Lipid- and phospho-regulation of CTP:Phosphocholine Cytidylyltransferase α association with nuclear lipid droplets.

Fatty acids stored in triacylglycerol-rich lipid droplets are assembled with a surface monolayer composed primarily of phosphatidylcholine (PC). Fatty acids stimulate PC synthesis by translocating CTP:phosphocholine cytidylyltransferase (CCT) α to the inner nuclear membrane, nuclear lipid droplets (nLD) and lipid associated promyelocytic leukemia (PML) structures (LAPS). Huh7 cells were used to identify how CCTα translocation onto these nuclear structures are regulated by fatty acids and phosphorylation of its serine-rich P-domain. Oleate treatment of Huh7 cells increased nLDs and LAPS that became progressively enriched in CCTα. In cells expressing the phosphatidic acid phosphatase Lipin1α or 1ß, the expanded pool of nLDs and LAPS had a proportional increase in associated CCTα. In contrast, palmitate induced few nLDs and LAPS and inhibited the oleate-dependent translocation of CCTα without affecting total nLDs. Phospho-memetic or phospho-null mutations in the P-domain revealed that a 70% phosphorylation threshold, rather than site-specific phosphorylation, regulated CCTα association with nLDs and LAPS. In vitro candidate kinase and inhibitor studies in Huh7 cells identified cyclin-dependent kinase (CDK) 1 and 2 as putative P-domain kinases. In conclusion, CCTα translocation onto nLDs and LAPS is dependent on available surface area and fatty acid composition, as well as threshold phosphorylation of the P-domain potentially involving CDKs.

Genome-wide characterization of plant CTP:phosphocholine cytidylyltransferases through evolutionary, biochemical, and structural analyses.

Phosphatidylcholine has essential functions in many eukaryotic cells, and its de novo biosynthesis is rate-limited by cytidine triphosphate:phosphocholine cytidylyltransferase (CCT). Although the biological and biochemical functions of CCT have been reported in mammals and several plants, this key enzyme has yet to be examined at a genome-wide level. As such, certain fundamental questions remain unanswered, including the evolutionary history, genetic and functional relationships, and structural variations among CCTs in the green lineage. In the current study, in-depth phylogenetic analysis, as well as the conservation and diversification in CCT gene structure and motif patterns, indicated that CCTs exist broadly in chlorophytes, bryophytes, lycophytes, monilophytes, gymnosperms, early-diverging angiosperms, monocots, and eudicots, and form eight relatively conserved clades. To further explore the potential function of selection pressure, we conducted extensive selection pressure analysis with a representative CCT gene, CCT1 from the model plant Arabidopsis thaliana (AthCCT1), and identified two positive selection sites, L59 and Q156. Site-directed mutagenesis and in vitro enzyme assays demonstrated that these positively selected sites were indeed important for the activity and substrate affinity of AthCCT1, and subsequent 3D structure analyses explained the potential biochemical mechanism. Taken together, our results unraveled the evolution and diversity of CCTs in the green lineage, as well as their association with the enzyme's biochemical and structural properties, and expanded our understanding of this important enzyme at the genome-wide level.

Identification of a nuclear localization signal in the Plasmodium falciparum CTP: phosphocholine cytidylyltransferase enzyme.

The phospholipid biosynthesis of the malaria parasite, Plasmodium falciparum is a key process for its survival and its inhibition is a validated antimalarial therapeutic approach. The second and rate-limiting step of the de novo phosphatidylcholine biosynthesis is catalysed by CTP: phosphocholine cytidylyltransferase (PfCCT), which has a key regulatory function within the pathway. Here, we investigate the functional impact of the key structural differences and their respective role in the structurally unique pseudo-heterodimer PfCCT protein in a heterologous cellular context using the thermosensitive CCT-mutant CHO-MT58 cell line. We found that a Plasmodium-specific lysine-rich insertion within the catalytic domain of PfCCT acts as a nuclear localization signal and its deletion decreases the nuclear propensity of the protein in the model cell line. We further showed that the putative membrane-binding domain also affected the nuclear localization of the protein. Moreover, activation of phosphatidylcholine biosynthesis by phospholipase C treatment induces the partial nuclear-to-cytoplasmic translocation of PfCCT. We additionally investigated the cellular function of several PfCCT truncated constructs in a CHO-MT58 based rescue assay. In absence of the endogenous CCT activity we observed that truncated constructs lacking the lysine-rich insertion, or the membrane-binding domain provided similar cell survival ratio as the full length PfCCT protein.

