An auto-inhibitory helix in CTP:phosphocholine cytidylyltransferase hijacks the catalytic residue and constrains a pliable, domain-bridging helix pair
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| Authors: | Ramezanpour, M; Lee, J; Taneva, SG; Tieleman, DP; Cornell, RB |
| Year: | 2018 |
| Journal: | J. Biol. Chem. 293 Article Link (DOI) PubMed |
| Title: | An auto-inhibitory helix in CTP:phosphocholine cytidylyltransferase hijacks the catalytic residue and constrains a pliable, domain-bridging helix pair |
| Abstract: | The activity of CTP:phosphocholine cytidylyltransferase (CCT), a key enzyme in phosphatidylcholine synthesis, is regulated by reversible interactions of a lipid-inducible amphipathic helix (domain M) with membrane phospholipids. When dissociated from membranes, a portion of the M domain functions as an auto-inhibitory (AI) element to suppress catalysis. The AI helix from each subunit binds to a pair of helices (E) that extend from the base of the catalytic dimer to create a four-helix bundle. The bound AI helices make intimate contact with loop L2, housing a key catalytic residue, Lys(122). The impacts of the AI helix on active-site dynamics and positioning of Lys(122) are unknown. Extensive MD simulations with and without the AI helix revealed that backbone carbonyl oxygens at the point of contact between the AI helix and loop L2 can entrap the Lys(122) side chain, effectively competing with the substrate, CTP. In silico, removal of the AI helices dramatically increased E dynamics at a predicted break in the middle of these helices, enabling them to splay apart and forge new contacts with loop L2. In vitro cross-linking confirmed the reorganization of the E element upon membrane binding of the AI helix. Moreover, when E bending was prevented by disulfide engineering, CCT activation by membrane binding was thwarted. These findings suggest a novel two-part auto-inhibitory mechanism for CCT involving capture of Lys(122) and restraint of the pliable E helices. We propose that membrane binding enables bending of the E helices, bringing the active site closer to the membrane surface. |
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