In the conversation titled “What about the mitochondria” this term ATP popped up in some quoted text.
This biological ATP is not the acronym for the Association of Tennis Professionals. 
Doesn’t this ATP thing deserve a separate conversation?
Substrate level phosphorylation. Just want to mention this right here.
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I searched the term you wrote and found it well explained in Wikipedia. Thanks for your kind assistance. I appreciate it.
Interesting ATP-related paper:
Evolution of the F0F1 ATP Synthase Complex in Light of the Patchy Distribution of Different Bioenergetic Pathways across Prokaryotes
Vassiliki Lila Koumandou, Sophia Kossida
2014 DOI: 10.1371/journal.pcbi.1003821
Bacteria and archaea are characterized by an amazing metabolic diversity, which allows them to persist in diverse and often extreme habitats. Apart from oxygenic photosynthesis and oxidative phosphorylation, well-studied processes from chloroplasts and mitochondria of plants and animals, prokaryotes utilize various chemo- or lithotrophic modes, such as anoxygenic photosynthesis, iron oxidation and reduction, sulfate reduction, and methanogenesis. Most bioenergetic pathways have a similar general structure, with an electron transport chain composed of protein complexes acting as electron donors and acceptors, as well as a central cytochrome complex, mobile electron carriers, and an ATP synthase. While each pathway has been studied in considerable detail in isolation, not much is known about their relative evolutionary relationships.
Our results indicate an ancient origin of this protein complex, and no clustering based on bioenergetic mode, which suggests that no special modifications are needed for the ATP synthase to work with different electron transport chains. Moreover, examination of the ATP synthase genetic locus indicates various gene rearrangements in the different bacterial lineages, ancient duplications of atpI and of the beta subunit of the F0 subcomplex, as well as more recent stochastic lineage-specific and species-specific duplications of all subunits.
if different bioenergetic pathways dispersed into different lineages by horizontal gene transfer, this did not involve the ATP synthase complex. Presumably, each species used its pre-existing ATP synthase complex and adapted it to utilize the proton gradient generated by vastly different ETCs.
A more thorough structural analysis would be needed to examine if certain structural modifications unite the ATP synthases of organisms using each bioenergetic pathway.
Given the ancient origin of the F0F1 ATPase, the phylogenetic trees can perhaps give clues as to the evolutionary relationships between different bacterial lineages. The branching order of bacterial lineages remains an issue unresolved through phylogenetic analysis
the ATP synthase cannot be used to reconstruct the origin of the diversity of bioenergetic modes in prokaryotes.
The F0F1 ATP synthase genetic locus is overall well conserved, although as demonstrated by multiple splits and duplications, in principle, the system is robust and flexible, as it can deal with a split between any subunits and/or a duplication of any subunit. The elucidation of the way in which certain species deal with these duplications, splits and losses, and the advantage any of these may confer, now requires further study.
Another interesting paper on the same topic:
Biophysical comparison of ATP synthesis mechanisms shows a kinetic advantage for the rotary process
Ramu Anandakrishnan, Zining Zhang, Rory Donovan-Maiye, and Daniel M. Zuckerman
PNAS October 2016 113 (40) 11220-11225;
DOI: 10.1073/pnas.1608533113
All living organisms—archaea, bacteria, and eukarya—use an intricate rotary molecular machine to synthesize ATP, the energy currency of the cell.
the rotary mechanism is faster than other possible mechanisms, particularly under challenging conditions, suggesting a possible evolutionary advantage.
The ATP synthase (F-ATPase) is a highly complex rotary machine that synthesizes ATP, powered by a proton electrochemical gradient.
the complexity of the F-ATPase may have resulted from positive selection for its kinetic advantage.
The rotary ATP synthase is universally used by living organisms as the primary mechanism for synthesizing ATP.
synthesis of ATP by a rotary mechanism—in which protons pass one at a time through the synthase—is more efficient than other mechanisms, particularly under challenging low-energy conditions.
Another interesting ATP Synthase paper:
Identification of cryptic subunits from an apicomplexan ATP synthase
eLife 2018;7:e38097 DOI: 10.7554/eLife.38097
The mitochondrial ATP synthase is a macromolecular motor that uses the proton gradient to generate ATP. Proper ATP synthase function requires a stator linking the catalytic and rotary portions of the complex.