Membrane Lipids Assist Catalysis by CTP: Phosphocholine Cytidylyltransferase.

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.

Differential dephosphorylation of CTP:phosphocholine cytidylyltransferase upon translocation to nuclear membranes and lipid droplets.

CTP:phosphocholine cytidylyltransferase-alpha (CCTα) and CCTß catalyze the rate-limiting step in phosphatidylcholine (PC) biosynthesis. CCTα is activated by association of its α-helical M-domain with nuclear membranes, which is negatively regulated by phosphorylation of the adjacent P-domain. To understand how phosphorylation regulates CCT activity, we developed phosphosite-specific antibodies for pS319 and pY359+pS362 at the N- and C-termini of the P-domain, respectively. Oleate treatment of cultured cells triggered CCTα translocation to the nuclear envelope (NE) and nuclear lipid droplets (nLDs) and rapid dephosphorylation of pS319. Removal of oleate led to dissociation of CCTα from the NE and increased phosphorylation of S319. Choline depletion of cells also caused CCTα translocation to the NE and S319 dephosphorylation. In contrast, Y359 and S362 were constitutively phosphorylated during oleate addition and removal, and CCTα-pY359+pS362 translocated to the NE and nLDs of oleate-treated cells. Mutagenesis revealed that phosphorylation of S319 is regulated independently of Y359+S362, and that CCTα-S315D+S319D was defective in localization to the NE. We conclude that the P-domain undergoes negative charge polarization due to dephosphorylation of S319 and possibly other proline-directed sites and retention of Y359 and S362 phosphorylation, and that dephosphorylation of S319 and S315 is involved in CCTα recruitment to nuclear membranes.

Genetic diseases of the Kennedy pathways for membrane synthesis.

The two branches of the Kennedy pathways (CDP-choline and CDP-ethanolamine) are the predominant pathways responsible for the synthesis of the most abundant phospholipids, phosphatidylcholine and phosphatidylethanolamine, respectively, in mammalian membranes. Recently, hereditary diseases associated with single gene mutations in the Kennedy pathways have been identified. Interestingly, genetic diseases within the same pathway vary greatly, ranging from muscular dystrophy to spastic paraplegia to a childhood blinding disorder to bone deformations. Indeed, different point mutations in the same gene (PCYT1; CCTα) result in at least three distinct diseases. In this review, we will summarize and review the genetic diseases associated with mutations in genes of the Kennedy pathway for phospholipid synthesis. These single-gene disorders provide insight, indeed direct genotype-phenotype relationships, into the biological functions of specific enzymes of the Kennedy pathway. We discuss potential mechanisms of how mutations within the same pathway can cause disparate disease.

Interdomain communication in the phosphatidylcholine regulatory enzyme, CCTα, relies on a modular αE helix.

CTP:phosphocholine cytidylyltransferase (CCT), the rate-limiting enzyme in phosphatidylcholine (PC) synthesis, is an amphitropic enzyme that regulates PC homeostasis. Recent work has suggested that CCTα activation by binding to a PC-deficient membrane involves conformational transitions in a helix pair (αE) that, along with a short linker of unknown structure (J segment), bridges the catalytic domains of the CCTα dimer to the membrane-binding (M) domains. In the soluble, inactive form, the αE helices are constrained into unbroken helices by contacts with two auto-inhibitory (AI) helices from domain M. In the active, membrane-bound form, the AI helices are displaced and engage the membrane. Molecular dynamics simulations have suggested that AI displacement is associated with hinge-like bending in the middle of the αE, positioning its C terminus closer to the active site. Here, we show that CCTα activation by membrane binding is sensitive to mutations in the αE and J segments, especially within or proximal to the αE hinge. Substituting Tyr-213 within this hinge with smaller uncharged amino acids that could destabilize interactions between the αE helices increased both constitutive and lipid-dependent activities, supporting a link between αE helix bending and stimulation of CCT activity. The solvent accessibilities of Tyr-213 and Tyr-216 suggested that these tyrosines move to new partially buried environments upon membrane binding of CCT, consistent with a folded αE/J structure. These data suggest that signal transduction through the modular αE helix pair relies on shifts in its conformational ensemble that are controlled by the AI helices and their displacement upon membrane binding.

