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Large-scale filament formation inhibits the activity of CTP synthetase.

Barry RM, Bitbol AF, Lorestani A, Charles EJ, Habrian CH, Hansen JM, Li HJ, Baldwin EP, Wingreen NS, Kollman JM, Gitai Z - Elife (2014)

Bottom Line: Structure-guided mutagenesis and mathematical modeling further indicate that coupling activity to polymerization promotes cooperative catalytic regulation.This previously uncharacterized regulatory mechanism is important for cellular function since a mutant that disrupts CtpS polymerization disrupts E. coli growth and metabolic regulation without reducing CTP levels.We propose that regulation by large-scale polymerization enables ultrasensitive control of enzymatic activity while storing an enzyme subpopulation in a conformationally restricted form that is readily activatable.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Princeton University, Princeton, United States.

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Determination of threshold concentration for CtpS polymerization in activity buffer.(A) Below 1 µM, the changes in light scattering (blue) are very low. At low [CtpS] the net changes in light scattering were both above and below zero, so the absolute values are plotted to allow comparison on a logarithmic plot (only the values for 0.05 µM and 0.075 µM CtpS were above zero). CtpS activity (red) is plotted on a logarithmic plot to compare the concentration at which polymerization and activity decreases begin. (B) The logarithmic plot of polymerization shows an inflection point at 1 µM. This predicts an approximate threshold concentration for polymerization between 1 µM and 2 µM.DOI:http://dx.doi.org/10.7554/eLife.03638.004
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fig1s1: Determination of threshold concentration for CtpS polymerization in activity buffer.(A) Below 1 µM, the changes in light scattering (blue) are very low. At low [CtpS] the net changes in light scattering were both above and below zero, so the absolute values are plotted to allow comparison on a logarithmic plot (only the values for 0.05 µM and 0.075 µM CtpS were above zero). CtpS activity (red) is plotted on a logarithmic plot to compare the concentration at which polymerization and activity decreases begin. (B) The logarithmic plot of polymerization shows an inflection point at 1 µM. This predicts an approximate threshold concentration for polymerization between 1 µM and 2 µM.DOI:http://dx.doi.org/10.7554/eLife.03638.004

Mentions: In order to identify the factors that control CtpS inhibition by assembly, we first confirmed that none of the substrates alone induced polymerization (Figure 2—figure supplement 1). We then directly tested our hypothesis that CtpS's product, CTP, a known inhibitor of CtpS activity, stimulates CtpS polymerization. In the absence of substrates (UTP, ATP, and glutamine), incubation with CTP caused CtpS to polymerize (Figure 2A). The threshold concentration for robust changes in light scattering by CtpS with saturating CTP (1–2 μM CtpS; Figure 2—figure supplement 2) agrees with the threshold concentration in the presence of substrates (1–2 μM CtpS; Figure 1—figure supplement 1). This result suggests that CTP alone is sufficient to influence polymerization and that the substrates and any other products of the enzymatic reaction are not necessary. To confirm that CTP stimulates CtpS assembly, we used ultracentrifugation as an independent assembly assay. Titrating with increasing amounts of CTP caused an increase in the amount of CtpS found in the pellet with respect to the 0 mM CTP condition (Figure 2B, Figure 2—figure supplement 3).10.7554/eLife.03638.011Figure 2.CTP is sufficient and necessary to stimulate CtpS polymerization.


Large-scale filament formation inhibits the activity of CTP synthetase.

Barry RM, Bitbol AF, Lorestani A, Charles EJ, Habrian CH, Hansen JM, Li HJ, Baldwin EP, Wingreen NS, Kollman JM, Gitai Z - Elife (2014)

Determination of threshold concentration for CtpS polymerization in activity buffer.(A) Below 1 µM, the changes in light scattering (blue) are very low. At low [CtpS] the net changes in light scattering were both above and below zero, so the absolute values are plotted to allow comparison on a logarithmic plot (only the values for 0.05 µM and 0.075 µM CtpS were above zero). CtpS activity (red) is plotted on a logarithmic plot to compare the concentration at which polymerization and activity decreases begin. (B) The logarithmic plot of polymerization shows an inflection point at 1 µM. This predicts an approximate threshold concentration for polymerization between 1 µM and 2 µM.DOI:http://dx.doi.org/10.7554/eLife.03638.004
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4126345&req=5

fig1s1: Determination of threshold concentration for CtpS polymerization in activity buffer.(A) Below 1 µM, the changes in light scattering (blue) are very low. At low [CtpS] the net changes in light scattering were both above and below zero, so the absolute values are plotted to allow comparison on a logarithmic plot (only the values for 0.05 µM and 0.075 µM CtpS were above zero). CtpS activity (red) is plotted on a logarithmic plot to compare the concentration at which polymerization and activity decreases begin. (B) The logarithmic plot of polymerization shows an inflection point at 1 µM. This predicts an approximate threshold concentration for polymerization between 1 µM and 2 µM.DOI:http://dx.doi.org/10.7554/eLife.03638.004
Mentions: In order to identify the factors that control CtpS inhibition by assembly, we first confirmed that none of the substrates alone induced polymerization (Figure 2—figure supplement 1). We then directly tested our hypothesis that CtpS's product, CTP, a known inhibitor of CtpS activity, stimulates CtpS polymerization. In the absence of substrates (UTP, ATP, and glutamine), incubation with CTP caused CtpS to polymerize (Figure 2A). The threshold concentration for robust changes in light scattering by CtpS with saturating CTP (1–2 μM CtpS; Figure 2—figure supplement 2) agrees with the threshold concentration in the presence of substrates (1–2 μM CtpS; Figure 1—figure supplement 1). This result suggests that CTP alone is sufficient to influence polymerization and that the substrates and any other products of the enzymatic reaction are not necessary. To confirm that CTP stimulates CtpS assembly, we used ultracentrifugation as an independent assembly assay. Titrating with increasing amounts of CTP caused an increase in the amount of CtpS found in the pellet with respect to the 0 mM CTP condition (Figure 2B, Figure 2—figure supplement 3).10.7554/eLife.03638.011Figure 2.CTP is sufficient and necessary to stimulate CtpS polymerization.

Bottom Line: Structure-guided mutagenesis and mathematical modeling further indicate that coupling activity to polymerization promotes cooperative catalytic regulation.This previously uncharacterized regulatory mechanism is important for cellular function since a mutant that disrupts CtpS polymerization disrupts E. coli growth and metabolic regulation without reducing CTP levels.We propose that regulation by large-scale polymerization enables ultrasensitive control of enzymatic activity while storing an enzyme subpopulation in a conformationally restricted form that is readily activatable.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Princeton University, Princeton, United States.

Show MeSH