal domain of ClpC1 from Mycobacterium Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M. M. J. et domains of AAA+ essential for 2017; Scopus Google Scholar). the of an to an in a large in the the for a hexameric While the of the the the hexameric mechanism of the first structure of a complex, the one obtained was from an mutant, to and protein degradation C. J. et and mechanism of the hexameric 2011; PubMed Scopus Google Scholar). the with to ATPase and protein degradation of and and that is with specific the This activation was that the is the active but is that is the activation for ClpC1 with in the M. M. J. et domains of AAA+ essential for 2017; Scopus Google Scholar). This the between these but that the is able to and protein degradation in with of was to the mechanism of action of antibiotics targeting ClpC1. an was the the of cyclomarin and is that cyclomarin and ecumicin largely their in the their in In as we have and has been (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 ecumicin in a in ClpC1 ATPase cyclomarin to other and degradation in with Although cyclomarin and ecumicin to a activation of ecumicin is a of of the and the a hexameric cyclomarin and ecumicin and a of activation and is observed for This the on protein that cyclomarin mechanism of action on a of the M. J. et activation of an AAA+ protease by the drug cyclomarin Chem. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). that was the the of cyclomarin with the of the the from the that the two are ecumicin and cyclomarin on we their action to the in is the The structures we determined further that the is a model for an active ClpC1 the of multiple structural features that we have in a the hexameric structure we has been observed for other members of the AAA+ a structure of as as S. J. S. S. et protein degradation in Full Text Full Text PDF PubMed Scopus Google Scholar, et of the AAA+ protease protein and Biol. PubMed Scopus Google Scholar). This that the we domain or the presence of a peptide in the by the and domains and in with the that ClpC1 is to bind and translocate protein In addition to the apo we determined structures the addition of cyclomarin or ecumicin antibiotics to cyclomarin-bound as as one of the two of ecumicin-bound are to the apo structure and have the sites for cyclomarin and The of the in mechanism of action largely but obtained with other AAA+ ATPases suggest that the substrate to and the of the ClpC1 to has been to activate the of ClpC1 A. T. J.M. Mycobacterium tuberculosis ClpC1 N-terminal domain is for J. Scopus Google Scholar). that cyclomarin a in the as for with in other where an ring to the ring is observed in and The of ecumicin-bound containing of the three domains the most to three to the other are that remain the of the in map of ecumicin-bound is to the structure of an for a with et for substrate and by the AAA+ PubMed Scopus Google Scholar). In this obtained at resolution the a substrate the the of the of addition of ecumicin was in a structure of the complex et of the N-terminal domain of ClpC1 in complex with the natural product ecumicin Biol. PubMed Scopus Google Scholar). The of the in the is to the in and in to the the of the sites of the in the is the resolution of the ecumicin-bound map we obtained to the or the of the the structures we obtained in the mechanism of action of cyclomarin and to of the cyclomarin and ecumicin in as as have that one of cyclomarin to the K. Brennich M. Kazmaier U. Lelievre J. Ballell L. Goldberg A. et al.The cyclomarin in the N-terminal domain of ClpC1 from Mycobacterium Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, of by cyclomarin Biol. Chem. Full Text Full Text PDF PubMed Scopus Google forming a a by the of and is cyclomarin, a with the N-terminal is the that cyclomarin in a in the of the and this is in an of in as as an of the domain by K. Brennich M. Kazmaier U. 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PubMed Scopus Google Scholar). for the to function as an in to interactions with the from these two of function the degradation of proteins proteins such as The to this a of degradation and a activation of degradation by are observed the addition of is the of the ATPase activation observed the addition of In we the first structure of an drug in its apo, cyclomarin-, and ecumicin-bound The ecumicin-bound ClpC1 structure to suggest a model for action on the of ecumicin-bound substrate degradation by the and into a an The are in by the ClpC1 and and as K. Brennich M. Kazmaier U. Lelievre J. Ballell L. Goldberg A. et al.The cyclomarin in the N-terminal domain of ClpC1 from Mycobacterium Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). ClpC1 ATPase and and degradation by complex as A. C. Akopian T. Kandror O. et of for of from PubMed Scopus Google Scholar). in a a to cyclomarin and ecumicin, and in The was the first of the respective in a of Mtb ClpC1 in complex with cyclomarin, and in complex with ecumicin a for at of of the the for at and and into and of a with a at the A of of of cyclomarin, and of ecumicin at a of to a of with a of to to with a of and or with a of and cyclomarin-bound ecumicin-bound and of for 2017; PubMed Scopus Google Scholar). was T. The N-terminal domain of unfoldase is and from the of Biol. PubMed Scopus Google Scholar). on the and was was T. M. T. C. A. et is a and for Biol. PubMed Scopus Google Scholar). a of to to a of of and as an for model and and cyclomarin-bound a was on particles from the a of to a of the the was for the cyclomarin-bound as as the ecumicin-bound particles as an for model and two and was a for the apo and cyclomarin-bound as as of the ecumicin-bound for model J. A. J.M. J. a for Biol. 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