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The Structure of the Catalytic Domain of the ATP Synthase from Mycobacterium Smegmatis Is a Target for Developing Antitubercular Drugs

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The Structure of the Catalytic Domain of the ATP Synthase from Mycobacterium Smegmatis Is a Target for Developing Antitubercular Drugs The structure of the catalytic domain of the ATP synthase from Mycobacterium smegmatis is a target for developing antitubercular drugs Alice Tianbu Zhanga,1, Martin G. Montgomerya,1, Andrew G. W. Leslieb, Gregory M. Cooka,c, and John E. Walkera,2 aThe Medical Research Council Mitochondrial Biology Unit, University of Cambridge, Cambridge Biomedical Campus, CB2 0XY Cambridge, United Kingdom; bThe Medical Research Council Laboratory of Molecular Biology, Cambridge Biomedical Campus, CB2 0QH Cambridge, United Kingdom; and cDepartment of Microbiology and Immunology, University of Otago, 9016 Dunedin, New Zealand Contributed by John E. Walker, December 17, 2018 (sent for review October 12, 2018; reviewed by Thomas M. Duncan and Wayne D. Frasch) The crystal structure of the F1-catalytic domain of the adeno- MDR-TB in adults. Its potential to shorten dramatically the sine triphosphate (ATP) synthase has been determined from My- treatment time for MDR-TB is highlighted by two recent studies. cobacterium smegmatis which hydrolyzes ATP very poorly. The In mouse models of TB, a combination of bedaquiline with PA- structure of the α3β3-component of the catalytic domain is similar 824, an antimycobacterial drug with a complex mode of action to those in active F1-ATPases in Escherichia coli and Geobacillus (6) and linezolid, a repurposed protein synthesis inhibitor, sig- stearothermophilus. However, its e-subunit differs from those in nificantly improved efficacy and relapse rates compared with the these two active bacterial F1-ATPases as an ATP molecule is not frontline regimen of rifampicin, isoniazid, and pyrazinamide (7, bound to the two α-helices forming its C-terminal domain, probably 8). In the Nix-TB phase III clinical trial using this three-drug because they are shorter than those in active enzymes and they lack combination, 74% of the patients with MDR-TB were culture an amino acid that contributes to the ATP binding site in active negative in 8 wk*. The most recent recommendations for the enzymes. In E. coli and G. stearothermophilus,theα-helices adopt treatment of MDR-TB, based on the balance of effectiveness an “up” state where the α-helices enter the α β -domain and pre- and harm and a preference for oral administration, now include 3 3 bedaquiline (9). Bedaquiline is effective against both actively vent the rotor from turning. The mycobacterial F1-ATPase is most similar to the F -ATPase from Caldalkalibacillus thermarum, which growing and nonreplicating cells of M. tuberculosis and acts by 1 inhibiting the ATP synthase (10, 11) thereby shutting off the also hydrolyzes ATP poorly. The βE-subunits in both enzymes are in the usual “open” conformation but appear to be occupied uniquely supply of cellular energy in the bacterium without noticeably by the combination of an adenosine 5′-diphosphate molecule with affecting the human enzyme found in the inner membranes of the mitochondria. Thus, these observations provide proof of no magnesium ion plus phosphate. This occupation is consistent principle that the mycobacterial ATP synthase is a suitable target with the finding that their rotors have been arrested at the same point in their rotary catalytic cycles. These bound hydrolytic prod- ucts are probably the basis of the inhibition of ATP hydrolysis. It can Significance be envisaged that specific as yet unidentified small molecules × 6 might bind to the F1 domain in Mycobacterium tuberculosis, pre- Last year, 1.6 10 people died from tuberculosis (TB), and vent ATP synthesis, and inhibit the growth of the pathogen. about 10 × 106 became infected with the causative bacte- rium Mycobacterium tuberculosis and 460,000 of them with Mycobacterium smegmatis | F1-ATPase | structure | inhibition | multidrug-resistant bacteria. Bedaquiline, a new anti-TB drug, tuberculosis developed to combat multidrug-resistant TB, kills M. tubercu- losis by preventing the operation of its molecular machine for n 2017, around 1.6 × 106 people died from tuberculosis (TB), generating adenosine triphosphate (ATP), the fuel of life, Iand Mycobacterium tuberculosis, the causative bacterium, is without affecting the equivalent human machine. Here, we now the second greatest killer of mankind by a single infectious describe the molecular structure of the module in the myco- agent, surpassed in its lethal impact only by human immunode- bacterial machine where ATP is generated. Differences be- tween this module and the equivalent human module can ficiency virus/acquired immunodeficiency syndrome (1). Of the now be exploited to develop new anti-TB drugs, unrelated to 10 × 106 people estimated to have developed TB in that single bedaquiline, that also may help to prevent and cure TB by year, 4.6% were resistant to both