Our findings highlight divergent features of the central metabolic machinery in apicomplexans, which may reveal new therapeutic opportunities.
The ATP synthase is a highly conserved protein complex found in the plasma membrane of bacteria, the inner membrane of mitochondria, and the thylakoid membrane of chloroplasts. The complex consists of two functionally distinct portions: the hydrophilic F1 and the membrane-bound Fo (Walker, 2013). The mechanism of this molecular motor is best understood for the mitochondrial ATP synthases of yeast and mammals. Within their mitochondria, the proton gradient generated by the electron transport chain (ETC) drives the rotation of a ring of csubunits in Fo and of the attached central stalk within F1. This rotation causes the conformational changes in the α and β subunits of F1 that mediate catalysis of ATP from ADP and inorganic phosphate (Pi) (Jonckheere et al., 2012). The stator, also known as the lateral stalk, is an essential component of the ATP synthase because it counteracts the rotation of the α and β subunits, enabling ATP synthesis (Dickson et al., 2006). It is therefore surprising that despite general conservation of the central subunits, the lateral elements of protozoan ATP synthases are structurally diverse, and these organisms appear to lack homologs for the stator subunits of yeast and mammals (Lapaille et al., 2010).
The elegant rotary mechanism of the mitochondrial ATP synthase requires a stator to impart the conformational changes on F1 that drive ATP catalysis. It has therefore been a long-standing conundrum that most apicomplexan genomes appear to code for only the core subunits of the ATP synthase but none of the stator subunits.
Our results reveal highly divergent aspects of the apicomplexan ATP synthase and demonstrate their importance for parasite viability.
Our work demonstrates that distant homology searches can complement the molecular characterization of protein complexes in apicomplexans as in other divergent eukaryotes.
Having provided strong evidence for the role of ICAP2 in the ATP synthase, more work will be needed to characterize the structure of the complex and understand how its unusual features mediate the specific adaptations of apicomplexan mitochondria.
The variable impact of the ATP synthase on the various stages of different apicomplexans may reflect the ability of these parasites to adapt to the changing environmental conditions they encounter across their life cycles.
To further understand the effects that lead to mitochondrial fragmentation, it will be interesting to compare uncoupling mutations, like ICAP2 loss, to disruption of the catalytic or proton-transporting functions of the T. gondii ATP synthase.
inhibiting oxidative phosphorylation at different steps is expected to have different consequences, which should be investigated further as we explore these pathways as therapeutic targets in apicomplexans.
Future studies into the structure and function of these divergent features will help us understand their contributions to apicomplexan adaptation. Unlike most organisms, all of the ATP synthase subunits are encoded in the nuclear genome, making T. gondii a highly tractable organism to study the evolution of this important protein complex.
I already told you what ATP stands for. Might I suggest that you consult a biology text, or perhaps one of those online courses you enjoy?
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Well if this thread is going to consist of nothing but quotations of papers detailing analysis of ATP synthase, I might aswell participate:
Finnigan GC, Hanson-Smith V, Stevens TH, Thornton JW. Evolution of increased
complexity in a molecular machine. Nature. 2012 Jan 9;481(7381):360-4. DOI:10.1038/nature10724
The vacuolar H±ATPase (V-ATPase) is a multisubunit protein complex that pumps protons across membranes to acidify subcellular compartments; this function is required for intracellular protein trafficking, coupled transport of small molecules and receptor-mediated endocytosis1.
Comparative genomic approaches suggest that the components of many molecular machines have appeared sequentially during evolution and that complexity increased gradually by incorporating new parts into simpler assemblies2–8.
…vertical approaches that combine computational phylogenetic analysis with gene synthesis and molecular assays allow changes in the sequence, structure and function of reconstructed ancestral proteins to be experimentally traced through time.9–11Here we apply this approach to characterize the evolution of a small molecular machine and dissect the mechanisms that caused it to increase in complexity.
To understand how the three-component ring evolved, we reconstructed ancestral V0 proteins from just before and after the increase in complexity, synthesized and functionally characterized them in a yeast genetic system, and used manipulative methods to identify the genetic and molecular mechanisms by which their functions changed.
