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• W2000896070 abstract "Amphitropic proteins are regulated by reversible membrane interaction. Anionic phospholipids generally promote membrane binding of such proteins via electrostatics between the negatively charged lipid headgroups and clusters of basic groups on the proteins. In this study of one amphitropic protein, a cytidylyltransferase (CT) that regulates phosphatidylcholine synthesis, we found that substitution of lysines to glutamine along both interfacial strips of the membrane-binding amphipathic helix eliminated electrostatic binding. Unexpectedly, three glutamates also participate in the selectivity for anionic membrane surfaces. These glutamates become protonated in the low pH milieu at the surface of anionic, but not zwitterionic membranes, increasing protein positive charge and hydrophobicity. The binding and insertion into lipid vesicles of a synthetic peptide containing the three glutamates was pH-dependent with an apparent pKathat varied with anionic lipid content. Glutamate to glutamine substitution eliminated the pH dependence of the membrane interaction, and reduced anionic membrane selectivity of both the peptide and the whole CT enzyme examined in cells. Thus anionic lipids, working via surface-localized pH effects, can promote membrane binding by modifying protein charge and hydrophobicity, and this novel mechanism contributes to the membrane selectivity of CT in vivo. Amphitropic proteins are regulated by reversible membrane interaction. Anionic phospholipids generally promote membrane binding of such proteins via electrostatics between the negatively charged lipid headgroups and clusters of basic groups on the proteins. In this study of one amphitropic protein, a cytidylyltransferase (CT) that regulates phosphatidylcholine synthesis, we found that substitution of lysines to glutamine along both interfacial strips of the membrane-binding amphipathic helix eliminated electrostatic binding. Unexpectedly, three glutamates also participate in the selectivity for anionic membrane surfaces. These glutamates become protonated in the low pH milieu at the surface of anionic, but not zwitterionic membranes, increasing protein positive charge and hydrophobicity. The binding and insertion into lipid vesicles of a synthetic peptide containing the three glutamates was pH-dependent with an apparent pKathat varied with anionic lipid content. Glutamate to glutamine substitution eliminated the pH dependence of the membrane interaction, and reduced anionic membrane selectivity of both the peptide and the whole CT enzyme examined in cells. Thus anionic lipids, working via surface-localized pH effects, can promote membrane binding by modifying protein charge and hydrophobicity, and this novel mechanism contributes to the membrane selectivity of CT in vivo. CTP:phosphocholine cytidylyltransferase phosphatidylcholine phosphatidylglycerol small unilamellar vesicles circular dichroism bovine serum albumin oleic acid green fluorescent protein wild type high performance liquid chromatography matrix-assisted laser desorption ionization Proteins that interact reversibly with cell membrane lipids usually have selectivity for negatively charged phospholipids (1Buckland A.G. Wilton D.C. Biochim. Biophys. Acta. 1999; 1483: 199-216Crossref Scopus (108) Google Scholar, 2Johnson J.E. Cornell R.B. Mol. Membr. Biol. 1999; 16: 217-235Crossref PubMed Scopus (239) Google Scholar). Some of these proteins show specificity for a particular anionic phospholipid. For example, protein kinase C binds preferentially to phosphatidylserine (3Newton A.C. Johnson J.E. Biochim. Biophys. Acta. 1998; 1376: 155-172Crossref PubMed Scopus (244) Google Scholar); MARCKS (4Wang J. Arbuzova A. Hangyas-Mihalyne G. McLaughlin S. J. Biol. Chem. 2001; 276: 5013-5019Google Scholar) and proteins with PH domains (5Lemon M.A. Ferguson K.M. Biochem. J. 2000; 350: 1-18Crossref PubMed Google Scholar) bind selectively to various phosphoinositides. However, many proteins exhibit non-selectivity with respect to the anionic phospholipid by a simple electrostatic interaction between clusters of basic residues on the protein and negatively charged lipid head groups (6Buser C.A. Kim J. McLaughlin S. Peitzsch R.M. Mol. Membr. Biol. 1995; 12: 69-75Crossref PubMed Scopus (40) Google Scholar). This binding affinity may be regulated by changes in membrane anionic lipid content, but more often by modification of the charge on the protein by a mechanism referred to as an “electrostatic switch” (7McLaughlin S. Aderem A. Trends Biochem. Sci. 1995; 20: 272-276Abstract Full Text PDF PubMed Scopus (616) Google Scholar). Phosphorylation of a basic patch on such proteins as MARCKS (7McLaughlin S. Aderem A. Trends Biochem. Sci. 1995; 20: 272-276Abstract Full Text PDF PubMed Scopus (616) Google Scholar), Src (8Murray D. Hermida-Matsumoto L. Buser C.A. Tsang J. Sigal C.T. Ben-Tal N. Honig B. Resh M.D. McLaughlin S. Biochemistry. 1998; 37: 2145-2159Crossref PubMed Scopus (160) Google Scholar), and ARNO (9Santy L.C. Frank S.R. Hatfield J.C. Casanova J.E. Curr. Biol. 1999; 9: 1173-1176Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) neutralizes positive charge. Alternatively, calcium binding to acidic residues in the C2 domain of protein kinase C and phospholipase A2 increases positive charge (10Murray D. Honig B Mol. Cell. 2002; 9: 145-154Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). Hisactophilin exhibits a variation on this theme, in which the protein charge may be modulated by cytosolic pH change (11Hanakam F. Gerisch G. Lotz S. Alt T. Seelig A. Biochemistry. 1996; 35: 11036-11044Crossref PubMed Scopus (69) Google Scholar). In this work, we provide an example in which increased anionic lipid composition modulates protein charge and hydrophobicity by influencing its protonation state and thereby increases membrane affinity. CTP:phosphocholine cytidylyltransferase (CT)1 catalyzes a key rate-limiting step in PC synthesis and contributes to maintenance of cell membrane PC homeostasis (12Cornell R.B. Northwood I.C. Trends Biochem. Sci. 2000; 25: 441-447Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). When the relative membrane PC content is altered, CT could respond by recognition of ensuing changes in the physical features of PC-deficient or PC-overloaded membranes, including changes in the negative surface charge density (13Cornell R.B. Biochem. Soc. Trans. 1998; 26: 539-544Crossref PubMed Scopus (27) Google Scholar). Thus, how CT responds to changes in surface charge has great bearing on the control of membrane phospholipid compositional homeostasis. There are three homologous mammalian CT isoforms, α, β1, and β2 (12Cornell R.B. Northwood I.C. Trends Biochem. Sci. 2000; 25: 441-447Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). Most of the biochemical characterization, including the work in this study, has been done with CTα. CTα activity is regulated by reversible membrane binding, which involves both electrostatic and hydrophobic interactions (12Cornell R.B. Northwood I.C. Trends Biochem. Sci. 2000; 25: 441-447Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 13Cornell R.B. Biochem. Soc. Trans. 1998; 26: 539-544Crossref PubMed Scopus (27) Google Scholar). It binds poorly to pure PC membranes in vitro, and its binding affinity increases in proportion to the negative surface charge of the membrane (14Cornell R.B. Biochemistry. 1991; 30: 5873-5880Crossref PubMed Scopus (60) Google Scholar, 15Arnold R.S. Cornell R.B. Biochemistry. 1996; 35: 9917-9924Crossref PubMed Scopus (85) Google Scholar). Importantly, its translocation to membranes in living cells also increases as a function of anionic lipid content (16Cornell R.B. Vance D.E. Biochim. Biophys. Acta. 1987; 919: 26-36Crossref PubMed Scopus (88) Google Scholar, 17Wang Y. MacDonald J.I.S. Kent C. J. Biol. Chem. 1993; 268: 5512-5518Abstract Full Text PDF PubMed Google Scholar, 18Weinhold P.A. Rounsifer M.E. Williams S.E. Brubaker P.G. Feldman D.A. J. Biol. Chem. 1984; 259: 10315-10321Abstract Full Text PDF PubMed Google Scholar). Membrane translocation is also accompanied by dephosphorylation of its C-terminal domain, but this event is subsequent to membrane binding (19Houweling M. Jamil H. Hatch G.M. Vance D.E. J. Biol. Chem. 1994; 269: 7544-7551Abstract Full Text PDF PubMed Google Scholar), and the effects of phosphorylation status on membrane partitioning can be overcome by raising the anionic lipid content of the membrane (20Wang Y. Kent C. J. Biol. Chem. 1995; 270: 17843-17849Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Based onin situ imaging with fluorescent antibodies or with GFP-tagged CTα, the enzyme is predominantly nuclear in many cells (reviewed in Ref. 12Cornell R.B. Northwood I.C. Trends Biochem. Sci. 2000; 25: 441-447Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar) and translocates to the nuclear envelope upon stimulation with exogenous fatty acids (12Cornell R.B. Northwood I.C. Trends Biochem. Sci. 2000; 25: 441-447Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 21Legace T.A. Storey M.K. Ridgeway N.D. J. Biol. Chem. 2000; 275: 14367-14374Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). However, in other cells and contexts CTα appears to be cytoplasmic and ER-bound (12Cornell R.B. Northwood I.C. Trends Biochem. Sci. 2000; 25: 441-447Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar,22Lykidis A. Baburina I. Jackowski S. J. Biol. Chem. 1999; 274: 26992-27001Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 23Delong C. Qin L. Cui Z. J. Biol. Chem. 2000; 275: 32325-32330Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 24Ridsdale R. Tseu I. Wang J. Post M. J. Biol. Chem. 2001; 276: 49148-49155Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). The reason for these cell-dependent differences in CTα localization is unresolved. A well characterized membrane-binding domain present in all CT isoforms (domain M) consists of a long amphipathic α-helix (Fig. 1 A and Refs. 25Dunne S.J. Cornell R.B. Johnson J.E. Glover N. Tracey A. Biochemistry. 1996; 35: 11975-11984Crossref PubMed Scopus (91) Google Scholar, 26Johnson J.E. Cornell R.B. Biochemistry. 1994; 33: 4327-4335Crossref PubMed Scopus (67) Google Scholar, 27Johnson J.E. Rao N.M. Hui S.W. Cornell R.B. Biochemistry. 1998; 37: 9509-9519Crossref PubMed Scopus (52) Google Scholar). Domain M can be subdivided further into: (i) an N-terminal polybasic region (subdomain N, residues 237–255), (ii) a central, net negative region containing three 11-mer repeats (the VEEKS subdomain, residues 256–288), and (iii) a C-terminal aromatic-rich region terminating in a predicted bend (Fig. 1 A). Peptides corresponding to either the entire domain M, subdomain N, or the VEEKS subdomain bind selectively to anionic lipids (27Johnson J.E. Rao N.M. Hui S.W. Cornell R.B. Biochemistry. 1998; 37: 9509-9519Crossref PubMed Scopus (52) Google Scholar). The non-polar face of domain M helix creates a ∼120o wedge containing 18 aliphatic or aromatic side chains. The polar face is rich in acidic side chains. One of the interfacial strips is exclusively basic, and the other is a mixture of acidic and basic residues (Fig. 1, B andC). Our goal is to characterize the determinants in domain M responsible for CT selectivity toward anionic lipids. Using a VEEKS subdomain peptide, we previously showed that mutating to alanine the three serines interrupting the nonpolar face increases peptide hydrophobicity and reduces (but does not eliminate) the selectivity for anionic lipids (27Johnson J.E. Rao N.M. Hui S.W. Cornell R.B. Biochemistry. 1998; 37: 9509-9519Crossref PubMed Scopus (52) Google Scholar). The N-terminal portion of domain M is the most highly conserved region of domain M (28Friesen J.A. Liu M. Kent C. Biochim. Biophys. Acta. 2001; 1533: 86-93Crossref PubMed Scopus (23) Google Scholar), and has the highest concentration of positive charge. Here we have probed the contribution of these basic amino acids to the electrostatic interaction with anionic phospholipids by en bloc substitution of 5 or 8 interfacial Lys/Arg with glutamine (Fig. 1 B). We found that the membrane affinity was progressively reduced upon progressive elimination of peptide positive charge. The electrostatic component of the membrane binding was virtually eliminated upon removal of positive charge from both interfaces flanking the hydrophobic face of the peptide. CT binding to anionic lipid vesicles is enhanced as the pH is lowered from 7.4 to 6.3 (15Arnold R.S. Cornell R.B. Biochemistry. 1996; 35: 9917-9924Crossref PubMed Scopus (85) Google Scholar). One hypothesis for this effect is that lowering the pH protonates the weakly acidic side chains in domain M, specifically the three interfacial glutamates, thereby neutralizing their negative charge and enhancing peptide hydrophobicity. We proposed (25Dunne S.J. Cornell R.B. Johnson J.E. Glover N. Tracey A. Biochemistry. 1996; 35: 11975-11984Crossref PubMed Scopus (91) Google Scholar) that the probability of protonation of these glutamates would be higher at the interface of an anionic membrane versus a zwitterionic membrane, because of the attraction of protons at the negative surface (Fig. 2). In this way, ironically, the positioning of three interfacial glutamic acid side chains could contribute to the selectivity for anionic phospholipids. To test this hypothesis we compared the binding characteristics of a wild-type VEEKS repeat peptide with a mutant analog, in which the interfacial glutamates were replaced with glutamine (Fig. 1 C). We found that the mutant peptide had reduced dependence on the anionic lipid content for binding, and that binding was pH-insensitive. These results imply that the interfacial glutamates contribute to the selectivity for anionic lipid. The same mutations were generated in the whole enzyme. The glutamine-substituted enzyme had higher affinity for membranes and, like the mutant peptide, was less dependent on high membrane surface charge for binding. Cell culture materials, restriction enzymes, and PCR primers were from Invitrogen. All other chemicals were reagent grade or better. All peptides were acetylated and aminated on their N and C termini. The syntheses of the 33-mer peptides corresponding to the wild-type subdomain N sequence (residues 236–268) and the VEEKS repeat sequence (residues 256–288) of rat CTα (29Kalmar G.B. Kay R.J. Lachance A. Aebersold R. Cornell R.B. Proc. Natl. Acad. Sci. 1990; 87: 6029-6033Crossref PubMed Scopus (129) Google Scholar) were described previously (27Johnson J.E. Rao N.M. Hui S.W. Cornell R.B. Biochemistry. 1998; 37: 9509-9519Crossref PubMed Scopus (52) Google Scholar). All other peptides were synthesized by Dr. Krystyna Piotrowska at the University of British Columbia Peptide Service Laboratory on an ABI model 431A synthesizer using Fmoc chemistry. The peptides were >98% pure after HPLC purification using a VYDAC C18 column. Their masses, determined by MALDI-mass spectroscopy, were within <2 daltons of their calculated mass, confirming the correct sequences. After lyophilization to remove the bound trifluoroacetic acid, the peptides were dissolved in water to a working stock concentration of 0.3–1 mm as confirmed by comparison to the fluorescence of a tryptophan standard. Egg PC and egg PG were from Northern Lipids (Vancouver, B.C.) or Avanti (Alabaster, Alabama). Stocks were quantitated (30Bartlett G.R. J. Biol. Chem. 1959; 234: 466-468Abstract Full Text PDF PubMed Google Scholar) and checked for purity by TLC as described (26Johnson J.E. Cornell R.B. Biochemistry. 1994; 33: 4327-4335Crossref PubMed Scopus (67) Google Scholar). Small unilamellar vesicles (SUV) were prepared by sonication as described (27Johnson J.E. Rao N.M. Hui S.W. Cornell R.B. Biochemistry. 1998; 37: 9509-9519Crossref PubMed Scopus (52) Google Scholar). Ionic strength of samples was varied by addition of 1–100 mm phosphate buffer stocks at pH 7.0 and addition of NaCl. Ionic strength was calculated using Equation 1, μ=½Σici(zi)2Equation 1 where c and z are the concentration and charge of each contributing ion, respectively. For pH titrations, phosphate buffers were prepared from pH 4.25 to 7.4 (50 mm) and pH 7.6 to 8.5 (40 mm). The ionic strength was equalized to 131 mm with NaCl. We accounted for the slight decrease in pH that occurred upon addition of 3 mm SUVs to samples for CD analysis (Fig. 6). SUVs of various compositions were incubated with peptide (10 μm) in phosphate buffer, pH 7.0, at 20 °C. Vesicle-bound peptide was trapped by centrifugation through Microcon-100 filters (Amicon; Beverly, MA) at 3000 × g for 10–30 min until one-half to three-fourths of the original volume had filtered (27Johnson J.E. Rao N.M. Hui S.W. Cornell R.B. Biochemistry. 