Remodeling of the interdomain allosteric linker upon membrane binding of CCTα pulls its active site close to the membrane surface.

The rate-limiting step in the biosynthesis of the major membrane phospholipid, phosphatidylcholine, is catalyzed by CTP:phosphocholine cytidylyltransferase (CCT), which is regulated by reversible membrane binding of a long amphipathic helix (domain M). The M domain communicates with the catalytic domain via a conserved ∼20-residue linker, essential for lipid activation of CCT. Previous analysis of this region (denoted as the αEC/J) using MD simulations, cross-linking, mutagenesis, and solvent accessibility suggested that membrane binding of domain M promotes remodeling of the αEC/J into a more compact structure that is required for enzyme activation. Here, using tryptophan fluorescence quenching, we show that the allosteric linker lies superficially on the membrane surface. Analyses with truncated CCTs show that the αEC/J can interact with lipids independently of the M domain. We observed strong FRET between engineered tryptophans in the αEC/J and vesicles containing dansyl-phosphatidylethanolamine that depended on the native J sequence. These data are incompatible with the extended conformation of the αE helix observed in the previously determined crystal structure of inactive CCT but support a bent αE helix conformation stabilized by J segment interactions. Our results suggest that the membrane-adsorbed, folded allosteric linker may partially cover the active site cleft and pull it close to the membrane surface, where cytidyl transfer can occur efficiently in a relatively anhydrous environment.

Arabidopsis CTP:phosphocholine cytidylyltransferase 1 is phosphorylated and inhibited by sucrose nonfermenting 1-related protein kinase 1 (SnRK1).

De novo phosphatidylcholine (PC) biosynthesis via the Kennedy pathway involves highly endergonic biochemical reactions that must be fine-tuned with energy homeostasis. Previous studies have shown that CTP:phosphocholine cytidylyltransferase (CCT) is an important regulatory enzyme in this pathway and that its activity can be controlled at both transcriptional and posttranslational levels. Here we identified an important additional mechanism regulating plant CCT1 activity. Comparative analysis revealed that Arabidopsis CCT1 (AtCCT1) contains catalytic and membrane-binding domains that are homologous to those of rat CCT1. In contrast, the C-terminal phosphorylation domain important for stringent regulation of rat CCT1 was apparently missing in AtCCT1. Instead, we found that AtCCT1 contains a putative consensus site (Ser-187) for modification by sucrose nonfermenting 1-related protein kinase 1 (SnRK1 or KIN10/SnRK1.1), involved in energy homeostasis. Phos-tag SDS-PAGE coupled with MS analysis disclosed that SnRK1 indeed phosphorylates AtCCT1 at Ser-187, and we found that AtCCT1 phosphorylation substantially reduces its activity by as much as 70%. An S187A variant exhibited decreased activity, indicating the importance of Ser-187 in catalysis, and this variant was less susceptible to SnRK1-mediated inhibition. Protein truncation and liposome binding studies indicated that SnRK1-mediated AtCCT1 phosphorylation directly affects the catalytic domain rather than interfering with phosphatidate-mediated AtCCT1 activation. Overexpression of the AtCCT1 catalytic domain in Nicotiana benthamiana leaves increased PC content, and SnRK1 co-expression reduced this effect. Taken together, our results suggest that SnRK1 mediates the phosphorylation and concomitant inhibition of AtCCT1, revealing an additional mode of regulation for this key enzyme in plant PC biosynthesis.

Disease-linked mutations in the phosphatidylcholine regulatory enzyme CCTα impair enzymatic activity and fold stability.