We found that the ancestral two-subunit ring can functionally replace the three-subunit ring of extant yeast. When the resurrected Anc.3–11 was transformed into yeast deficient for Vma3 ( vma3 Δ) or Vma11( vma11 Δ), growth in the presence of elevated CaCl2 was rescued, indicating that the functions of the present-day Vma3 and Vma11 proteins were already present before the duplication that generated them (Fig. 2a).
Similar experiments with the components of the ancestral three-component ring show that after the duplication of Anc.3–11, its descendants Anc.3 and Anc.11 both became necessary for a functional complex because of complementary losses of ancestral functions.
Hey, so an irreducibly complex function evolved. Previously conditional components became critical after some initially neutral mutations occurred.
Taken together, these data indicate that the specificity of the ring arrangement and the obligate roles of Vma3 and Vma11 evolved by complementary loss of asymmetric interactions with other members of the ring (Fig. 3g, h).
How complexity and novel functions evolve has been a longstanding question in evolutionary biology25–27, because mutations that compromise existing functions are far more frequent than those that generate new ones28. Our results indicate that the architectural complexity of molecular assemblies can evolve because of a few simple, relatively high-probability mutations that degrade ancestral interfaces but leave other functions intact.
Although mutations that enhanced the functions of individual ring components may have occurred during evolution, our data indicate that simple degenerative mutations are sufficient to explain the historical increase in complexity of a crucial molecular machine. There is no need to invoke the acquisition of ‘novel’ functions caused by low-probability mutational combinations.
How fascinating!
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Very interesting paper indeed. Thanks.
Maybe now other contributors here would be encouraged to participate as well?
Here’s more information related to the the interesting paper you referenced:
Some assembly required: Contributions of Tom Stevens' lab to the V‐ATPase field
Laurie A. Graham, Gregory C. Finnigan, Patricia M. Kane 2018 DOI: 10.1111/tra.12559
The mechanisms by which a new subunit could be evolved within the V‐ATPase enzyme (or other multisubunit molecular machines) were not known. Therefore, in collaboration with Joe Thornton and colleagues, the Stevens lab computationally predicted and synthesized the most recent common ancestor to the Vma3 and Vma11 proteins (Anc.3‐11) as well as the ancestor to the more distantly related Vma16 subunit (Anc.16)—roughly estimated to be nearly a billion years ago. Following this “ancestral gene reconstruction,” the lab experimentally tested their function(s) in budding yeast.42 Incredibly, replacement of the entire yeast proteolipid system (deletion of all 3 native subunits) with the ancestral variants allowed for a functional V‐ATPase complex in vivo. Moreover, a mutational analysis of additional reconstructed variants revealed that inclusion of a small number of (loss‐of‐function) point mutations were sufficient to “ratchet” the newly evolved Vma11p ancestor into position as an obligate member of the structure.42 This work highlighted a possible general evolutionary mechanism for how biological complexity might arise by relatively “simple” molecular processes. A similar evolutionary approach was used to create an “ancestral a subunit,” a protein that had characteristics of both yeast subunit a isoforms Vph1p and Stv1p.
Here’s more information about the main author.
Fascinating.
Where can we look at the structures of Anc.3, Anc.11, Anc.16 compared to Vma3, Vma11, Vma16 respectively?
Perhaps you biologists can provide a link to that information?
Thanks.
Fascinating. I had not read about this work on the a-subunit before. Incredibly the n-terminals of the ancestral a-subunits inferred from extant species:
… is only 38 and 53% identical to the N-termini of Stv1p and Vph1p, respectively (Figure 2A).
Nevertheless they are still able to resurrect an ancestor so substantially different in sequence from it’s modern descendants, find that it is functional and can perform the functions of both extant descendants.
Even despite significant uncertainty about the ancestral state at a substantial number of residues, all mutants resurrected were still functional proteins:
Our ancestral reconstruction included 134 residues that were “poorly” supported by our maximum likelihood phylogenetic algorithm (posterior probability, <0.8) and had plausible secondary alternate states (posterior probability, >0.2). Posterior probability (PP) is a measure of the confidence of each ancestral state expressed as a probability (Hanson-Smith et al. , 2010
). Given the statistical support for alternative amino acids within the Anc.a sequence, we sampled 50 independent, single–amino acid substitutions to Anc.a; sites were chosen randomly across the entire protein sequence. Point mutations were introduced into the protein sequence of Anc.a, and each alternative state was tested in vph1Δ stv1Δ yeast by growth assays on calcium and zinc. We determined that our reconstruction was robust to uncertainty, as all 50 changes to Anc.a still allowed for full complementation (Supplemental Table S2).
I continue to be astonished that it is possible to reconstruct evolutionary events at the molecular scale that happened billions of years ago, in the laboratory, to test evolutionary predictions and to understand how they happened.
I don’t think they have been characterized in anything but secondary structure.
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Ok. Thanks.
I understood that they reverse-engineered Anc.3, Anc.11, Anc.16 which they put back in the system -replacing the modern versions Vma3, Vma11, Vma16- to test for affected functionality. But perhaps I misunderstood that?
Here’s another interesting paper on this topic:
The c-ring stoichiometry of ATP synthase is adapted to cell physiological requirements of alkaliphilic Bacillus pseudofirmus OF4
Laura Preiss, Adriana L. Klyszejko, David B. Hicks, Jun Liu, Oliver J. Fackelmayer, Özkan Yildiz, Terry A. Krulwich, and Thomas Meier
PNAS May 7, 2013 110 (19) 7874-7879; doi: 10.1073/pnas.1303333110
Alkaliphilic Bacillus species have evolved several adaptations to cope with the bioenergetic challenge of ATP synthesis at a low pmf . One of these adaptations is the stretch of AxAxAxA instead of GxGxGxG in the c-subunit sequence encoding the inner ring of α-helices of the c-ring. This alteration was proposed to enhance both the c-ring stoichiometry and its size in alkaliphilic Bacillus species (19, 20, 26). Our observations here concur with the expectation that a higher c-ring stoichiometry (i.e, increased i ) would be advantageous at a low overall pmf in alkaliphiles (21, 32), because the phosphorylation potential for ATP synthesis (ΔGp) at thermodynamic equilibrium equals i × pmf . The common theme of the enlarged c-ring stoichiometries found in alkaliphilic Bacillus species (c13) and the contrasting smaller c10rings found in neutralophilic Bacillus species [e.g., Bacillus PS3 (33), Geobacillus kaustophilus (Fig. S6)] further support this proposed evolutionary adaptation mechanism. The alanine motif is a necessary, but insufficient, adaptation of alkaliphilic Bacillus bacteria. It has a direct influence on the c-ring stoichiometry and its indigenous property to determine the ATP synthase i value, and thus directly modulates the cell’s physiology and bioenergetics, facilitating growth at pH >10. Remarkably, and in agreement with previous work (25), this observation also suggests that i can be adapted by just one or two selected mutations. This property enables adaptation to new environmental challenges, a process that can occur within a rather short evolutionary time frame.
[Emphasis added]
Cited by this recent paper:
Convergent evolution of unusual complex I homologs with increased proton pumping capacity: energetic and ecological implications.
Chadwick GL, et al. ISME J. 2018.
ISME J. 2018 Nov;12(11):2668-2680. doi: 10.1038/s41396-018-0210-1. Epub 2018 Jul 10.
Phylogenetic analyses reveal that these modified complexes appear to have arisen independently multiple times in a remarkable case of convergent molecular evolution.
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Hey, why no love for NADH?
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No you understood that correctly. That is what they did.
But recreating the ancestral versions of these molecules and using them in place of their modern descendants does not require that those molecules are characterized in their 3dimensional structure.
Essentially they are just inferring what the DNA sequence encoding the protein used to be like billions of years ago, then they synthesize such a DNA sequence, and splice it into the genome of an organism that had the modern versions of those genes deleted. The organism will then build this protein like it would any other protein, by transcribing this DNA sequence into RNA, and then translating this RNA into amino acid sequence.
They first test the organism after having the modern descendant proteins deleted to see how this affects the organism. They show that this negatively affects the organism because the functions that depended on this deleted gene no longer functions.
Then they splice in the reconstructed ancestor gene, and test the organism again, and show that this ancestor gene has successfully “resurrected” the function of the deleted gene.
They also do some labeling to work to demonstrate that the reconstructed ancestor gene is actually expressed in the cell, and produces a bona fide protein.
