Interdomain and Membrane Interactions of CTP:Phosphocholine Cytidylyltransferase Revealed via Limited Proteolysis and Mass Spectrometry (original) (raw)
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Membrane Binding Modulates the Quaternary Structure of CTP:Phosphocholine Cytidylyltransferase
Journal of Biological Chemistry, 2004
CTP:phosphocholine cytidylyltransferase (CCT), a key enzyme that controls phosphatidylcholine synthesis, is regulated by reversible interactions with membranes containing anionic lipids. Previous work demonstrated that CCT is a homodimer. In this work we show that the structure of the dimer interface is altered upon encountering membranes that activate CCT. Chemical cross-linking reactions were established which captured intradimeric interactions but not random CCT dimer collisions. The efficiency of capturing covalent cross-links with four different reagents was diminished markedly upon presentation of activating anionic lipid vesicles but not zwitterionic vesicles. Experiments were conducted to show that the anionic vesicles did not interfere with the chemistry of the cross-linking reactions and did not sequester available cysteine sites on CCT for reaction with the cysteine-directed cross-linking reagent. Thus, the loss of cross-linking efficiency suggested that contact sites at the dimer interface had increased distance or reduced flexibility upon binding of CCT to membranes. The regions of the enzyme involved in dimerization were mapped using three approaches: 1) limited proteolysis followed by cross-linking of fragments, 2) yeast two-hybrid analysis of interactions between select domains, and 3) disulfide bonding potential of CCTs with individual cysteine to serine substitutions for the seven native cysteines. We found that the Nterminal domain (amino acids 1-72) is an important participant in forming the dimer interface, in addition to the catalytic domain (amino acids 73-236). We mapped the intersubunit disulfide bond to the cystine 37 pair in domain N and showed that this disulfide is sensitive to anionic vesicles, implicating this specific region in the membrane-sensitive dimer interface. CTP:phosphocholine cytidylyltransferase (CCT) 1 is a key regulatory enzyme in phosphatidylcholine (PC) biosynthesis. It
Biochemistry, 1998
We are probing the mechanism of the lipid selective membrane interactions of CTP: phosphocholine cytidylyltransferase (CT). We have proposed that the membrane binding domain of CT (domain M) consists of a continuous amphipathic R-helix between residues ∼240-295 [Dunne, S. J., et al. (1996) Biochemistry 35, 11975-11984]. This study examined the secondary structure and membrane binding properties of synthetic peptides derived from domain M: a 62mer peptide encompassing the entire domain (Pep62), a 33mer corresponding to the N-terminal portion (PepNH1), and two 33mers corresponding to the three C-terminal 11mer repeats, one with the wild-type sequence (Pep33Ser), and one with the three serines in the nonpolar face substituted with alanine (Pep33Ala). Peptide secondary structure was analyzed by circular dichroism, and lipid interactions were analyzed by a direct vesicle binding assay, by effects of lipid vesicles on peptide tryptophan fluorescence, and by monolayer surface pressure changes. All peptides bound to vesicles as R-helices with selectivity for anionic lipids. Binding involved intercalation of the peptide tryptophan into the hydrophobic membrane core. PepNH1, the peptide with the highest positive charge density, showed strong selectivity for anionic lipids. PepNH1 and Pep33Ser did not bind to PC vesicles; however, the more hydrophobic peptides, Pep33Ala and Pep62, did bind to PC vesicles, with apparent partition coefficients for PC that were only ∼1 order of magnitude lower than those for PC/PG (1/1). Our results suggest that the polar serines interrupting the nonpolar face of the amphipathic helix serve to lower the lipid affinity and thereby enhance selectivity for anionic lipids. Although diacylglycerol is an activator of the enzyme, none of the peptides responded differentially to PC/diacylglycerol vesicles versus pure PC vesicles, suggesting that domain M alone is not sufficient for the enzyme's response to diacylglycerol. Increases in surface pressure at an air-water interface indicated that the domain M peptides had strong surface-seeking tendencies. This supports a binding orientation for domain M parallel to the membrane surface. Binding of CT peptides to spread lipid monolayers caused surface pressure reductions, suggesting condensation of lipids in the formation of lipid-peptide complexes. At low monolayer surface pressures, Pep62 interacted equally with anionic and zwitterionic phospholipids. This suggests that one determinant of the selectivity for anionic lipids is the lipid packing density (area per molecule). CTP:phosphocholine