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MRC Mitochondrial Biology Unit
SCIENTIFIC METHOD & EXPERIMENTAL DESIGN
Bioenergetics of the Mitochondrion:
Structure & Function of ATP Synthase
MRC Mitochondrial Biology Unit
Max Perutz 1914-2002
“In Science truth always wins”
MRC Mitochondrial Biology Unit
Boris Ephrussi 1901-1979
Discovered cytoplasmic inheritance
“We cannot discover the truth of an hypothesis by counting the number of people who believe it.”
MRC Mitochondrial Biology Unit
The Scientific Method: version 1
• Hypothesis
• Experiment to test hypothesis
• Result; accept, reject or modify hypothesis
• Conclusion
MRC Mitochondrial Biology Unit
The Scientific Method v2: accretion of information
• Collect accurate observations
• Gradually build up a detailed picture
• “Until it becomes unreasonable to hold an opposing view” A. Klug
• Test features experimentally (and by calculation and modelling)
MRC Mitochondrial Biology Unit
Then Now
MRC Mitochondrial Biology Unit
Peter Mitchell (1920-1992)
MRC Mitochondrial Biology Unit
MRC Mitochondrial Biology Unit
Metabolism in mitochondria
MRC Mitochondrial Biology Unit
MRC Mitochondrial Biology Unit
MRC Mitochondrial Biology Unit
MRC Mitochondrial Biology Unit
Subunit compositions of F-ATPases
Eubacteria and chloroplasts Mitochondria
The differences provide opportunities for drug discovery
MRC Mitochondrial Biology Unit
ATP synthase rotor ring sizes 8-17
stator F1 highly conserved
peripheral stalk highly variable
All ATP synthase conserve central features
MRC Mitochondrial Biology Unit
ATP synthase from Trypanosoma brucei has an elaborated canonical F1-domain and conventional catalytic sites. Montgomery, M. G., Gahura, O., Leslie, A. G. W., Zikova, E. & Walker, J. E. (2018). Proc. Natl. Acad. Sci. U. S. A. 115, 2102-2107.
Fundamental features of F-ATPases are highly conserved
MRC Mitochondrial Biology Unit
What do we still need to know about the F-ATPase?
• 1. How is rotation generated during ATP hydrolysis?
• 2. How is rotation generated during ATP synthesis?
• 3. How is the enzyme regulated?
• 4. How is the enzyme assembled?
• 5. Is the permeability transition pore associated?
MRC Mitochondrial Biology Unit
1. How rotation is generated from ATP hydrolysis
• “Single molecule” rotational data mainly from bacteria
• Structural data almost entirely from mitochondria
• Reconciliation of these two data sets
MRC Mitochondrial Biology Unit
MRC Mitochondrial Biology Unit
Kinosita, Yoshida, Noji & colleagues
MRC Mitochondrial Biology Unit
MRC Mitochondrial Biology Unit
Structural definition of phosphate release stepp
IF1 stops the enzyme at the catalytic dwell
Thiophosphate stops F1-ATPase at the phosphate release dwell
MRC Mitochondrial Biology Unit
Active mammalian IF1 is an antiparallel
α-helical dimer in crystals
130 Å
N CCN
MRC Mitochondrial Biology Unit
pH regulates oligomeric state and inhibitory activity of IF1
Inactive tetramer converted to active dimer by decreasing pH from 8.0 to 6.5
Cabezon, Butler, Runswick & Walker, J Biol Chem (2000) 275, 25460-25464
MRC Mitochondrial Biology Unit
pH and Ca2+ regulate the oligomeric state and inhibitory activity of IF1
Boreikaite, Wicky, Watt, Clarke & Walker, PNAS (2019) 116,10354-10359
MRC Mitochondrial Biology Unit
• Inhibits ATP hydrolysis, but not synthesis
• Inhibitory region intrinsically disordered when free, structured when bound
• Inhibitory activity regulated by pH and [Ca2+]