But they don’t physically characterize the protein in 3dimensional structure. They don’t do NMR or x-ray crystallography or any of that sort of work which would show what the shape of the ancestor protein is actually like, compared to the modern descendants. It is reasonable to infer, however, that it is very much like it’s modern descendants. The overall secondary structure of the protein (which can be inferred from merely knowing the DNA sequence that encodes it) has been preserved over this incredible span of time.
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Excellent explanation. Thanks.
Definitely my lack of strong biology education doesn’t help. I appreciate your assistance with understanding this topic.
I’m trying to understand the part of their paper where they explain (1) how they infer what the DNA sequence encoding the protein used to be like billions of years ago and (2) how different was the old DNA sequence that encoded for Anc.3, Anc.11, Anc.16, compared to the modern DNA sequence that encodes for Vma3, Vma11, Vma16.
Again, help from a more knowledgeable person is required in this case. Perhaps this is how a multidisciplinary team works in biology these days?
Thanks.
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This part is explained in the methods section " In silico reconstruction of ancestral protein sequences".
In general, such work would proceed like this:
A basic requirement is to gather protein sequences from several different organisms to use as the basis for the inference. Having these, they can use them to infer a phylogenetic tree from an alignment. You can find many great explanations of protein sequence alignments from just googling the term “multiple sequence alignment”. Wikipedia has an article:
With an alignment, they proceed to infer a phylogenetic tree, for example using the maximum likelihood algorithm(this is also the algorithm they actually used). A good explanation for how that algorithm works can be viewed in this video: Maximum likelihood phylogenetic algorithm
Once they have their phylogenetic tree, they can use it to infer the ancestral states. A good video explaining how that is done can be watched here: https://www.youtube.com/watch?v=Siau2o_egGI
Once they have inferred the ancestral state, they proceed to contact a private company (in this case GenScript) and get them to synthesize the actual molecules for them (they basically just write them “we need some plasmids of this or that type, containing protein coding DNA sequences which should look like this [AGCT…etc atc]”), the company makes these DNA sequences for them, which the researchers then use in their experiments.
and (2) how different was the old DNA sequence that encoded for Anc.3, Anc.11, Anc.16, compared to the modern DNA sequence that encodes for Vma3, Vma11, Vma16.
The interesting fact would be the protein sequences, since they are the ones actually performing the function in the cell. The DNA sequence merely encodes these proteins. An overview of the different sequences (shown aligned so you can see the differences) can be seen in supplementary material 3:
Eyeballing estimate, they’re between 35% and 50% similar. You can count the highlighed black residues, they’re the identical ones.
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Thanks for the detailed explanation including illustrations and videos.
The sequences seem quite different but still functionally similar in this case?
That’s very interesting.
Just realized that I mistakenly called Anc.3-11 as Anc.3 and Anc.11 which seems wrong.
Does the modern version have separate Sc.3 and Sc.11 while the ancestor had them together as Anc.3-11?
In the figure does the pink background denote the matching between Sc.3 and Anc.3-11 and the green background indicates the matching between Sc.11 and Anc.3-11 in the cases where Sc.3 and Sc.11 didn’t match?
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Yes. These sequences have diverged quite a lot over this span of time, but they have retained their function.
Does the modern version have separate Sc.3 and Sc.11 while the ancestor had them together as Anc.3-11?
The Sc.3 and Sc.11 proteins evolved from a common ancestral protein, which is called Anc.3-11 in the paper. So yes, the ancestral organism had two genes encoding two different versions of the hexameric ring protein, each called Anc.16 (the ancestor of Vma16) and Anc.3-11(the common ancestor of Vma.3 and Vma.11) respectively.