cytidylyltransferase (CT), 1 a key ratedetermining enzyme for PC biosynthesis, is regulated by reversible membrane binding. Membrane binding and enzyme activation can be regulated by fluctuations in the membrane content of acidic lipid, and/or diacylglycerol (DAG), or by changes in the phosphorylation state of CT (1-3). Activation of the enzyme in vitro by lipid vesicles is dependent on the mole percent of acidic lipid or DAG mixed with PC (4-8). Diacylglycerol or enzyme dephosphorylation lowers the percent of acidic lipid required for activation (3, 9). Both electrostatic and hydrophobic interactions mediate the membrane binding process (7-10). Mammalian CT is organized into at least three discrete domains. The N-terminal two-thirds of the protein forms a protease-insensitive domain (11), which has been proposed to house the catalytic site on the basis of homologies to other cytidylyltransferases (12-14), and mutational analysis (15, 16). The C-terminal region of the protein is highly phos
Structure of the Membrane Binding Domain of CTP:Phosphocholine Cytidylyltransferase
Biochemistry, 1996
It has been proposed that the domain of the regulatory enzyme, CTP:phosphocholine cytidylyltransferase, which mediates reversible binding of the enzyme to membranes, is an amphipathic R-helix of approximately 60 amino acid residues and that this domain is adjacent to the putative active site domain of this enzyme. Circular dichroism indicated that the secondary structures of two overlapping peptides spanning this region were predominantly R-helical in the presence of PG vesicles or sodium dodecyl sulfate micelles. Interproton distances were obtained from two-dimensional NMR spectroscopic measurements to solve the structures of these two peptides. The C-terminal 22 amino acid peptide segment (corresponding to Val267-Ser288) was a well-defined R-helix over its length. The N-terminal 33-mer (corresponding to Asn236-Glu268) was composed of an R-helix from Glu243 to Lys266, a well-structured bend of about 50°at Tyr240-His241-Leu242, and an N-terminal four-residue helix. It is proposed that the three residues involved in generating the bend act as the hinge between the catalytic and regulatory domains. The nonpolar faces of the 33-mer and 22-mer were interrupted by Ser260, Ser271, and Ser282. These residues may serve to limit the hydrophobicity and facilitate reversible and lipid-selective membrane binding. The hydrophobic faces of the helices were flanked by a set of basic amino acid residues on one side and basic amino acid residues interspersed with glutamates on the other. The disposition of these side chains gives clues to the basis for the specificities of these peptides for anionic surfaces. † This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (A.S.T.) and the Medical Research Council of Canada (R.B.C. and a studentship to J.E.J.). ‡ The structure information has been deposited with the Brookhaven Protein Data Bank under the filenames 1PEH, 1PEI, R1PEHMR, and R1PEIMR.
Journal of Biological Chemistry, 2008
CTP:phosphocholine cytidylyltransferase (CCT), a rate-limiting enzyme in phosphatidylcholine synthesis, is regulated by reversible membrane interactions mediated by an amphipathic helical domain (M) that binds selectively to anionic lipids. CCT is a dimer; thus the functional unit has two M domains. To probe the functional contribution of each domain M we prepared a CCT heterodimer composed of one full-length subunit paired with a CCT subunit truncated before domain M that was also catalytically dead. We compared this heterodimer to the fulllength homodimer with respect to activation by anionic vesicles, vesicle binding affinities, and promotion of vesicle aggregation. Surprisingly for all three functions the dimer with just one domain M behaved similarly to the dimer with two M domains. Full activation of the wild-type subunit was not impaired by loss of one domain M in its partner. Membrane binding affinities were the same for dimers with one versus two M domains, suggesting that the two M domains of the dimer do not engage a single bilayer simultaneously. Vesicle cross-bridging was also unhindered by loss of one domain M, suggesting that another motif couples with domain M for cross-bridging anionic membranes. Mutagenesis revealed that the positively charged nuclear localization signal sequence constitutes that second motif for membrane cross-bridging. We propose that the two M domains of the CCT dimer engage a single bilayer via an alternating binding mechanism. The tethering function involves the cooperation of domain M and the nuclear localization signal sequence, each engaging separate membranes. Membrane binding of a single M domain is sufficient to fully activate the enzymatic activity of the CCT dimer while sustaining the low affinity, reversible membrane interaction required for regulation of CCT activity.
