A 22-mer Segment in the Structurally Pliable Regulatory Domain of Metazoan CTP: Phosphocholine Cytidylyltransferase Facilitates Both Silencing and Activating Functions

Simon Fraser University, Canada.
Journal of Biological Chemistry (Impact Factor: 4.57). 09/2012; 287(46). DOI: 10.1074/jbc.M112.402081
Source: PubMed


CTP: phosphocholine cytidylyltransferase (CCT), an amphitropic enzyme that regulates phosphatidylcholine synthesis, is composed of a catalytic head domain, and a regulatory tail. The tail region has dual functions as a regulator of membrane binding/enzyme activation, and as an inhibitor of catalysis in the unbound form of the enzyme, suggesting conformational plasticity. These functions are well conserved in CCTs across diverse phyla, although the sequences of the tail regions are not. CCT regulatory tails of diverse origins are composed of a long membrane lipid-inducible amphipathic helix (m-AH) followed by a highly disordered segment, reminiscent of the Parkinson disease-linked protein, αsynuclein, which shares a novel sequence motif with vertebrate CCTs. To unravel features required for silencing we created chimeric enzymes by fusing the catalytic domain of rat CCTα to the regulatory tail of CCTs from Drosophila, C. elegans, or S. cerevisiae, or to αsynuclein. Only the tail domains of the two invertebrate CCTs were competent for both suppression of catalytic activity and for activation by lipid vesicles. Thus both functions of the m-AH can tolerate significant changes in length and sequence. We identified a highly amphipathic 22-residue segment in the m-AH with features conserved among animal CCTs but not yeast CCT or alpha-synuclein. Deletion of this segment from rat CCT increased the lipid-independent V(max) by 10-fold, equivalent to the effect of deleting the entire tail, and severely weakened membrane binding affinity. However, membrane binding was required for additional increases in catalytic efficiency. Thus full activation of CCT may require not only loss of a silencing conformation in the m-AH but a gain of an activating conformation, promoted by membrane binding.

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Available from: Rosemary Cornell, Nov 22, 2014
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    • "Abbreviations: ceramide transport protein, CERT; cholesterol, Chol; phosphatidylinositol 4-phosphate, PI4P; phosphocholine, PCho; phosphatidylserine synthase I/II, PSS1/2. k cat and a > 10-fold decrease in the K m for CTP333435. In vitro and in vivo studies have identified two distinct classes of membrane lipid activators of CCT. "

    Full-text · Chapter · Jan 2016
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    • "Electrostatic interactions at the boundaries of the hydrophobic face also promote membrane binding of CCTα [32]. The inhibition and activating functions of domain M are localized to a conserved 22-amino acid segment that interacts with the catalytic domain and membranes [33]. In the absence of membranes , this segment of the amphipathic helix silences catalytic activity by association with the active site helix αE; association with membranes relieves inhibition, resulting in a ~ 100-fold elevation in "
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    ABSTRACT: 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-crosslinking 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 which flank the active site and over-lap 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 to 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.
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