The membrane-binding domain of an amphitropic enzyme suppresses catalysis by contact with an amphipathic helix flanking its active site.

CTP:phosphocholine cytidylyltransferase (CCT), the regulatory enzyme in the synthesis of phosphatidylcholine, is activated by binding membranes using a lipid-induced amphipathic helix (domain M). Domain M functions to silence catalysis when CCT is not membrane engaged. The silencing mechanism is unknown. We used photo-cross-linking and mass spectrometry to identify contacts between domain M and other CCT domains in its soluble form. Each of four sites in domain M forged cross-links to the same set of peptides that flank the active site and overlap at helix αE at the base of the active site. These cross-links were broken in the presence of activating lipid vesicles. Mutagenesis of domain M revealed that multiple hydrophobic residues within a putative auto-inhibitory (AI) motif contribute to the contact with helix αE and silencing. Helix αE was confirmed as the docking site for domain M by deuterium exchange analysis. We compared the dynamics and fold stability of CCT domains by site-directed fluorescence anisotropy and urea denaturation. The results suggest a bipartite structure for domain M: a disordered N-terminal portion and an ordered C-terminal AI motif with an unfolding transition identical with that of helix αE. Reduction in hydrophobicity of the AI motif decreased its order and fold stability, as did deletion of the catalytic domain. These results support a model in which catalytic silencing is mediated by the docking of an amphipathic AI motif onto the amphipathic helices αE. An unstructured leash linking αE with the AI motif may facilitate both the silencing contact and its membrane-triggered disruption.