• Inhibitory activity not regulated by phosphorylation of Ser-14
Properties of IF1
MRC Mitochondrial Biology Unit
Bason, Montgomery, Leslie & Walker 2015 PNAS 112, 6009-6014
Thiophosphate inhibited bovine F1-ATPase
MRC Mitochondrial Biology Unit
MRC Mitochondrial Biology Unit
2. How is rotation driven by the pmf?
• mechanical coupling mechanism
• the proton pathway
MRC Mitochondrial Biology Unit
W. Junge
MRC Mitochondrial Biology Unit
H+/ATP = ring symmetry/ 3
MRC Mitochondrial Biology Unit
Morales et al, 2015 Vinothkumar et al, 2016 Zhou et al, 2015
MRC Mitochondrial Biology Unit
MRC Mitochondrial Biology Unit
MRC Mitochondrial Biology Unit
MRC Mitochondrial Biology Unit
MRC Mitochondrial Biology Unit
MRC Mitochondrial Biology Unit
MRC Mitochondrial Biology Unit
MRC Mitochondrial Biology Unit
3. Assembly of human ATP synthase
MRC Mitochondrial Biology Unit
MRC Mitochondrial Biology Unit
MRC Mitochondrial Biology Unit
MRC Mitochondrial Biology Unit
ATP synthase is assembled from modules
• ATPase evolved in a modular fashion
• ATPase made of three structural modules
• Achieved by different routes in bacteria, yeast and mammals
MRC Mitochondrial Biology Unit
Walker, J. E. & Cozens, A. L. (1986). Evolution of ATP synthase. Chemica Scripta 26B, 263-272.
Modular evolution of ATP synthase
MRC Mitochondrial Biology Unit
Modular assembly of yeast ATP synthase
A Tzagoloff and colleagues
MRC Mitochondrial Biology Unit
Studies of assembly of human ATP synthase
• Remove each subunit selectively by CRISPR-cas9
• Characterise vestigial ATPase by SDS-PAGE, NGE and SILAC
• Examine effect on growth of clonal cells
d
γ
δ εα α
β
β
β
IF1
c eg
b
F6
OSCP
8
d
γ
δ εα α
β
β
β
c
b
F6
OSCP
8
IF1
γ
δ εα α
β
β
β
IF1
c8
d
γ
δ εα α
β
β
β
IF1
c eg
b
F6
OSCP
8 f
d
γ
δ εα α
β
β
β
IF1
eg
b
F6
OSCP
f
d
γ
δ εα α
β
β
β
IF1
c eg
b
F6
OSCP
8 f
d
γ
δ εα α
β
β
β
c eg
b
F6
OSCP
8
ATP6
ATP8 f6.8P
L
d
γ
δ εα α
β
β
β
c eg
b
F6
OSCP
8
ATP6
ATP8 f
6.8P
LDAP
IT
Δb (or OSCP)
ρ0
Δe (or g)
Δc
Δf
Δ6.8kDΔDAPIT
Monomer Monomer Monomer
Monomer
Monomer
Monomer/dimerDimerOligomer
Complete
d
γ
δ εα α
β
β
β
c eg
b
F6
OSCP
8
ATP6
ATP8 f
6.8P
LDAP
IT
Effect of removal of yeast subunit g on mitochondrial morphology
Control mitochondria Δg mitochondria
Paumard, … & Velours, EMBO J (2002) 21, 221-230
MRC Mitochondrial Biology Unit
Harner et al (2016), ELife 5e18853
MICOS, mitochondrial inner membrane complex for maintenance of cristae junctions
MRC Mitochondrial Biology Unit
MRC Mitochondrial Biology Unit
The mitochondrial
permeability transition pore
MRC Mitochondrial Biology Unit
MRC Mitochondrial Biology Unit
Mitochondrial permeability transition pore
• 1976, Hunter et al
• Allows molecules < 1.5 kDa to pass; diameter 2 nm
• Consequences: mitochondrial depolarization, uncoupling of ox-phos, mitochondrial swelling
• Proposed components: VDAC, ANT
• Inducers: Ca2+, adenine nucleotides, phosphate, oxidative stress
• Inhibitor: Cyclosporin A
MRC Mitochondrial Biology Unit
Defining features of the PTP
• Opened by elevation of matrix Ca2+
• Opening inhibited by cyclosporin A binding to cyclophilin D
• Opening leads to entry of water and swelling of mitochondria
• Pore estimated to be about 2-2.9 nm in diameter
MRC Mitochondrial Biology Unit
