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J. Biol. Chem., Vol. 280, Issue 20, 19613-19624, May 20, 2005

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Interdomain and Membrane Interactions of CTP:Phosphocholine Cytidylyltransferase Revealed via Limited Proteolysis and Mass Spectrometry^*

Michael J. Bogan, George R. Agnes, Frederic Pio¶, and Rosemary B. Cornell¶||

From the Departments of Chemistry and ^¶Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada

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 CCT were probed by monitoring accessibility to proteases. Time-limited digestion with chymotrypsin or arginine C of soluble CCT (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 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 Phe²³⁴-Leu³⁰³ were inaccessible in CCT_mem, suggesting burial in the membrane. Surprisingly, residues Leu²⁷⁴-Leu³⁰³ 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.

Received for publication, December 14, 2004 , and in revised form, February 10, 2005.

^* This work was supported by grants from the Canadian Institutes for Health Research (to R. B. C.), the Natural Sciences and Engineering Research Council (to G. R. A. and to F. P.), and Waters Technologies Inc. (to G. R. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplementary Fig. 1.

Recipient of a Natural Sciences and Engineering Research Council post-graduate award. Present address: Lawrence Livermore National Laboratory, Physics and Advanced Technologies Directorate, Medical Technology Program, Livermore, CA 94550.

^|| To whom correspondence should be addressed: Dept. of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, BC V5A 1S6, Canada. Tel.: 604-291-3709; E-mail: cornell{at}sfu.ca.

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