rifampicin and isoniazid and inhibiting the formation of ATP. are classed as multidrug resistant (MDR), and 8.5% of the MDR-TB cases were extensively drug resistant (XDR). Only Author contributions: G.M.C. and J.E.W. designed research; A.T.Z. and M.G.M. performed 55% of the MDR-TB and 30% of the XDR-TB cases were research; A.T.Z., M.G.M., A.G.W.L., G.M.C., and J.E.W. analyzed data; J.E.W. wrote the treated successfully. These alarming statistics serve to emphasize paper; and J.E.W. supervised project. the urgent need to develop new drugs that are effective and fast Reviewers: T.M.D., SUNY Upstate Medical University; and W.D.F., Arizona State University. acting against drug-resistant strains of M. tuberculosis. Prefera- The authors declare no conflict of interest. bly, they should be effective also against latent M. tuberculosis Published under the PNAS license. infections where the bacteria lie dormant in infected humans in a Data deposition: The atomic coordinates and structure factors have been deposited in the nonreplicating state before emerging as an active infection. It has Protein Data Bank, www.wwpdb.org (PDB ID code 6FOC). been estimated that between a quarter and a third of the world’s See Commentary on page 3956. population are latently infected (2). However, the impact of this 1A.T.Z. and M.G.M. contributed equally to this work. latency has been questioned recently as nearly everyone who falls 2To whom correspondence should be addressed. Email: [email protected]. seriously ill with TB does so within 2 y of being infected, and la- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. tent infections rarely become active even in old age (3). 1073/pnas.1817615116/-/DCSupplemental. In 2012, the U.S. Food and Drug Administration approved the Published online January 25, 2019. use of a novel oral drug bedaquiline (SIRTURO) for the treat- *Conradie F, et al. (2017) The NIX-TB trial of pretomanid, bedaquiline and linezoid to ment of MDR-TB (4, 5), and bedaquiline received fast-track treat XDR-TB. Conference on RetroViruses and Opportunistic Infections 2017, Seattle, approval as a component of a combination therapy for treating abstr 80LB. 4206–4211 | PNAS | March 5, 2019 | vol. 116 | no. 10 www.pnas.org/cgi/doi/10.1073/pnas.1817615116 Downloaded by guest on September 28, 2021 for developing new drugs to combat tuberculosis. A rational Appendix,Fig.S3andTableS2). It had a very low ATP hy- approach to the design of new drugs in addition to bedaquiline to drolase activity (0.07 U/mg), and, in contrast to some other SEE COMMENTARY inhibit the mycobacterial ATP synthase requires ideally that the latent F1-ATPases, for example, the enzyme from Caldalka- structures and mechanistic and regulatory mechanisms of the libacillus thermarum (16), this low activity could not be stim- human and mycobacterial ATP synthases be understood. The hu- ulated by lauryldimethylamine oxide (LDAO). However, when man enzyme is very similar in both respects to the well-studied bo- the mycobacterial enzyme was treated briefly with trypsin, the vine enzyme, which therefore provides an excellent surrogate. specific activity increased by 100-fold to 7 U/mg. Although Mycobacterial ATP synthases have been less studied, and only the characterization of the proteolytic fragments (SI Appendix, structure of the c ring in the membrane domain of the enzyme’s Table S3) did not provide a clear indication of the mechanism rotor in the enzyme from the nonpathogenic organism Mycobacte- of activation, it is worth noting that the e-subunit had been rium phlei has been established (12). It is here that bedaquiline binds degraded almost completely after 2 min with a corresponding (12), presumably impeding the turning of the rotor in the intact significant increase in activity. In E. coli F1-ATPase and enzyme. It has been proposed that it also binds at a secondary site in F1Fo-ATPase from M. smegmatis, activation by trypsinolysis the e-subunit (13, 14). Before, the work described here, the structure has been attributed to removal of the e-subunit (17). The activity of its F1-catalytic domain was not known in any mycobacterial ATP of F1-ATPase from M. smegmatis uncovered by trypsinolysis was synthase, and there was no molecular understanding of why the doubled by the addition of LDAO (SI Appendix,Fig.S4). mycobacterial enzymes are barely capable of hydrolyzing ATP (15), whereas, for example, the enzymes from facultative anaerobes, such Structure Determination. Hexagonal crystals of the complex con- as Escherichia coli can both synthesize and hydrolyze ATP. Here, we taining all four subunits (SI Appendix, Fig. S5) have the unit-cell describe the structure of the inhibited state of the catalytic domain of parameters a = b = 105.2, c = 628.6 Å with α = β = 90.0° and γ = the ATP synthase from another nonpathogenic Mycobacterium, 120.0° and belong to space group P3121 with one F1-ATPase in Mycobacterium smegmatis. It is an excellent surrogate for the cata- the asymmetric unit (see SI Appendix, Table S4 for a summary of lytic domain of the F1-ATPase from M.
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