Induction of the PTP
• Treat cells with thapsigargin
• Permeabilize cytoplasmic membrane with the ionophore ferutinin and introduce exogenous Ca2+
• Permeabilize cells with digitonin and introduce pulses of Ca2+
MRC Mitochondrial Biology Unit
Pore opening and cyclophilin D
• peptidylprolyl cis-trans isomerase
• mitochondrial version of a larger family
• modulates PTP, but is not a component
• binding of cyclosporin A inhibits pore opening
MRC Mitochondrial Biology Unit
MRC Mitochondrial Biology Unit
The Scientific Method
• Observation: mitochondria have a permeability transition pore
• Hypothesis: dimeric ATP synthase or components provide the PTP
• Experiment: systematically remove dimeric ATPase; test the pore
• Result: the pore survives removal of dimeric ATPase
• Conclusion: the dimeric ATPase does not provide the pore
• See: Walker et al (2020) www.pnas.org/cgi/doi/10.1073/pnas.1921409117
MRC Mitochondrial Biology Unit
Topic 1: The membrane rotors of ATP synthases in various species have a range of symmetries from 8-17. For example for humans it is 8; for Synechococcus it is 15. What are the bioenergetic consequences, and why do you think that the enzymes have evolved in this way?
• Walker, J. E. (2013). The Keilin Lecture 2012: The ATP synthase: the understood, the uncertain and the unknown. Biochem. Soc. Trans. 41, 1-16.
• Watt, I. N., Montgomery, M. G., Runswick, M. J., Leslie, A. G. W. & Walker, J. E. (2010). Bioenergetic cost of making an adenosine triphosphate molecule in animal mitochondria. Proc. Natl. Acad. Sci. U. S. A. 107, 16823-16827.
• Walpole, T. B., Palmer, D. N., Jiang, H., Ding. S., Fearnley, I. M., & Walker, J.E. (2015). Conservation of complete trimethylation of lysine-43 in the rotor ring of c-subunits of metazoan ATP synthases. Mol. Cell. Proteomics. 14, 828-840.
MRC Mitochondrial Biology Unit
Topic 2. In the past 20 years or so, bedaquiline is the only new drug introduced into clinical practice for treatment of tuberculosis. It inhibits the mycobacterial ATP synthase, but how? Given the urgent need for developing new drugs against multiply or even totally resistant bacterial pathogens, what features of the ATP synthase make it an attractive target for development of new drugs against bacterial pathogens, and what are the draw-backs? Remember, ideally the drug should not affect the human enzyme
• Walker, J. E. (2013). The Keilin Lecture 2012: The ATP synthase: the understood, the uncertain and the unknown. Biochem. Soc. Trans. 41, 1-16
• Bason, J., Montgomery, M. G., Leslie A. G. W., & Walker, J. E. (2014). Pathway of binding of the intrinsically disordered mitochondrial inhibitor protein to F1-ATPase. Proc. Natl. Acad. Sci. U. S. A. 11, 11305-11310.
• Andries et al. (2005). A diarylquinoline drug active on the ATP synthase from Mycobacterium tuberculosis. Science, 307, 223-227.
• Preiss et al (2015). Structure of the mycobacterial ATP synthase Fo rotor ring in complex with the anti-TB drug bedaquiline. Sci. Adv. 1:e1500106
• Zhang et al (2019). The structure of the catalytic domain the ATP synthase from Mycobacterium smegmatis is a target fro developing antitubercular drugs. Proc. Natl. Acad. Sci. U. S. A. 116, 4206-4211.