The Journal of Biological Chemistry, 2008
CTP:phosphocholine cytidylyltransferase (CCT), a rate-limiting enzyme in phosphatidylcholine synthesis, is regulated by reversible membrane interactions mediated by an amphipathic helical domain (M) that binds selectively to anionic lipids. CCT is a dimer; thus the functional unit has two M domains. To probe the functional contribution of each domain M we prepared a CCT heterodimer composed of one full-length subunit paired with a CCT subunit truncated before domain M that was also catalytically dead. We compared this heterodimer to the fulllength homodimer with respect to activation by anionic vesicles, vesicle binding affinities, and promotion of vesicle aggregation. Surprisingly for all three functions the dimer with just one domain M behaved similarly to the dimer with two M domains. Full activation of the wild-type subunit was not impaired by loss of one domain M in its partner. Membrane binding affinities were the same for dimers with one versus two M domains, suggesting that the two M domains of the dimer do not engage a single bilayer simultaneously. Vesicle cross-bridging was also unhindered by loss of one domain M, suggesting that another motif couples with domain M for cross-bridging anionic membranes. Mutagenesis revealed that the positively charged nuclear localization signal sequence constitutes that second motif for membrane cross-bridging. We propose that the two M domains of the CCT dimer engage a single bilayer via an alternating binding mechanism. The tethering function involves the cooperation of domain M and the nuclear localization signal sequence, each engaging separate membranes. Membrane binding of a single M domain is sufficient to fully activate the enzymatic activity of the CCT dimer while sustaining the low affinity, reversible membrane interaction required for regulation of CCT activity.
Journal of Biological Chemistry, 2008
CTP:phosphocholine cytidylyltransferase (CCT), a rate-limiting enzyme in phosphatidylcholine synthesis, is regulated by reversible membrane interactions mediated by an amphipathic helical domain (M) that binds selectively to anionic lipids. CCT is a dimer; thus the functional unit has two M domains. To probe the functional contribution of each domain M we prepared a CCT heterodimer composed of one full-length subunit paired with a CCT subunit truncated before domain M that was also catalytically dead. We compared this heterodimer to the fulllength homodimer with respect to activation by anionic vesicles, vesicle binding affinities, and promotion of vesicle aggregation. Surprisingly for all three functions the dimer with just one domain M behaved similarly to the dimer with two M domains. Full activation of the wild-type subunit was not impaired by loss of one domain M in its partner. Membrane binding affinities were the same for dimers with one versus two M domains, suggesting that the two M domains of the dimer do not engage a single bilayer simultaneously. Vesicle cross-bridging was also unhindered by loss of one domain M, suggesting that another motif couples with domain M for cross-bridging anionic membranes. Mutagenesis revealed that the positively charged nuclear localization signal sequence constitutes that second motif for membrane cross-bridging. We propose that the two M domains of the CCT dimer engage a single bilayer via an alternating binding mechanism. The tethering function involves the cooperation of domain M and the nuclear localization signal sequence, each engaging separate membranes. Membrane binding of a single M domain is sufficient to fully activate the enzymatic activity of the CCT dimer while sustaining the low affinity, reversible membrane interaction required for regulation of CCT activity.
Journal of Biological Chemistry
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. This work was supported by grants from the Canadian Institutes of Health Research (to R. B. C. and D. P. T.) and supported in part by the Canada Research Chairs program. The authors declare that they have no conflicts of interest with the contents of this article. This article was selected as one of our Editors' Picks. This article contains Figs. S1-S10.
