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.
Highlights
Active cells are continually undergoing membrane phospholipid turnover, membrane expansion, and trafficking of components between organelles via vesicular transport
The three forms of CTP:phosphocholine cytidylyltransferase (CCT) were digested in parallel, and the fragments were separated by electrophoresis
Bands 13–17 migrating ahead of the 21-kDa marker were not produced upon digestion of CCT236 and did not react with either the anti-N or anti-cat domain antibody (Fig. 2, B and C), indicating that they derive from the C-terminal domains, M and/or P
Summary
Region showed that it is a continuous amphipathic ␣-helix in a membrane-mimetic environment [10]. Previous analysis by limited proteolysis with chymotrypsin and mapping the major SDS-PAGE-resolved fragments by epitope-specific antibodies gave rise to the proposal that CCT consists of a protease-resistant compact head (up to residue 225) and a protease-sensitive, flexible, exposed tail [14]. The accessibility of the interdomain contact points involved in the regulatory conformational switch should be low in the soluble form and high in the membrane-bound form. Based on this premise the present work identifies domain N rather than domain C as the best candidate for a site of contact with the C-terminal region of domain M
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