| 10 Mar 2026
Bioengineering the Ice Worm Complex V ATP6 subunit to Increase Energy Production
Abstract. Glacier ice worms, Mesenchytraeus solifugus, are among a few animals that complete their life cycle in hydrated glacier ice. These worms are distinguished from congener species by relatively high intracellular ATP levels, likely associated with a lateral gene transfer event adding 18 amino acids (aa) to the carboxy terminal of the Complex V FO ATP6 subunit, a major regulator of ATP synthesis. By examining the kinetic profiles of respective 13/18 aa natural variants fused with E. coli AtpB (prokaryotic counterpart of ATP6) in the context of ice worm evolution, we conclude that the 18 aa variant predates the 13 aa variant, but the latter likely outcompetes the former in natural populations. Utilizing the 13 aa variant as a template, a panel of genetically engineered extension mutants were constructed and tested by Michaelis–Menten kinetics as a function of increasing [ADP]. Our data show that Vmax can be increased significantly over natural 13/18 aa variants, and suggest a functional mechanism by which a dynamic tether transfers protons from the FO exit pore to the mitochondrial lumen via the protonation/deprotonation of distal histidine residue(s).
Summary:
This manuscript characterizes the function of ATP synthase containing the C-terminal 13/18 aa extension variants found in Mesenchytraeus solifugus ATP6. This work shows that fusion protein of E. coli AtpB containing the 13 aa extension from M. solifugus confers increased ATP synthesis rate in a similar manner as the 18 aa extension fusion protein shown in a previous study. The authors also explored the ATP synthesis activities of engineered mutants derived from the 13 aa variant sequence, in which alternating histidine residues were added or substituted in the sequence. The results showed that some of the engineered mutants outperform the wild-type 13 aa variant.
The authors conclude that the presence of histidine residues and the flexibility of the extension region contribute to an increase in proton transfer by the FO motor, which results in the increased ATP synthesis rate. This conclusion is consistent with and provides significant insight into the unique characteristic of M. solifugus, in which the ATP level is higher compared to similar species. The increased ATP synthesis rate of the engineered mutants contributes to the overall understanding of the enzyme mechanism, and also has biomedical implications.
Overall, the strengths of this work are the clear demonstration that ATP6 C-terminal 13 aa extension causes elevated ATP synthesis rate, consistency of the results with the observed high ATP production by M. solifugus, and the mutagenesis work that characterizes how the histidine-rich extension region enhances the ATP synthesis rate. The major weakness of this study is the quality of the Western blot for Mutants 3 and 6 and the reliance on that result to conclude that those two mutants have the highest ATP synthesis rates, which must be addressed before publication.
Major comments
1. Mutants 3 and 6 are shown to have the highest normalized ATP synthesis rates. However, these are the two mutants that showed very faint Western blot bands that were almost faded to the background (supplementary figure A1). Therefore, it is concerning whether the ATP synthesis rates for these mutants were accurate upon normalization. If the ATP synthesis activities of Mutants 3 and 6 are not accurate, the conclusions made by the authors in lines 308 and 328 (regarding flexibility being more critical than the number of histidine residues) must be re-evaluated.
To address this, the authors should provide:
2. Line 491 - The authors suggest that Mutants 3 and 6 had lower expression of ATP synthase because “fewer ATP synthase complexes were necessary to meet cellular energy demands”. It is also possible that expression levels were low because the mutant protein was unstable, not because the cell did not need as many ATP synthase complexes.
To address this, the authors should provide:
3. To truly substantiate the mechanism proposed in the final paragraph, a proton flux assay (such as an ACMA quenching assay) would directly quantify the relationship between the increased ATP synthesis rates and the proton transfer rates through the FO This would significantly add to the mechanistic understanding of the unique characteristics of this ATP synthase species and the function of the FO motor in general. However, it is understandable if this is outside of the scope of this study.
4. Line 286 – The authors suggest that the 13 aa variant became dominant over the 18 aa in certain regions through natural selection for smaller mitochondrial genome size. However, it is not clear if the 15 bp difference in mitochondrial genome size could impose a significant selective pressure. The authors should acknowledge genetic drift as an equally plausible mechanism for fixation in this section.
Minor Comments: