title: Interdomain and membrane interactions of CTP:phosphocholine cytidylyltransferase revealed via limited proteolysis and mass spectrometry.
abstract: CTP:phosphocholine cytidylyltransferase (CCT) is a multi-domain enzyme that regulates phosphatidylcholine synthesis. It converts to an active form upon binding cell membranes, and interdomain dissociations have been hypothesized to accompany this process. To identify these interdomain and membrane interactions, the tertiary structures of three forms of CCTalpha were probed by monitoring accessibility to proteases. Time-limited digestion with chymotrypsin or arginine C of soluble CCTalpha (CCT(sol)), phospholipid vesicle-bound CCT (CCT(mem)), and a soluble constitutively active CCT truncated at amino acid 236 generated complex mixtures of peptides that were resolved and identified by gel electrophoresis/immunoblotting and by matrix-assisted laser desorption/ionization-mass spectrometry, with or without coupling to capillary liquid chromatography. Identification of cleavage sites enabled assembly of peptide bond accessibility maps for each CCT form. Our results reveal a approximately 80-residue core within the catalytic domain (domain C) as the most inaccessible region in all three forms and the C-terminal phosphorylation domain as the most accessible. Membrane binding has little effect on the protease accessibility of these domains. To map the protease sites onto the catalytic domain, its three-dimensional structure was modeled from the atomic coordinates of glycerol-phosphate cytidylyltransferase (Protein Data Bank code 1COZ). The protease inaccessibility of most sites in domain C could be explained by burial or location within secondary structural elements. The accessibility of the N-terminal domain (domain N) was enhanced upon membrane binding. Residues Phe(234)-Leu(303) were inaccessible in CCT(mem), suggesting burial in the membrane. Surprisingly, residues Leu(274)-Leu(303) of this domain were also inaccessible in CCT(sol). We propose that this region is buried by interdomain contacts with domain N in CCT(sol). Membrane binding and burial of domain M in the lipid bilayer may disrupt this interaction, leading to increased exposure of sites in domain N.
authors.affiliation: Department of Chemistry, Simon Fraser University, Burnaby, British Columbia, Canada.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Bogan
authors.forename: George R
authors.initials: GR
authors.lastname: Agnes
authors.forename: Frederic
authors.initials: F
authors.lastname: Pio
authors.forename: Rosemary B
authors.initials: RB
authors.lastname: Cornell
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D019916:Choline-Phosphate Cytidylyltransferase / Q000737:chemistry* / Q000235:genetics; D002918:Chymotrypsin; D004591:Electrophoresis, Polyacrylamide Gel; D066298:In Vitro Techniques; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D010449:Peptide Mapping; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D010446:Peptide Fragments; D011994:Recombinant Proteins; D019916:Choline-Phosphate Cytidylyltransferase; D002918:Chymotrypsin