MRC Mitochondrial Biology Unit
Topic 3. The permeability transition in human mitochondria refers to the opening of a non-specific membrane channel, known as the permeability transition pore (PTP) in the inner membrane. Opening is triggered by calcium ions, or reactive oxygen species, among other effectors, leading to swelling of the organelle, disruption of the inner membrane, loss of ATP synthesis and cell death. It is an uncharacterized part of cell death by necrosis and possibly apoptosis. Past proposals that the pore is provided by the voltage dependent ion channel in the outer membrane plus the adenine nucleotide translocase in the inner membrane have not withstood scrutiny. A recent proposal is that the pore is associated with the dimeric ATP synthase. How would you test this hypothesis? Remember, you will probably need to assay the functional PTP in a cellular context.
• Dimers of mitochondrial ATP synthase form the permeability transition pore. Georgia et al. (2013) PNAS 110, 5887-5892.
• An uncoupling channel within the c-subunit of the ATP synthase is the permeability transition pore Alavian et al. (2014) PNAS 111, 10580-10585.
MRC Mitochondrial Biology Unit
MRC Mitochondrial Biology Unit
Topic 1: The membrane rotors of ATP synthases in various species have a range of symmetries from 8-17. For example for humans it is 8; for Synechococcus it is 15. What are the bioenergetic consequences, and why do you think that the enzymes have evolved in this way?
• Walker, J. E. (2013). The Keilin Lecture 2012: The ATP synthase: the understood, the uncertain and the unknown. Biochem. Soc. Trans. 41, 1-16.
• Watt, I. N., Montgomery, M. G., Runswick, M. J., Leslie, A. G. W. & Walker, J. E. (2010). Bioenergetic cost of making an adenosine triphosphate molecule in animal mitochondria. Proc. Natl. Acad. Sci. U. S. A. 107, 16823-16827.
• Walpole, T. B., Palmer, D. N., Jiang, H., Ding. S., Fearnley, I. M., & Walker, J.E. (2015). Conservation of complete trimethylation of lysine-43 in the rotor ring of c-subunits of metazoan ATP synthases. Mol. Cell. Proteomics. 14, 828-840.
MRC Mitochondrial Biology Unit
c-ring symmetries and H+:ATP ratios
• 8-fold symmetry in metazoans
• 10-fold symmetry in yeast
• 10-15-fold in eubacteria
• 14-fold in chloroplasts
• Therefore, H+/ATP = 2.7-5.0, depending on species
MRC Mitochondrial Biology Unit
P/O ratio in mammalian mitochondria
e- donor P/O calc P/O exp
NADH 10/3.7 = 2.7 2.5
Succinate 6/3.7 = 1.6 1.5
NADH 2e- to O2 = 10 H+ pumped out Succinate 2e- to O2 = 6 H+ pumped out ADP/ATP exchange + Pi/H+ symport = 1 H+ in
ATP synthesis H+/ATP = 8/3 = 2.7ATP synthesis H+/ATP = 3.7
Moles ADP phosphorylated/2e- to O2 = P/O ratio
MRC Mitochondrial Biology Unit
MRC Mitochondrial Biology Unit
MRC Mitochondrial Biology Unit
MRC Mitochondrial Biology Unit
MRC Mitochondrial Biology Unit
MRC Mitochondrial Biology Unit
Topic 2. In the past 20 years or so, bedaquiline is the only new drug introduced into clinical practice for treatment of tuberculosis. It inhibits the mycobacterial ATP synthase, but how? Given the urgent need for developing new drugs against multiply or even totally resistant bacterial pathogens, what features of the ATP synthase make it an attractive target for development of new drugs against bacterial pathogens, and what are the draw-backs? Remember, ideally the drug should not affect the human enzyme
• Walker, J. E. (2013). The Keilin Lecture 2012: The ATP synthase: the understood, the uncertain and the unknown. Biochem. Soc. Trans. 41, 1-16
• Bason, J., Montgomery, M. G., Leslie A. G. W., & Walker, J. E. (2014). Pathway of binding of the intrinsically disordered mitochondrial inhibitor protein to F1-ATPase. Proc. Natl. Acad. Sci. U. S. A. 11, 11305-11310.
