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Responsible Editor | Xi tuberculosis was first discovered in humans in the Mediterranean region about 9,000 years ago [1].
In the descriptions of Eastern and Western poets and writers, tuberculosis is often given a romantic color.
This "romantic disease", even in the era when antibiotics are popularized, can still cause about 1.
5 million deaths every year [2], which is the same as the new crown that has ravaged the world this year (COVID19, as of December 10, 2020, the number of deaths is 1.
57 million.
[3]) The comparison is not too much.
Not only that, the resistance of tuberculosis to first-line antibiotics is also increasing year by year [2], making the development of a new generation of tuberculosis antibiotics more important.
Bedaquiline (bedaquiline, BDQ, Sirturo, TMC207, R207910), which was approved for marketing in many regions of the world in 2012-14, is the first approved after rifamycins (rifamycins) was used for clinical treatment in 1966 New tuberculosis antibiotics [4].
In the few years after approval, BDQ has become one of the safest and most effective drugs for the treatment of multidrug-resistant and extensively drug-resistant TB [5].
Unlike all previous tuberculosis antibiotics, the target of BDQ is tuberculosis ATP synthase [6].
ATP synthase is a macromolecular complex widely found in bacteria, chloroplasts and mitochondria.
It is the energy currency of cells-the production factory of ATP.
BDQ inhibits ATP synthase and limits the synthesis of ATP by Mycobacterium tuberculosis, thereby killing Mycobacterium tuberculosis.
Recently, the John Rubinstein team of Hospital for Sick Children in Canada published an article Structure of mycobacterial ATP synthase bound to the tuberculosis drug bedaquiline in Nature.
The author uses single-particle cryo-electron microscopy to analyze the series structure of Mycobacterium smegmatis (Mycobacterium smegmatis) ATP synthase binding to BDQ, and explains how BDQ binds to Mycobacterium ATP synthase with high affinity and ATP synthesis How does enzyme autoinhibition allow mycobacteria to preserve energy in an oxygen-deficient environment and increase the probability of survival.
The main function of ATP synthase is to synthesize ATP using electrochemical proton motive force.
But what is interesting is that when proton power disappears, ATP synthase can in turn hydrolyze ATP and consume energy.
Mycobacterium tuberculosis is a strictly aerobic bacterium, but it can survive in a low-consumption dormant state for many years in a hypoxic host, until it is activated under the right conditions to cause disease.
Therefore, it is particularly important to strictly control the hydrolysis of ATP synthase for Mycobacterium tuberculosis that lacks energy [7].
By observing the structure of M.
smegmatis ATP synthase, the authors found that there is a 35 amino acid extension at the C-terminus of subunit α (picture 1 and video 1 below).
It hooks on the rotor part of ATP synthase like a hook, so that it can only turn to the direction of ATP synthesis, thus ensuring that its hydrolysis function is limited to the minimum.
The authors then knocked out the hook part of ATP synthase and found that the ATP synthase after knocking out the hook showed normal ATP hydrolysis activity.
Previous experiments successfully proved that the binding site of BDQ is close to the subunit c of ATP synthase [6, 8].
What is puzzling is that although BDQ can inhibit the synthesis of ATP by ATP synthase at nanomolar (nM) concentration [8,9], its affinity for subunit c is only micromolar (μM) concentration [9,10].
It shows that the binding point of BDQ and ATP synthase may not only include subunit c [10].
The structure shows that the binding of BDQ caused a great conformational change of ATP synthase.
Among the 9 subunits c of mycobacterial ATP synthase, 7 can bind to BDQ.
Five of these sites only contain subunit c, and the remaining two sites include subunit a in addition to subunit c (Figures C and D below), which greatly increases the number of these two sites Van der Waals interface between BDQ and ATP synthase.
The authors suspect that these two sites with the participation of subunit a are the reason why BDQ can inhibit ATP synthase at nanomolar concentrations.
To test this hypothesis, the authors used a desalting column to wash the ATP synthase bound to excess BDQ and analyzed its structure.
