Large-scale filament formation inhibits the activity of CTP synthetase.
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.
Affiliation: Department of Molecular Biology, Princeton University, Princeton, United States.Show MeSH
Mentions: To validate the filament structure and its mechanistic implications, we generated structure-guided mutants in the CtpS polymerization interface. Two discrete segments constitute the novel filament assembly contacts: the linker region α-helix 274–284, and the short α-helix 330–336 of the GATase domain (Figure 3D,E). Though the exact amino acid sequences at the inter-tetramer assembly interfaces are not well conserved, relative to the rest of CtpS, both sites feature many charged or hydrophobic residues available for potential polymerization stabilization across species (Figure 6—figure supplement 1). We previously demonstrated that in E. coli, an mCherry-CtpS fusion faithfully reproduces the filamentous localization of native CtpS (as assayed by immunofluorescence) (Ingerson-Mahar et al., 2010). As an initial screen for CtpS assembly, we therefore introduced four mutations in the linker region α-helix and surrounding residues (E277R, F281R, N285D, and E289R) into mCherry-CtpS (Figure 6A). All four polymerization interface mutants disrupted mCherry-CtpS localization, exhibiting a diffuse localization pattern rather than linear filaments (Figure 6B).10.7554/eLife.03638.022Figure 6.Linker helix residues form a polymerization interface.
Affiliation: Department of Molecular Biology, Princeton University, Princeton, United States.