Investigation of the dimer interface of CTP:phosphocholine cytidylyltransferase and its modulation by lipids

CTP:phosphocholine cytidylyltransferase (CCT) is the rate-limiting enzyme in phosphatidylcholine synthesis. CCT is an amphipathic homodimer, whose activity is regulated by membrane binding - a process resulting in conformational restructuring of the dimer interface. I used site-directed mutagenesis of domain N lysines in CCT, followed by lysine specific chemical cross-linking in the presence and absence of activating lipids, to locate sites of dimer interactions and to pinpoint where conformational rearrangement occurs. Analysis revealed that none of the lysines in the N-terminus are required for cross-linking. Thus lysinemediated cross-links involve other CCT domains. I found that CCT 236, despite lacking a membrane-binding domain, appears to undergo conformational changes that disrupt the dimer interface in the presence of activating lipids. The mechanism causing lipid-induced rearrangements of both CCT 367 and CCT 236 dimer interfaces remains unknown, but appear to be different. Moreover, the two CCTs are activated by different lipids suggesting different activation mechanisms.