CTP:phosphocholine cytidylyltransferase (CCT) is the key regulatory enzyme in phosphatidylcholine (PC) synthesis and is activated by binding to PC-deficient membranes. Mutations in the gene encoding CCTα (PCYT1A) cause three distinct pathologies in humans: lipodystrophy, spondylometaphyseal dysplasia with cone-rod dystrophy (SMD-CRD), and isolated retinal dystrophy. Previous analyses showed that for some disease-linked PCYT1A variants steady state levels of CCTα and PC synthesis were reduced in patient fibroblasts, but other variants impaired PC synthesis with little effect on CCT levels. To explore the impact on CCT stability and function we expressed WT and mutant CCTs in COS-1 cells, which have very low endogenous CCT. Over-expression of two missense variants in the catalytic domain (V142M and P150A) generated aggregated enzymes that could not be refolded after solubilization by denaturation. Other mutations in the catalytic core that generated CCTs with reduced solubility could be purified. Five variants destabilized the catalytic domain-fold as assessed by lower transition temperatures for unfolding, and three of these manifested defects in substrate Km values. A mutation (R223S) in a signal-transducing linker between the catalytic and membrane-binding domains also impaired enzyme kinetics. E280del, a single amino acid deletion in the autoinhibitory helix increased the constitutive (lipid-independent) enzyme activity ∼4-fold. This helix also participates in membrane binding, and surprisingly E280del enhanced the enzyme's response to anionic lipid vesicles ∼4-fold. These in vitro analyses on purified mutant CCTs will complement future measurements of their impact on PC synthesis in cultured cells and in tissues with a stringent requirement for CCTα.

Structural determinants of the catalytic mechanism of Plasmodium CCT, a key enzyme of malaria lipid biosynthesis.

The development of the malaria parasite, Plasmodium falciparum, in the human erythrocyte, relies on phospholipid metabolism to fulfil the massive need for membrane biogenesis. Phosphatidylcholine (PC) is the most abundant phospholipid in Plasmodium membranes. PC biosynthesis is mainly ensured by the de novo Kennedy pathway that is considered as an antimalarial drug target. The CTP:phosphocholine cytidylyltransferase (CCT) catalyses the rate-limiting step of the Kennedy pathway. Here we report a series of structural snapshots of the PfCCT catalytic domain in its free, substrate- and product-complexed states that demonstrate the conformational changes during the catalytic mechanism. Structural data show the ligand-dependent conformational variations of a flexible lysine. Combined kinetic and ligand-binding analyses confirm the catalytic roles of this lysine and of two threonine residues of the helix αE. Finally, we assessed the variations in active site residues between Plasmodium and mammalian CCT which could be exploited for future antimalarial drug design.

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

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.

Rational Design of an Efficient Halotolerant Enzymatic System for In Vitro One-Pot Synthesis of Cytidine Diphosphate Choline.

Salt accumulation often impedes cytidine diphosphate choline (CDP-choline) in vitro biosynthetic process. In this work a halotolerant in vitro enzymatic system is developed to solve this problem. It applies a halotolerant choline-phosphate cytidylyltransferase (CCT) obtained from rational design instructed by a unique strategy, which refers to one of the features of naturally occurring halophilic enzymes. By increasing acidic residues on protein surface where is most variable with respect to amino acid in the sequence alignment with other CCT, the mutants are obtained. The mutants represent higher catalytic activities and IC50 values (inhibit activity by 50%) at high-salt concentrations. Furthermore, when the halotolerant CCT is applied to in vitro one-pot biosynthesis of CDP-choline, the maximum titer and productivity are 161 ± 3.5 mM and 6.2 ± 0.1 mM L-1 h-1 , respectively. When acetate concentration increases, it still keeps relatively high reaction rate and is 2.2-fold higher than process using wild-type CCT (3.87 mM L-1 h-1 comparing with 1.74 mM L-1 h-1 ). This halotolerant system has great potential for industrial use, and the rational design concept can be applied to modify other enzymes, addressing the salt accumulation problem in in vitro systems, and gives insight into resolving by-product inhibition during reaction.

Molecular Mechanism of Action of Antimalarial Benzoisothiazolones: Species-Selective Inhibitors of the Plasmodium spp. MEP Pathway enzyme, IspD.

