title: A 22-mer segment in the structurally pliable regulatory domain of metazoan CTP: phosphocholine cytidylyltransferase facilitates both silencing and activating functions.
abstract: 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, α-synuclein, which we show 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, Caenorhabditis elegans, or Saccharomyces cerevisiae or to α-synuclein. 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 silencing and activating 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 α-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.
authors.affiliation: Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada.
authors.forename: Ziwei
authors.initials: Z
authors.lastname: Ding
authors.forename: Svetla G
authors.initials: SG
authors.lastname: Taneva
authors.forename: Harris K H
authors.initials: HK
authors.lastname: Huang
authors.forename: Stephanie A
authors.initials: SA
authors.lastname: Campbell
authors.forename: Lucie
authors.initials: L
authors.lastname: Semenec
authors.forename: Nansheng
authors.initials: N
authors.lastname: Chen
authors.forename: Rosemary B
authors.initials: RB
authors.lastname: Cornell
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D020134:Catalytic Domain; D019916:Choline-Phosphate Cytidylyltransferase / Q000737:chemistry / Q000502:physiology*; D019295:Computational Biology / Q000379:methods; D003570:Cytidine Triphosphate / Q000737:chemistry*; D004789:Enzyme Activation; D020868:Gene Silencing; D007700:Kinetics; D008055:Lipids / Q000737:chemistry; D008969:Molecular Sequence Data; D010713:Phosphatidylcholines / Q000737:chemistry; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D017386:Sequence Homology, Amino Acid; D051844:alpha-Synuclein / Q000737:chemistry
chemical_list: D008055:Lipids; D010713:Phosphatidylcholines; D051844:alpha-Synuclein; D003570:Cytidine Triphosphate; D019916:Choline-Phosphate Cytidylyltransferase