1998; 37: 9509-9519Crossref PubMed Scopus (52) Google Scholar). Samples of the filtrate were diluted 2-fold in phosphate and/or NaCl to equalize ionic strength, and adjusted to pH 10 with NaOH. The free peptide concentration was analyzed by derivatization with fluorescamine (0.4 mm, Sigma Chemical) (31Castell J.V. Cervera M. Marco R. Anal. Biochem. 1979; 99: 379-391Crossref PubMed Scopus (107) Google Scholar). For binding assays with the VEEKS-repeat peptides, samples of the filtrate were diluted 1:1 with methanol, and the peptide concentration was measured via tryptophan fluorescence (excitation, 280 nm; emission, 345 nm) to increase sensitivity by a factor of ∼5. For each analysis, standard curves were conducted with known concentrations of the appropriate peptide. A dimensionless partition coefficient (Kx) was calculated using samples where the percent-bound peptide ranged between 16 and 70% unless otherwise stated, using Equation 2, Kx=([P]b/[P]f)([H2O]/[L])Equation 2 where [P]b/[P]f = molar ratio of bound peptide/free peptide, and [H2O]/[L] = molar ratio of water (55.5m) to accessible lipid (0.6 × total [lipid]). SUVs of various compositions were incubated with peptide (3 μm) at 20 °C in phosphate buffer as described for at least 5 min prior to spectral acquisition. Tryptophan fluorescence spectra were acquired as described previously (27Johnson J.E. Rao N.M. Hui S.W. Cornell R.B. Biochemistry. 1998; 37: 9509-9519Crossref PubMed Scopus (52) Google Scholar). Tyrosine fluorescence of Pep-5KQ (15 μm) was monitored at 304 nm (excitation, 280 nm). The increase in peptide fluorescence at 304 nm in the presence of vesicles was normalized to F304 of peptide in buffer. All spectra were acquired at 20 °C on an SLM 4800C or PTI model QM-1 spectrofluorometer. Spectra were smoothed, and the contribution of the lipid was subtracted. To calculate Kx values from fluorescence measurements, the ratio of bound/total peptide was estimated from the fluorescence measurements by (F −Fo)/(F max −Fo), where F = fluorescence increase or blue shift of sample containing peptide + lipid;Fo = fluorescence value of lipid-free peptide;F max = fluorescence value at saturating lipid contents. CD spectra were acquired at 20 °C as described previously (27Johnson J.E. Rao N.M. Hui S.W. Cornell R.B. Biochemistry. 1998; 37: 9509-9519Crossref PubMed Scopus (52) Google Scholar). Peptides (30 μm) were mixed with lipid vesicles or trifluoroethanol for at least 5 min prior to spectral acquisition. Spectra were smoothed and the contribution of the buffer and/or lipid was subtracted. The CD values were converted to mean residue molar ellipticity (θ; deg cm2dmol−1), and the percent helix was estimated from θ222 nm as described (27Johnson J.E. Rao N.M. Hui S.W. Cornell R.B. Biochemistry. 1998; 37: 9509-9519Crossref PubMed Scopus (52) Google Scholar). To generate a mutant CTα containing glutamines at codons 257, 268, and 279, we designed the following mutagenic primers: CL1, 5′-cGGATCCaaATCGATAGATCTcatccagaagtggcaggagaagtCCCGGGagttcattggaagt-3′; CL2; 5′-cGGATCCAGATCTATCGATttctcctgcactttctgcacaaattctttcgacttttcctgcacatctttcacttt-3′. The mutagenic nucleotides are in bold and the engineered restriction sites, BamHI, BglII, ClaI, and SmaI (uppercase) created silent mutations. Two PCR reactions were performed using Pfu polymerase with wild-type rat CTα cDNA (32MacDonald J. Kent C. Prot. Express Purif. 