Among the 7 binding sites after washing, the occupancy of the 5 sites containing only subunit c decreased significantly, while the occupancy of the 2 sites containing subunit a remained basically unchanged, indicating that BDQ Its nanomolar affinity comes from the common contact with subunits a and c (pictured below, video 2).
Interestingly, when the authors tested the inhibitory effect of BDQ with M.
smegmatis ATP synthase that knocked out the subunit α hook, Nanomolar concentration can inhibit ATP synthase from hydrolyzing BDQ of ATP, but at micromolar concentration, the inhibitory effect is lost.
This is similar to the results of previous experiments using inverted membrane vesicles to detect the inhibition of ATP synthesis by BDQ [11], which means that at micromolar concentrations, BDQ can "decouple" ATP synthase.
It is now known that the nanomolar concentration of BDQ can limit the growth of mycobacteria, while killing mycobacteria requires the concentration of BDQ to reach the micromolar level, which means that the decoupling of BDQ at the micromolar concentration may be very important for its bactericidal effect [11 ,12].
Unfortunately, although the authors added 200uM BDQ when analyzing the structure, they still failed to observe the ATP synthase decoupled by BDQ.
Understanding the mystery of BDQ decoupling ATP synthase requires more experimental follow-up.
Guo Hui and Gautier Courbon, doctoral students of the Department of Medical Biophysics at the University of Toronto, are the co-first authors of the article.
Original link: https:// Further reading: Science | Subcellular localization of antibiotics reveals the mechanism of anti-tuberculosis; ScienceBased on the development of anti-tuberculosis drug targets, Rao Zihe The academician team has spent many years analyzing the atomic resolution structure of mycobacterium respiratory supercomplex III2IV2SOD2.
Plate maker: SY reference scroll up to read 1.
Hershkovitz, I.
et al.
Detection and molecular characterization of 9000-year-old Mycobacterium tuberculosis from a neolithic settlement in the Eastern mediterranean.
PLoS One 3, 1–6 (2008).
2.
World Health Organization.
Global Tuberculosis Report 2019.
(World Health Organization, 2019).
3.
Johns Hopkins University Coronavirus Resource Center.
COVID-19 dashboard by the Center for Systems Science and Engineering (CSSE) at Johns Hopkins University.
2020 (https://coronavirus.
jhu.
edu/map.
html.
opens in new tab).
4.
Mahajan, R.
Bedaquiline: First FDA-approved tuberculosis drug in 40 years.
Int.
J.
Appl.
Basic Med.
Res.
3, 1 (2013).
5.
World Health Organization.
Consolidated Guidelines on Tuberculosis Treatment.
(World Health Organization, 2019).
6.
Andries, K.
et al.
A Diarylquinoline Drug Active on the ATP Synthase of Mycobacterium tuberculosis.
Pharmacogenomics 307, 223–227 (2005).
7 .
Lu, P.
, Lill, H.
& Bald, D.
ATP synthase in mycobacteria: Special features and implications for a function as drug target.
Biochim.
Biophys.
Acta-Bioenerg.
1837, 1208–1218 (2014).
8.
Preiss, L.
et al.
Structure of the mycobacterial ATP synthase Forotor ring in complex with the anti-TB drug bedaquiline.
Sci.
Adv.
1, 1–9 (2015).
9.
Koul, A.
et al.
Diarylquinolines target subunit c of mycobacterial ATP synthase.
Proteins Struct.
Funct.
Genet.
3, 323–324 (2007).
10.
Haagsma, AC et al.
Probing the interaction of the diarylquinoline TMC207 with its target mycobacterial ATP synthase.
PLoS One 6, 1–7 (2011).
11.
Hards, K.
et al.
Bactericidal mode of action of bedaquiline.
J.
Antimicrob.
Chemother.
70, 2028 –2037 (2015).
12.
Hards, K.
et al.
Ionophoric effects of the antitubercular drug bedaquiline.
Proc.
Natl.