• Andries et al. (2005). A diarylquinoline drug active on the ATP synthase from Mycobacterium tuberculosis. Science, 307, 223-227.
• Preiss et al (2015). Structure of the mycobacterial ATP synthase Fo rotor ring in complex with the anti-TB drug bedaquiline. Sci. Adv. 1:e1500106
• Zhang et al (2019). The structure of the catalytic domain the ATP synthase from Mycobacterium smegmatis is a target fro developing antitubercular drugs. Proc. Natl. Acad. Sci. U. S. A. 116, 4206-4211.
MRC Mitochondrial Biology Unit
Inhibition of Staph. aureus by penicillin exuded from the mould
Fleming’s experiment in 1928
MRC Mitochondrial Biology Unit
Chronology of penicillin development
• 1928 Fleming’s observation
• 1939 Florey, Chain start investigation of penicillin
• 1940 Abraham: penicillin resistant Staph aureus
• 1943 Penicillin used in clinical practice
MRC Mitochondrial Biology Unit
Nobel Prize in Physiology or Medicine 1945“for the discovery of penicillin and its curative effect in various
infectious diseases”
MRC Mitochondrial Biology Unit
MRC Mitochondrial Biology Unit
Emergence of antibiotic resistance
MRC Mitochondrial Biology Unit
MRC Mitochondrial Biology Unit
Annual death by antimicrobial resistance (AMR)
Tetanus60000
Cholera110000
Measles130000
AMR700000
Diabetes1500000
Cancers8200000
Diarrhea1300000
Car traffic accidents1300000
AMR in 205010000000
WHO. Review on Antimicrobial Resistance. 2014
MRC Mitochondrial Biology Unit
Annual deaths from AMR by 2050
MRC Mitochondrial Biology Unit
Spread of antibiotic resistance
MRC Mitochondrial Biology Unit
MRC Mitochondrial Biology Unit
In the USA, animals consume twice as many medically important antibiotics as humans
MRC Mitochondrial Biology Unit
1940 1950 1960 1970 1980 1990 2000 2010
Sulfa drugsPenicillin
TetracyclineMacroglidesAminoglycosidesChloramphenicol
Discovery gap
Antibiotic era Increasing drug resistance
GlycopeptidesStreptomycinsQuinolinesCephalosporins
OxazolidoninesMutilinsLipopeptides
MRC Mitochondrial Biology Unit
Tuberculosis
!Disease of the respiratory system; can spread to brain, kidneys and bones
!Highly contagious via coughing
! Symptoms are chest pain, fatigue, weight loss, fever
! 2 billion people infected; 1.6 million estimated deaths in 2017
! 60% untreated die
MRC Mitochondrial Biology Unit
Why is there a resurgence of TB ?
