Lipid-induced conformational switch in the membrane binding domain of @entity93 : XXXX cytidylyltransferase: a circular dichroism study.

@entity13607 @entity1949 @entity6579 @entity93 @entity2434 @entity2945 @entity3634
@entity93 : @entity2945 cytidylyltranferase (CCT) regulates @entity2434 (PC) biosynthesis. Its activity is controlled by reversible interactions with membrane lipids, mediated by an internal segment referred to as domain M. Although domain M peptides adopt an amphipathic alpha-helical structure when membrane bound, the structure of this domain in the context of the whole enzyme in the lipid-free and lipid-bound state is unknown. Here we derive lipid-induced secondary structural changes in @entity13607 using circular dichroism and three deconvolution programs. The analysis of two fragments, CCT236 (CCT1-236, housing the catalytic domain) and a synthetic domain M peptide (CCT237-293) aided the assignment of structural change to specific domains. To carry out this study, we developed a micellar lipid activating system that would avoid generation of CCT-induced lipid vesicle aggregates that interfere with the CD analysis. @entity6579 / @entity3634 (LPC/PG) mixed micelles supported full activation of CCT and caused an increase in the alpha-helix content of full-length CCT from 25 to 41%, at the expense of all other conformations. LPC/PG also induced an increase in alpha-helix content of the domain M peptide from 7 to 85% at the expense of all other conformers. This lipid system did not significantly affect the secondary structure of CCT236, nor did it affect the @entity1949 pattern of this region within full-length CCT, suggesting that the region containing the catalytic domain changes very little upon membrane activation of CCT. Our data suggest that lipids trigger a conformational switch in domain M from a mixed structure to an alpha-helix, thus creating a hydrophobic face for membrane insertion. Our results negate the idea that domain M is entirely helical in both the soluble and membrane-bound forms of CCT.