The methylerythritol phosphate (MEP) pathway is an essential metabolic pathway found in malaria parasites, but absent in mammals, making it a highly attractive target for the discovery of novel and selective antimalarial therapies. Using high-throughput screening, we have identified 2-phenyl benzo[d]isothiazol-3(2H)-ones as species-selective inhibitors of Plasmodium spp. 2-C-methyl-D-erythritol-4-phosphate cytidyltransferase (IspD), the third catalytic enzyme of the MEP pathway. 2-Phenyl benzo[d]isothiazol-3(2H)-ones display nanomolar inhibitory activity against P. falciparum and P. vivax IspD and prevent the growth of P. falciparum in culture, with EC50 values below 400 nM. In silico modeling, along with enzymatic, genetic and crystallographic studies, have established a mechanism-of-action involving initial non-covalent recognition of inhibitors at the IspD binding site, followed by disulfide bond formation through attack of an active site cysteine residue on the benzo[d]isothiazol-3(2H)-one core. The species-selective inhibitory activity of these small molecules against Plasmodium spp. IspD and cultured parasites suggests they have potential as lead compounds in the pursuit of novel drugs to treat malaria.

Membrane lipid compositional sensing by the inducible amphipathic helix of CCT.

The amphipathic helical (AH) membrane binding motif is recognized as a major device for lipid compositional sensing. We explore the function and mechanism of sensing by the lipid biosynthetic enzyme, CTP:phosphocholine cytidylyltransferase (CCT). As the regulatory enzyme in phosphatidylcholine (PC) synthesis, CCT contributes to membrane PC homeostasis. CCT directly binds and inserts into the surface of bilayers that are deficient in PC and therefore enriched in lipids that enhance surface charge and/or create lipid packing voids. These two membrane physical properties induce the folding of the CCT M domain into a ≥60 residue AH. Membrane binding activates catalysis by a mechanism that has been partially deciphered. We review the evidence for CCT compositional sensing, and the membrane and protein determinants for lipid selective membrane-interactions. We consider the factors that promote the binding of CCT isoforms to the membranes of the ER, nuclear envelope, or lipid droplets, but exclude CCT from other organelles and the plasma membrane. The CCT sensing mechanism is compared with several other proteins that use an AH motif for membrane compositional sensing. This article is part of a Special Issue entitled: The cellular lipid landscape edited by Tim P. Levine and Anant K. Menon.

Human ISPD Is a Cytidyltransferase Required for Dystroglycan O-Mannosylation.

A unique, unsolved O-mannosyl glycan on α-dystroglycan is essential for its interaction with protein ligands in the extracellular matrix. Defective O-mannosylation leads to a group of muscular dystrophies, called dystroglycanopathies. Mutations in isoprenoid synthase domain containing (ISPD) represent the second most common cause of these disorders, however, its molecular function remains uncharacterized. The human ISPD (hISPD) crystal structure showed a canonical N-terminal cytidyltransferase domain linked to a C-terminal domain that is absent in cytidyltransferase homologs. Functional studies demonstrated cytosolic localization of hISPD, and cytidyltransferase activity toward pentose phosphates, including ribulose 5-phosphate, ribose 5-phosphate, and ribitol 5-phosphate. Identity of the CDP sugars was confirmed by liquid chromatography quadrupole time-of-flight mass spectrometry and two-dimensional nuclear magnetic resonance spectroscopy. Our combined results indicate that hISPD is a cytidyltransferase, suggesting the presence of a novel human nucleotide sugar essential for functional α-dystroglycan O-mannosylation in muscle and brain. Thereby, ISPD deficiency can be added to the growing list of tertiary dystroglycanopathies.

Molecular Mechanism for the Thermo-Sensitive Phenotype of CHO-MT58 Cell Line Harbouring a Mutant CTP:Phosphocholine Cytidylyltransferase.

