Journal of Molecular Biology
Volume 432, Issue 18, 21 August 2020, Pages 5023-5042
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Review
Membrane Lipids Assist Catalysis by CTP: Phosphocholine Cytidylyltransferase

https://doi.org/10.1016/j.jmb.2020.03.024Get rights and content

Highlights

  • CCT is activated by membrane binding.

  • CCT seeks negatively charged surfaces and lipid packing voids for insertion of an amphipathic helix.

  • The amphipathic helix is a silencing device in the CCT soluble form.

  • Membrane binding facilitates remodeling of a malleable inter-domain allosteric linker.

  • Linker folding pulls the active site close to the membrane and shields it from solvent.

Abstract

While most of the articles in this issue review the workings of integral membrane enzymes, in this review, we describe the catalytic mechanism of an enzyme that contains a soluble catalytic domain but appears to catalyze its reaction on the membrane surface, anchored and assisted by a separate regulatory amphipathic helical domain and inter-domain linker. Membrane partitioning of CTP: phosphocholine cytidylyltransferase (CCT), a key regulatory enzyme of phosphatidylcholine metabolism, is regulated chiefly by changes in membrane phospholipid composition, and boosts the enzyme's catalytic efficiency > 200-fold. Catalytic enhancement by membrane binding involves the displacement of an auto-inhibitory helix from the active site entrance-way and promotion of a new conformational ensemble for the inter-domain, allosteric linker that has an active role in the catalytic cycle. We describe the evidence for close contact between membrane lipid, a compact allosteric linker, and the CCT active site, and discuss potential ways that this interaction enhances catalysis.

Section snippets

CCT Activity Is Regulated by Reversible Membrane Binding

CCT is the rate-limiting and regulatory enzyme in the Kennedy pathway for the synthesis of phosphatidylcholine (PC) (Figure 1(a)), the dominant pathway for de novo PC synthesis in nearly all eukaryotic cells [2]. Its substrates and products are water-soluble, and the CCT active site is accessible to solvent. However, CCT requires a means to respond to changing concentrations of the pathway end-product, PC. This is accomplished by a membrane–lipid sensor domain (domain M), linked to the

Regulation of the Equilibrium between Soluble and Membrane-Bound Forms

The membrane partitioning of CCT is regulated by the membrane lipid composition and by the phosphorylation status of the region C-terminal to domain M (Figure 1(b)). A potential third influencing factor is the available protein-free space for embedding into the membrane surface. The features of the membrane and the amphipathic helical M domain that are responsible for stable binding and the antagonism of binding by phosphorylation have been reviewed recently [3]. We provide an overview here,

The CCT-Catalyzed Reaction Is Driven by Electrostatic Forces

The CCT catalytic domain is a modified Rossman fold with a β-sheet scaffold of five parallel β strands flanked by six α-helices, and an active site at the base of the β sheet [74]. In metazoan CCTs, this fold is capped by an N-terminal extension of ~ 35 ordered residues that interact with the catalytic fold and stabilize the dimer [74]. The job of the enzyme, beyond capturing the substrates, is to promote a U-shape of CTP, convert the α-phosphate from tetrahedral to the trigonal-planar geometry

Inhibition Mechanism

The activity of purified CCTα is very low (~ 50 M−1 s−1) and rises to ≥ 10,000 in the presence of added lipid, typically added in the form of small sonicated vesicles containing acidic lipid. The change is a reflection of a ~ 10-fold drop in the Km for CTP, but no change in the Km for phosphocholine, and a 20- to 30-fold increase in kcat [10,11,20,89]. The catalytic silencing device is housed in the most conserved and hydrophobic region of the M domain–a 22mer segment corresponding to residues

Membrane binding redirects the conformational ensemble of the allosteric inter-domain linker

Domain M has dual roles in regulation. It is inhibitory in the soluble form (CCTsol), as described above, and activating in the membrane form (CCTmem). The key evidence for dual roles is that deletion of domain M or specifically the AI segment is only partially activating [20,90], which is incompatible with a strictly auto-inhibitory function. For optimal membrane binding and activation, both the leash and the AI segment must be present [11,20,91,92]. How does the membrane-bound domain M

Benefits of a Reaction on the Membrane Surface: Electrostatic Enhancement of Key Catalytic Residues

A general strategy for catalysis, especially for reactions like CCT that rely on electrostatic enhancement, is the sequestration of active sites from water upon substrate binding to create a low-dielectric medium for the reaction. The charged and polarizable groups that catalyze the reaction can then forge ion pairs or H-bonds with substrates without interference from water and potentially neutralizing counter-ions [[94], [95], [96]]. This is often brought about by the movement of mobile loops

Similarities between the Activation Mechanism of CCT and Other Membrane-Bound Enzymes

Does the plasticity of the allosteric linker in CCT reflect a mechanism for activation that is common to other membrane enzymes with soluble catalytic domains? The enzyme with the most similar membrane activation mechanism is another enzyme of phospholipid metabolism, neutral sphingmyelinase (nSMase2). nSMase2 is activated by PS and other anionic phospholipids [103], and uses a membrane-associated allosteric linker, the juxta-membrane (JX) segment, to tether the catalytic domain to an integral

Membrane Participation Is Intrinsic to the CCT-Catalyzed Reaction, But to What Extent?

Membrane lipids participate in the CCT reaction on several levels. Firstly, the PC-deficient membrane captures CCT by providing a surface for folding and insertion of the domain M amphipathic helix. Increased membrane surface charge likely attracts the positively charged leash intervening between the J segment and the AI, and may selectively recruit dephosphorylated species [1] (Figure 5(b)). The leash–membrane interaction may assist in the dissociation of the AI helix, leading to αE helix

Acknowledgments

I thank Dr. Svetla Taneva for a critique of the manuscript. I would like to thank several Research Associates for their many contributions over the years to the analysis of the lipid activation of CCT: Dr. Jaeyong Lee, Dr. Svetla Taneva, Dr. Joanne Johnson, and Ziwei Ding. The research cited from my lab was funded by grants from CIHR and NSERC.

Declaration of Interest: None.

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