The present and ancestral situation is described in the paper:
The vacuolar H±ATPase (V-ATPase) is a multisubunit protein complex that pumps protons across membranes to acidify subcellular compartments; this function is required for intracellular protein trafficking, coupled transport of small molecules and receptor-mediated endocytosis1. V-ATPase dysfunction has been implicated in human osteoporosis, in acquired drug resistance in human tumours, and in pathogen virulence12–14. A key subcomplex of the V-ATPase is the V0protein ring, a hexameric assembly that uses a rotary mechanism to move protons across organelle membranes (Fig. 1a)15,16. Although the V-ATPase is found in all eukaryotes, the V0 ring varies in subunit composition among lineages. In animals and most other eukaryotes, the ring consists of one subunit of Vma16 protein and five copies of its paralogue, Vma3 (Fig. 1b)1. In Fungi, the ring consists of one Vma16 subunit, four copies of Vma3 and one Vma11 subunit, arranged in a specific orientation17. All three proteins are required for V-ATPase function in Fungi18,19, but the mechanisms are unknown by which both Vma3 and Vma11 became obligate components with specific positional roles in the complex.
So they investigate the evolution that led to the requirement for three different versions of the hexameric ring protein in Fungi. Other eukaryotes only have two different versions of the protein.
In the figure does the pink background denote the matching between Sc.3 and Anc.3-11 and the green background indicates the matching between Sc.11 and Anc.3-11 in the cases where Sc.3 and Sc.11 didn’t match?
Yes.
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The text you quoted is self-explanatory. Thanks.
What would happen if the uncolored AAs are replaced in Sc.3 Sc.11 Sc.16 with the corresponding AAs in their ancestors Anc.3-11 Anc.16? Would the system stop working?
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For most of them, probably not. While the effect of every single possible mutational combination wasn’t characterized (there are many millions, so that would be practically impossible), much of that mutational diversity actually exists in living populations “out there” in the wild right now. That diversity is partly what made it possible to infer ancestor states in the first place. If you look at the phylogeny from Supplement S2 you can see they used about 200 different protein sequences from many different species, and all of these protein sequences will be different from each other.
But your question does raise an important point about mutations and mutational pathways. To say that the modern descendants evolved from Anc.3-11 and Anc.16 is not to say that any arbitrarily picked mutation will work in conjunction with any other found in the ancestor. There are probably some of them which had to happen in a particular order. It is not an unusual result in protein evolution that some historical substitutions have the effect of making other, later substitutions possible. And if that historical substitution had not happened first, the later one might have been deleterious.
There have been studies done using similar types of work to assess the kinds of mutational “pathways” by which modern proteins evolved from their distant ancestors (though not to my knowledge on ATPase), and even to elucide whether other such “pathways” exist in addition to the pathway through which the extant proteins evolved.
See for example:
Tyler N. Starr, Lora K. Picton, and Joseph W. Thornton. Alternate evolutionary histories in the sequence space of an ancient protein. Nature. 2017 Sep 21; 549(7672): 409–413. doi: 10.1038/nature23902
Abstract
To understand why molecular evolution turned out as it did, we must characterize not only the path that evolution followed across the space of possible molecular sequences but also the many alternative trajectories that could have been taken but were not. A large-scale comparison of real and possible histories would establish whether the outcome of evolution represents a unique or optimal state driven by natural selection or the contingent product of historical chance events1; it would also reveal how the underlying distribution of functions across sequence space shaped historical evolution2,3. Here we combine ancestral protein reconstruction4 with deep mutational scanning5–10 to characterize alternate histories in the sequence space around an ancient transcription factor, which evolved a novel biological function through well-characterized mechanisms11,12. We found hundreds of alternative protein sequences that use diverse biochemical mechanisms to perform the derived function at least as well as the historical outcome. These alternatives all require prior permissive substitutions that do not enhance the derived function, but not all require the same permissive changes that occurred during history. We found that if evolution had begun from a different starting point within the network of sequences encoding the ancestral function, outcomes with different genetic and biochemical forms would likely have resulted; this contingency arises from the distribution of functional variants in sequence space and epistasis between residues. Our results illuminate the topology of the vast space of possibilities from which history sampled one path, highlighting how the outcome of evolution depends on a serial chain of compounding chance events.
Can be accessed for free here: Alternate evolutionary histories in the sequence space of an ancient protein - PMC
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Excellent explanation again. Your helpful contribution in this topic are very appreciated.
Is this paper:
Curr Opin Struct Biol. 2018 Jun;50:18-25. doi: 10.1016/j.sbi.2017.10.009. Epub 2017 Nov 5.
somehow relevant to the discussed case too?
Yes, directly. Compensatory mutations and epistasis are names for the very phenomena I refer to.
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