Control and elimination of malaria still represents a major public health challenge. Emerging parasite resistance to current therapies urges development of antimalarials with novel mechanism of action. Phospholipid biosynthesis of the Plasmodium parasite has been validated as promising candidate antimalarial target. The most prevalent de novo pathway for synthesis of phosphatidylcholine is the Kennedy pathway. Its regulatory and often also rate limiting step is catalyzed by CTP:phosphocholine cytidylyltransferase (CCT). The CHO-MT58 cell line expresses a mutant variant of CCT, and displays a thermo-sensitive phenotype. At non-permissive temperature (40°C), the endogenous CCT activity decreases dramatically, blocking membrane synthesis and ultimately leading to apoptosis. In the present study we investigated the impact of the analogous mutation in a catalytic domain construct of Plasmodium falciparum CCT in order to explore the underlying molecular mechanism that explains this phenotype. We used temperature dependent enzyme activity measurements and modeling to investigate the functionality of the mutant enzyme. Furthermore, MS measurements were performed to determine the oligomerization state of the protein, and MD simulations to assess the inter-subunit interactions in the dimer. Our results demonstrate that the R681H mutation does not directly influence enzyme catalytic activity. Instead, it provokes increased heat-sensitivity by destabilizing the CCT dimer. This can possibly explain the significance of the PfCCT pseudoheterodimer organization in ensuring proper enzymatic function. This also provide an explanation for the observed thermo-sensitive phenotype of CHO-MT58 cell line.

Composite aromatic boxes for enzymatic transformations of quaternary ammonium substrates.

Cation-π interactions to cognate ligands in enzymes have key roles in ligand binding and enzymatic catalysis. We have deciphered the key functional role of both charged and aromatic residues within the choline binding subsite of CTP:phosphocholine cytidylyltransferase and choline kinase from Plasmodium falciparum. Comparison of quaternary ammonium binding site structures revealed a general composite aromatic box pattern of enzyme recognition sites, well distinguished from the aromatic box recognition site of receptors.

The curvature sensitivity of a membrane-binding amphipathic helix can be modulated by the charge on a flanking region.

Membrane-induced amphipathic helices (m-AH) can act as membrane curvature sensors by binding preferentially to hydrophobic lipid packing defects enriched in curved surfaces. Reliance on hydrophobicity and membrane curvature for binding is enhanced when electrostatic interactions are weak. We probed the role of modifying membrane and protein charge on the curvature sensing of two m-AH-containing proteins, CTP:phosphocholine cytidylyltransferase (CCT) and α-synuclein (α-syn). The m-AH domains in both proteins are flanked by disordered tails with multiple phosphoserines (CCT) or acidic residues (α-syn), which we mutated to glutamate or serine to modify protein charge. Analysis of binding to vesicles of varying curvature showed that increasing the negative charge of the tail region decreased the binding strength and augmented the curvature dependence, especially for CCT. We attribute this to charge repulsion. Conversely, increasing the membrane negative charge dampened the curvature dependence. Our data suggest that discrimination of curved versus flat membranes with high negative charge could be modulated by phosphorylation.

Structural basis for autoinhibition of CTP:phosphocholine cytidylyltransferase (CCT), the regulatory enzyme in phosphatidylcholine synthesis, by its membrane-binding amphipathic helix.

CTP:phosphocholine cytidylyltransferase (CCT) interconverts between an inactive soluble and active membrane-bound form in response to changes in membrane lipid composition. Activation involves disruption of an inhibitory interaction between the αE helices at the base of the active site and an autoinhibitory (AI) segment in the regulatory M domain and membrane insertion of the M domain as an amphipathic helix. We show that in the CCT soluble form the AI segment functions to suppress kcat and elevate the Km for CTP. The crystal structure of a CCT dimer composed of the catalytic and AI segments reveals an AI-αE interaction as a cluster of four amphipathic helices (two αE and two AI helices) at the base of the active sites. This interaction corroborates mutagenesis implicating multiple hydrophobic residues within the AI segment that contribute to its silencing function. The AI-αE interaction directs the turn at the C-terminal end of the AI helix into backbone-to-backbone contact with a loop (L2) at the opening to the active site, which houses the key catalytic residue, lysine 122. Molecular dynamics simulations suggest that lysine 122 side-chain orientations are constrained by contacts with the AI helix-turn, which could obstruct its engagement with substrates. This work deciphers how the CCT regulatory amphipathic helix functions as a silencing device.