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J. Biol. Chem., Vol. 279, Issue 27, 28817-28825, July 2, 2004

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Membrane Binding Modulates the Quaternary Structure of CTP:Phosphocholine Cytidylyltransferase^*

Mingtang Xie¶, Jillian L. Smith¶, Ziwei Ding||, Daqing Zhang, and Rosemary B. Cornell**

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

CTP:phosphocholine cytidylyltransferase (CCT), a key enzyme that controls phosphatidylcholine synthesis, is regulated by reversible interactions with membranes containing anionic lipids. Previous work demonstrated that CCT is a homodimer. In this work we show that the structure of the dimer interface is altered upon encountering membranes that activate CCT. Chemical cross-linking reactions were established which captured intradimeric interactions but not random CCT dimer collisions. The efficiency of capturing covalent cross-links with four different reagents was diminished markedly upon presentation of activating anionic lipid vesicles but not zwitterionic vesicles. Experiments were conducted to show that the anionic vesicles did not interfere with the chemistry of the cross-linking reactions and did not sequester available cysteine sites on CCT for reaction with the cysteine-directed cross-linking reagent. Thus, the loss of cross-linking efficiency suggested that contact sites at the dimer interface had increased distance or reduced flexibility upon binding of CCT to membranes. The regions of the enzyme involved in dimerization were mapped using three approaches: 1) limited proteolysis followed by cross-linking of fragments, 2) yeast two-hybrid analysis of interactions between select domains, and 3) disulfide bonding potential of CCTs with individual cysteine to serine substitutions for the seven native cysteines. We found that the N-terminal domain (amino acids 1–72) is an important participant in forming the dimer interface, in addition to the catalytic domain (amino acids 73–236). We mapped the intersubunit disulfide bond to the cystine 37 pair in domain N and showed that this disulfide is sensitive to anionic vesicles, implicating this specific region in the membrane-sensitive dimer interface.

Received for publication, March 24, 2004

^* This work was supported by Canadian Institutes for Health Research Grant 12134. 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.

These authors contributed equally to this work.

^¶ Present address: Dept. of Molecular Cell and Development Biology, University of California Los Angeles, Los Angeles, CA 90095-1606.

^|| Present address: Zhanchunyuan 4-6-60, Haidian, Beijing 100083, People's Republic of China.

To whom correspondence should be addressed: Dept. of Molecular Biology and Biochemistry, Simon Fraser University, 8888 University Dr., Burnaby, BC V5A 1S6, Canada. Tel.: 604-291-3709; Fax: 604-291-5583; E-mail: cornell{at}sfu.ca.

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