Abstract
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 CCTα activation by binding to a PC-deficient membrane involves conformational transitions in a helix pair (αE) that, along with a short linker of unknown structure (J segment), bridges the catalytic domains of the CCTα dimer to the membrane-binding (M) domains. In the soluble, inactive form, the αE 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 αE, positioning its C terminus closer to the active site. Here, we show that CCTα activation by membrane binding is sensitive to mutations in the αE and J segments, especially within or proximal to the αE hinge. Substituting Tyr-213 within this hinge with smaller uncharged amino acids that could destabilize interactions between the αE helices increased both constitutive and lipid-dependent activities, supporting a link between αE 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 αE/J structure. These data suggest that signal transduction through the modular αE helix pair relies on shifts in its conformational ensemble that are controlled by the AI helices and their displacement upon membrane binding.
Highlights
CTP:phosphocholine cytidylyltransferase (CCT), the ratelimiting enzyme in phosphatidylcholine (PC) synthesis, is an amphitropic enzyme that regulates PC homeostasis
We hypothesize that a similar conformational change in the ␣E helices may occur upon membrane engagement via the M domain and that this is required for activation. We explored this hypothesis by (i) examining the impacts of mutations at conserved sites in the ␣E on lipid activation and (ii) probing the environment of conserved tyrosines and engineered tryptophans within the ␣E hinge, ␣EC, and J segment in various CCT forms: the soluble form (CCTsol), the membrane-bound form (CCTmem), and a fragment encompassing the catalytic domain and allosteric linker (CCT-236)
This work is the first report of the participation of residues in the ␣E and J segments in the regulation of catalysis upon membrane binding via domain M
Summary
The kcat/Km(CTP) value measured for CCT when membrane-bound (9800 sϪ1 MϪ1; Fig. 3A) is similar to that obtained for the constitutively active GCT (13,500 sϪ1 MϪ1) [20] with its short ␣E helix. When we truncated CCT at residue 212 to create a short ␣E helix like GCT, the enzyme activity was completely obliterated (Fig. 3, A and B). The transition temperature for unfolding (Tm) for CCT-212, as monitored by Sypro Orange fluorescence, was reduced 10 °C, from 55 °C (full-length) to 45 °C (CCT-212) (Table 1). These data suggest a requirement for some or all of the C-terminal region for CCT catalytic function and catalytic domain stability
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