Membrane binding modulates the quaternary structure of CTP:phosphocholine cytidylyltransferase.
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.
Xie
Mingtang
M
Department of Molecular Biology and Biochemistry, Simon Fraser University, 8888 University Drive, Burnaby, British Columbia V5A 1S6, Canada.
Smith
Jillian L
JL
Ding
Ziwei
Z
Zhang
Daqing
D
Cornell
Rosemary B
RB
D000818:Animals; D000838:Anions; D015153:Blotting, Western; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D019916:Choline-Phosphate Cytidylyltransferase / Q000737:chemistry*; D002918:Chymotrypsin / Q000737:chemistry; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry; D003553:Cystine / Q000737:chemistry; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D050356:Lipid Metabolism; D008055:Lipids / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010736:Phosphoglycerate Mutase / Q000737:chemistry; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D051381:Rats; D013997:Time Factors; D020798:Two-Hybrid System Techniques
D000838:Anions; D003432:Cross-Linking Reagents; D004220:Disulfides; D008055:Lipids; D003553:Cystine; D019916:Choline-Phosphate Cytidylyltransferase; D002918:Chymotrypsin; C117608:alpha-chymotrypsin; D010736:Phosphoglycerate Mutase; D003545:Cysteine