Acad.
Sci.
115, 7326–7331 (2018).
In the descriptions of Eastern and Western poets and writers, tuberculosis is often given a romantic color.
This "romantic disease", even in the era when antibiotics are popularized, can still cause about 1.
5 million deaths every year [2], which is the same as the new crown that has ravaged the world this year (COVID19, as of December 10, 2020, the number of deaths is 1.
57 million.
[3]) The comparison is not too much.
Not only that, the resistance of tuberculosis to first-line antibiotics is also increasing year by year [2], making the development of a new generation of tuberculosis antibiotics more important.
Bedaquiline (bedaquiline, BDQ, Sirturo, TMC207, R207910), which was approved for marketing in many regions of the world in 2012-14, is the first approved after rifamycins (rifamycins) was used for clinical treatment in 1966 New tuberculosis antibiotics [4].
In the few years after approval, BDQ has become one of the safest and most effective drugs for the treatment of multidrug-resistant and extensively drug-resistant TB [5].
Unlike all previous tuberculosis antibiotics, the target of BDQ is tuberculosis ATP synthase [6].
ATP synthase is a macromolecular complex widely found in bacteria, chloroplasts and mitochondria.
It is the energy currency of cells-the production factory of ATP.
BDQ inhibits ATP synthase and limits the synthesis of ATP by Mycobacterium tuberculosis, thereby killing Mycobacterium tuberculosis.
Recently, the John Rubinstein team of Hospital for Sick Children in Canada published an article Structure of mycobacterial ATP synthase bound to the tuberculosis drug bedaquiline in Nature.
The author uses single-particle cryo-electron microscopy to analyze the series structure of Mycobacterium smegmatis (Mycobacterium smegmatis) ATP synthase binding to BDQ, and explains how BDQ binds to Mycobacterium ATP synthase with high affinity and ATP synthesis How does enzyme autoinhibition allow mycobacteria to preserve energy in an oxygen-deficient environment and increase the probability of survival.
The main function of ATP synthase is to synthesize ATP using electrochemical proton motive force.
But what is interesting is that when proton power disappears, ATP synthase can in turn hydrolyze ATP and consume energy.
Mycobacterium tuberculosis is a strictly aerobic bacterium, but it can survive in a low-consumption dormant state for many years in a hypoxic host, until it is activated under the right conditions to cause disease.
Therefore, it is particularly important to strictly control the hydrolysis of ATP synthase for Mycobacterium tuberculosis that lacks energy [7].
By observing the structure of M.
smegmatis ATP synthase, the authors found that there is a 35 amino acid extension at the C-terminus of subunit α (picture 1 and video 1 below).
It hooks on the rotor part of ATP synthase like a hook, so that it can only turn to the direction of ATP synthesis, thus ensuring that its hydrolysis function is limited to the minimum.
The authors then knocked out the hook part of ATP synthase and found that the ATP synthase after knocking out the hook showed normal ATP hydrolysis activity.
Previous experiments successfully proved that the binding site of BDQ is close to the subunit c of ATP synthase [6, 8].
What is puzzling is that although BDQ can inhibit the synthesis of ATP by ATP synthase at nanomolar (nM) concentration [8,9], its affinity for subunit c is only micromolar (μM) concentration [9,10].
It shows that the binding point of BDQ and ATP synthase may not only include subunit c [10].
The structure shows that the binding of BDQ caused a great conformational change of ATP synthase.
Among the 9 subunits c of mycobacterial ATP synthase, 7 can bind to BDQ.
Five of these sites only contain subunit c, and the remaining two sites include subunit a in addition to subunit c (Figures C and D below), which greatly increases the number of these two sites Van der Waals interface between BDQ and ATP synthase.
The authors suspect that these two sites with the participation of subunit a are the reason why BDQ can inhibit ATP synthase at nanomolar concentrations.
To test this hypothesis, the authors used a desalting column to wash the ATP synthase bound to excess BDQ and analyzed its structure.