Biochemistry, 2001
The CTP:phosphocholine cytidylyltransferase (CCT) governs the rate of phosphatidylcholine (PtdCho) biosynthesis, and its activity is governed by interaction with membrane lipids. The carboxyterminus was dissected to delineate the minimum sequences required for lipid responsiveness. The helical domain is recognized as a site of lipid interaction, and all three tandem R-helical repeats from residues 257 through 290 were found to be required for regulation of enzymatic activity by this domain. Truncation of the carboxy-terminus to remove one or more of the R-helical repeats yielded catalytically compromised proteins that were not responsive to lipids but retained sufficient activity to accelerate PtdCho biosynthesis when overexpressed in vivo. The role of the helical region in lipid-activation was tested further by excising residues 257 through 309 to yield a protein that retained a 57-residue carboxy terminal domain fused to the catalytic core. This construct tested the hypothesis that the helical region inhibits activity in the absence of lipid rather than activates the enzyme in the presence of lipid. This hypothesis predicts constitutive activity for CCTR[∆257-309]; however, this protein was tightly regulated by lipid with activities comparable to the full-length CCTR, in both the absence and presence of lipid. Activation of CCTR[∆257-309] was dependent exclusively on anionic lipids, whereas full-length CCTR responded to either anionic or neutral lipids. Phosphatidic acid delivered in Triton X-100 micelles was the preferred activator of the second lipid-activation domain. These data demonstrate that CCTR can be regulated by lipids by two independent domains: (i) the three amphipathic R-helical repeats that interact with both neutral and anionic lipid mixtures and (ii) the last 57 residues that interact with anionic lipids. The results show that both domains are inhibitory in the absence of lipid and activating in the presence of lipid. Removal of both domains results in a nonresponsive, dysregulated enzyme with reduced activity. The data also demonstrate for the first time that the 57-residue carboxy-terminal domain in CCTR participates in lipid-mediated regulation and is sufficient for maximum activation of enzyme activity.
Journal of Biological Chemistry
of CTPwhosphocholine cytidylyltransferase (CT; EC 2.7.7.15) produced several distinct fragments which were mapped to the N terminus of CT using antibodies directed against the N and C terminus and the conserved central domain. A time c o m e of chymotrypsin proteolysis showed a progression in digestion as follows: 42 + 39 -* 35 + 30 + 28 + 26 m a . The binding of CT and of the chymotrypsin fragments to lipid vesicles was assessed by floatation analysis. The ability of the fragments to bind to activating lipid vesicles correlated w i t h the presence of a putative amphipathic a-helix, helix-1, between residues 236 and 293. Fragments lacking this helix could, however, bind to phosp~tidylcholin~sphingosine vesicles, which inhibit CT activity, and were capable of dimer formation. The degree of resistance to c h y m o t~s i n degradation increased when CT was bound to the strongly activating lipid vesicles phoEphatidylcho~~ oleic acid
Chemical Cross-linking Reveals a Dimeric Structure for CTP: Phosphocholine Cytidylyltransferase
Journal of Biological Chemistry, 1989
5 104-5 110). Sodium dodecyl sulfate-poIyacrylamide gel electrophoresis with or without 8-mercaptoethanol revealed a single major band of 42,000 daltons. This band corresponds to the 45-kDa catalytic subunit isolated by Feldman and Weinhold (Feldman, D. A., and Weinhold, P. A. (1987) J. Biol. Chem. 262, 9075-9081). A minor component of 84,000 daltons was intensified in nonreducing gels when the sulfhydryl reducing agent, dithiothreitol, was removed from the enzyme preparation by dialysis. Reduction with dithiothreitol and electrophoresis in the second dimension showed that this 84-kDa protein was derived from the 42-kDa protein. This result suggested that the 42 kDa protein can be converted to an 84-kDa protein by disulfide bond formation. Reaction with the thiolcleavable cross-linking reagents, dithiobis(succimidy1 propionate) or dimethyL3,3'-dithiobispropionirnidate, converted the 42-kDa cytidylyltransferase subunit into a diffuse band approximately twice its molecular mass. Disulfide reduction and electrophoresis in the second dimension showed that this band was derived exclusively from the 42-kDa subunit. This cross-linking pattern was observed when cytidylyltransferase was bound to a Triton X-100 micelle or when bound to a membrane vesicle containing phosphatidylcholine, oleic acid, and Triton X-100. Reaction of the fully reduced enzyme with glutaraldehyde also generated a cross-linked dimer. All three cross-linking reagents inactivated the enzyme. Reduction of the disulfide cross-linkers with dithiothreitol partially reactivated the transferase. When Triton was removed from the enzyme preparation by DEAE-Sepharose chromatography, reaction of the detergent-depleted enzyme with glutaraldehyde generated a band corresponding to a hexamer and higher molecular weight aggregates. The dimeric form was regenerated by addition of either Triton X-100 or phosphatidylcholine-oleic acid vesicles. We conclude that the purified, native cytidylyltransferase, when bound to a detergent micelle or membrane vesicle, is a dimer composed of two noncovalently linked 42-kDa subunits. In the absence of a membrane or micelle, the dimers self-aggregate in a reversible manner.