!Poor adherence to TB control programs
!Emergence of drug resistance - MDR, XRD, TDR
!Widespread synergy with HIV
!No quick and accurate diagnostics
!Outdated drugs and vaccines
MRC Mitochondrial Biology Unit
Dormancy Resuscitation
MMP, matrix metallo-protease
MRC Mitochondrial Biology Unit
Control of TB requires
• New drugs against novel targets
• New vaccines - BCG (Bacillus Calmette-Guerin) was introduced in 1921
• Early diagnosis
MRC Mitochondrial Biology Unit
Shortens anti-tuberculosis treatment (8 weeks) and effective (bactericidal-sterilizing) in patients with drug-susceptible or drug-resistant TB
Most significant discovery in TB research – last 50 years
2 weeks
FDCs
2-4 months6-30 months
MRC Mitochondrial Biology Unit
Bedaquiline (SirturoTM)
• First approved anti-TB drug with novel action in 40 years
• Inhibits ATP synthase by binding to the rotor
• Highly specific for mycobacteria
• Active against both dormant and replicating states
• Active against MDR and XDR mycobacteria
MRC Mitochondrial Biology Unit
Bedaquiline is not a wonder drug
• FDA approval based on phase 2 trials (440 people)
• recommended only for MDR-, XDR- and TDR-TB
• cardiovascular risk
• very lipophilic; human half-life 5-6 months
• patients susceptible to phospholipidosis
• can cause death (10% unexplained)
• need more inhibitors of mycobacterial ATPase
MRC Mitochondrial Biology Unit
Zhang, Montgomery, Leslie, Cook & Walker, PNAS (2019) 116, 4206-4211
The catalytic domain of ATP synthase from M. smegmatis as a drug target
MRC Mitochondrial Biology Unit
Topic 3. The permeability transition in human mitochondria refers to the opening of a non-specific membrane channel, known as the permeability transition pore (PTP) in the inner membrane. Opening is triggered by calcium ions, or reactive oxygen species, among other effectors, leading to swelling of the organelle, disruption of the inner membrane, loss of ATP synthesis and cell death. It is an uncharacterized part of cell death by necrosis and possibly apoptosis. Past proposals that the pore is provided by the voltage dependent ion channel in the outer membrane plus the adenine nucleotide translocase in the inner membrane have not withstood scrutiny. A recent proposal is that the pore is associated with the dimeric ATP synthase. How would you test this hypothesis? Remember, you will probably need to assay the functional PTP in a cellular context.
• Dimers of mitochondrial ATP synthase form the permeability transition pore. Georgia et al. (2013) PNAS 110, 5887-5892.
• An uncoupling channel within the c-subunit of the ATP synthase is the permeability transition pore Alavian et al. (2014) PNAS 111, 10580-10585.
MRC Mitochondrial Biology Unit
Human HAP1 cells
• near haploid human cell line used with CRISPR-cas9 • derived from myeloid leukaemia • fragment of chr15 in chr19, and reciprocal translocation chr 9 and 22• ATP5G1-G3 on chrs 17, 12 and 2
MRC Mitochondrial Biology Unit
ATP5G1 & ATP5G2 Dyer & Walker, Biochem J (1993) 293, 51-64 ATP5G3 Yan et al (1994) Genomics 24, 375-377
MRC Mitochondrial Biology Unit
Expression of subunit c in human HAP1cells
MRC Mitochondrial Biology Unit
Identification of a HAP1 clone devoid of subunit c
J He, H Ford, J Carrol, S Ding, I M Fearnley & J E Walker, PNAS (2017) 114, 3409-3414
MRC Mitochondrial Biology Unit
MRC Mitochondrial Biology Unit
MRC Mitochondrial Biology Unit
MRC Mitochondrial Biology Unit
Mitochondrial F-ATPase and the PTP: conclusions
• subunit c is not involved in forming the PTP
• nor are subunits ATP6 and ATP8
MRC Mitochondrial Biology Unit
MRC Mitochondrial Biology Unit
Ca2+ retention capacity in permeabilised HAP1-WT, Δb and ΔOSCP cells
HAP1-WT
HAP1-Δb
HAP1-ΔOSCP
-CsA +CsA
MRC Mitochondrial Biology Unit
Summary of findings
• Removal of subunit c and [ATP6 + ATP8] has no effect on the pore
• Removal of e, f, g, 6.8PL or DAPIT has no effect on the pore
• Removal of b or OSCP has no effect on the pore
• In δ[c + δ] cells, there is no ATP synthase, but still a PTP
• ….. pore size is same as in WT-cells
• ….. inhibition by cyclosporin A, BKA and CATR as in WT-cells
• ATP SYNTHASE DOES NOT PROVIDE THE PTP
MRC Mitochondrial Biology Unit