Interdomain communication in the phosphatidylcholine regulatory enzyme, CCT{alpha}, relies on a modular {alpha}E helix [Enzymology] (Journal of Biological Chemistry)

CTP:phosphocholine cytidylyltransferase (CCT), the rate-limiting enzyme in phosphatidylcholine (PC) synthesis, is an amphitropic enzyme that regulates PC homeostasis. Recent work has suggested that CCT? activation by binding to a PC-deficient membrane involves conformational transitions in a helix pair (?E) that, along with a short linker of unknown structure (J segment), bridges the catalytic domains of the CCT? dimer to the membrane-binding (M) domains. In the soluble, inactive form, the ?E helices are constrained into unbroken helices by contacts with two auto-inhibitory (AI) helices from domain M. In the active, membrane-bound form, the AI helices are displaced and engage the membrane. Molecular dynamics simulations have suggested that AI displacement is associated with hinge-like bending in the middle of the ?E, positioning its C terminus closer to the active site. Here, we show that CCT? activation by membrane binding is sensitive to mutations in the ?E and J segments, especially within or proximal to the ?E hinge. Substituting Tyr-213 within this hinge with smaller uncharged amino acids that could destabilize interactions between the ?E helices increased both constitutive and lipid-dependent activities, supporting a link between ?E helix bending and stimulation of CCT activity. The solvent accessibilities of Tyr-213 and Tyr-216 suggested that these tyrosines move to new partially buried environments upon membrane binding of CCT, consistent with a folded ?E/J structure. These data suggest that signal transduction through the modular ?E helix pair relies on shifts in its conformational ensemble that are controlled by the AI helices and their displacement upon membrane binding.