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J Biol Chem. 2018 May 4;293(18):7070-7084. doi: 10.1074/jbc.RA118.002053. Epub 2018 Mar 8.

An auto-inhibitory helix in CTP:phosphocholine cytidylyltransferase hijacks the catalytic residue and constrains a pliable, domain-bridging helix pair.

Author information

1
From the Centre for Molecular Simulation, Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4 and.
2
the Departments of Molecular Biology and Biochemistry and.
3
From the Centre for Molecular Simulation, Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4 and tieleman@ucalgary.ca.
4
the Departments of Molecular Biology and Biochemistry and cornell@sfu.ca.
5
Chemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada.

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 α helices (αE) 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, Lys122 The impacts of the AI helix on active-site dynamics and positioning of Lys122 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 Lys122 side chain, effectively competing with the substrate, CTP. In silico, removal of the AI helices dramatically increased αE 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 αE element upon membrane binding of the AI helix. Moreover, when αE 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 Lys122 and restraint of the pliable αE helices. We propose that membrane binding enables bending of the αE helices, bringing the active site closer to the membrane surface.

KEYWORDS:

CTP:phosphocholine cytidylyltransferase; allosteric regulation; auto-inhibition; lipid-protein interaction; membrane activation; molecular dynamics; nucleotide transferase; protein cross-linking; protein dynamic; protein-membrane interaction; regulation of catalysis

PMID:
29519816
PMCID:
PMC5936808
DOI:
10.1074/jbc.RA118.002053
[Indexed for MEDLINE]
Free PMC Article

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