Among the 7 binding sites after washing, the occupancy of the 5 sites containing only subunit c decreased significantly, while the occupancy of the 2 sites containing subunit a remained basically unchanged, indicating that BDQ Its nanomolar affinity comes from the common contact with subunits a and c (pictured below, video 2).
Interestingly, when the authors tested the inhibitory effect of BDQ with M.
smegmatis ATP synthase that knocked out the subunit α hook, Nanomolar concentration can inhibit ATP synthase from hydrolyzing BDQ of ATP, but at micromolar concentration, the inhibitory effect is lost.
This is similar to the results of previous experiments using inverted membrane vesicles to detect the inhibition of ATP synthesis by BDQ [11], which means that at micromolar concentrations, BDQ can "decouple" ATP synthase.
It is now known that the nanomolar concentration of BDQ can limit the growth of mycobacteria, while killing mycobacteria requires the concentration of BDQ to reach the micromolar level, which means that the decoupling of BDQ at the micromolar concentration may be very important for its bactericidal effect [11 ,12].
Unfortunately, although the authors added 200uM BDQ when analyzing the structure, they still failed to observe the ATP synthase decoupled by BDQ.
Understanding the mystery of BDQ decoupling ATP synthase requires more experimental follow-up.
Guo Hui and Gautier Courbon, doctoral students of the Department of Medical Biophysics at the University of Toronto, are the co-first authors of the article.
Original link: https:// Further reading: Science | Subcellular localization of antibiotics reveals the mechanism of anti-tuberculosis; ScienceBased on the development of anti-tuberculosis drug targets, Rao Zihe The academician team has spent many years analyzing the atomic resolution structure of mycobacterium respiratory supercomplex III2IV2SOD2.
Plate maker: SY reference scroll up to read 1.
Hershkovitz, I.
et al.
Detection and molecular characterization of 9000-year-old Mycobacterium tuberculosis from a neolithic settlement in the Eastern mediterranean.
PLoS One 3, 1–6 (2008).
2.
World Health Organization.
Global Tuberculosis Report 2019.
(World Health Organization, 2019).
3.
Johns Hopkins University Coronavirus Resource Center.
COVID-19 dashboard by the Center for Systems Science and Engineering (CSSE) at Johns Hopkins University.
2020 (https://coronavirus.
jhu.
edu/map.
html.
opens in new tab).
4.
Mahajan, R.
Bedaquiline: First FDA-approved tuberculosis drug in 40 years.
Int.
J.
Appl.
Basic Med.
Res.
3, 1 (2013).
5.
World Health Organization.
Consolidated Guidelines on Tuberculosis Treatment.
(World Health Organization, 2019).
6.
Andries, K.
et al.
A Diarylquinoline Drug Active on the ATP Synthase of Mycobacterium tuberculosis.
Pharmacogenomics 307, 223–227 (2005).
7 .
Lu, P.
, Lill, H.
& Bald, D.
ATP synthase in mycobacteria: Special features and implications for a function as drug target.
Biochim.
Biophys.
Acta-Bioenerg.
1837, 1208–1218 (2014).
8.
Preiss, L.
et al.
Structure of the mycobacterial ATP synthase Forotor ring in complex with the anti-TB drug bedaquiline.
Sci.
Adv.
1, 1–9 (2015).
9.
Koul, A.
et al.
Diarylquinolines target subunit c of mycobacterial ATP synthase.
Proteins Struct.
Funct.
Genet.
3, 323–324 (2007).
10.
Haagsma, AC et al.
Probing the interaction of the diarylquinoline TMC207 with its target mycobacterial ATP synthase.
PLoS One 6, 1–7 (2011).
11.
Hards, K.
et al.
Bactericidal mode of action of bedaquiline.
J.
Antimicrob.
Chemother.
70, 2028 –2037 (2015).
12.
Hards, K.
et al.
Ionophoric effects of the antitubercular drug bedaquiline.
Proc.
Natl.
Acad.
Sci.
115, 7326–7331 (2018).
