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Lipid-induced conformational switch in the membrane binding domain of CTP:phosphocholine cytidylyltransferase: a circular dichroism study.
- Source :
-
Biochemistry [Biochemistry] 2003 Oct 14; Vol. 42 (40), pp. 11768-76. - Publication Year :
- 2003
-
Abstract
- CTP:phosphocholine cytidylyltranferase (CCT) regulates phosphatidylcholine (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 CCTalpha 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. Lysophosphatidylcholine/phosphatidylglycerol (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 proteolytic fragmentation 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.
- Subjects :
- Animals
Circular Dichroism
Enzyme Activation
Enzyme Stability
Isoenzymes chemistry
Lipid Bilayers chemistry
Micelles
Peptide Fragments chemistry
Protein Conformation
Protein Structure, Secondary
Protein Structure, Tertiary
Rats
Recombinant Proteins chemistry
Choline-Phosphate Cytidylyltransferase chemistry
Membrane Proteins chemistry
Phospholipids chemistry
Subjects
Details
- Language :
- English
- ISSN :
- 0006-2960
- Volume :
- 42
- Issue :
- 40
- Database :
- MEDLINE
- Journal :
- Biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 14529288
- Full Text :
- https://doi.org/10.1021/bi035234k