1993; 4: 1-7Crossref PubMed Scopus (32) Google Scholar) inserted into the SalI site of pBSKS (+) as a template. The CL1 mutagenic primer was paired with the vector reverse T7 primer, and the CL2 mutagenic primer was paired with the vector forward T3 primer to generate PCR products of 910 and 430 bp, respectively. These PCR products were cut with ClaIand SalI and inserted into the appropriate sites of pBSKS (+). The accuracy of the resulting constructs was confirmed by sequencing. The two PCR fragments were joined at the ClaI site to generate CT-3EQ. CT-3EQ was moved to the expression vector pAX142 (33Cornell R.B. Kalmar G.B. Kay R.J. Johnson M.A. Sanghera J.S. Pelech S.L. Biochem. J. 1995; 310: 699-708Crossref PubMed Scopus (66) Google Scholar) using SalI. COS-1 cells were cultured and transfected with pAX-142 constructs as described (34Walkey C.J. Kalmar G.B. Cornell R.B. J. Biol. Chem. 1994; 269: 5742-5749Abstract Full Text PDF PubMed Google Scholar) except that the seeding density was 1 × 106 cells/10-cm dish, and the cells were glycerol-shocked for 2.5 min following rather than preceding treatment with 100 μm chloroquine for 3 h at 37 °C. Cells were transfected with 3 μg of pAX142-CTα-WT per 10-cm dish for 20 h and 10 μg of pAX142-CTα-3EQ for 60 h to achieve equivalent expression levels. To enrich cells with oleic acid they were incubated for 1 h at 37 °C with media containing 1 mm sodium oleate (Sigma Chemical) and 0.5–10 mg ml−1 BSA (fatty acid free; Calbiochem) to achieve OA/BSA molar ratios of 133:6.6. The oleate was prepared as a 10× sonicated stock in phosphate-buffered saline and was co-sonicated with the BSA prior to addition to cells. At the highest OA:BSA ratio, the viability of the cells after 1 h was >90%. The transfected cells were harvested with phosphate-buffered saline containing 2.5 mm EDTA, and homogenized by sonication for 2 × 15 s at 4 °C in 0.4 ml 10 mm Tris, pH 7.4, 1 mm EDTA, 3 mm MgCl2, 0.5 mm phenylmethylsulfonyl fluoride, 2 mmdithiothreitol. The protein concentration of the homogenates was ∼5 mg ml−1 (35Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (215575) Google Scholar). Electrophoresis and Western blotting using an antibody against the N-terminal 17 amino acids of CTα was as described (36Veitch D.V. Gilham D. Cornell R.B. Eur. J. Biochem. 1998; 255: 227-234Crossref PubMed Scopus (46) Google Scholar). After adding K2HPO4 to a final concentration of 0.2 m, the homogenates were centrifuged at 100,000 × g for 1 h at 4 °C to generate a particulate and soluble fraction. The particulate fraction was resonicated in homogenization buffer/0.2 M K2HPO4 as above. Aliquots of both fractions were assayed for CT activity for 15 min under optimum conditions of substrates (15Arnold R.S. Cornell R.B. Biochemistry. 1996; 35: 9917-9924Crossref PubMed Scopus (85) Google Scholar) and lipid activator (200 μm egg PC/oleic acid (1:1)). The lipids in the particulate fractions were extracted (37Bligh E.G. Dyer W.J. Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42666) Google Scholar) and analyzed for phospholipid phosphorus content (30Bartlett G.R. J. Biol. Chem. 1959; 234: 466-468Abstract Full Text PDF PubMed Google Scholar). Fatty acid, cholesterol, and triacylglycerol were separated by TLC on precharred silica H plates (Analtech) using hexane/diethyl ether/acetic acid (60:40:1). The TLC plates were scanned and densitometry was performed with Scion-Image software by reference to standards of oleic acid, cholesterol, and triolein, which were spotted on each TLC plate. Linear regression analysis of the plots of density versus nmol of standard lipid between 0–20 nmol gave r = 0.93–0.995. The mol fraction oleic acid was calculated as mol fatty acid/(mol PL + mol cholesterol + mol triacylglycerol + mol fatty acid). The contribution of the interfacial lysines and glutamates to membrane binding were analyzed using peptides in which the motifs are best represented. The role of the interfacial basic strip was investigated using peptides corresponding to amino acids 236–268 of rat CTα (29Kalmar G.B. Kay R.J. Lachance A. Aebersold R. Cornell R.B. Proc. Natl. Acad. Sci. 1990; 87: 6029-6033Crossref PubMed Scopus (129) Google Scholar). The membrane interactions of the wild-type version of this peptide have been previously characterized (27Johnson J.E. Rao N.M. Hui S.W. Cornell R.B. Biochemistry. 1998; 37: 9509-9519Crossref PubMed Scopus (52) Google Scholar), and its structure in complex with SDS micelles has been solved (25Dunne S.J. Cornell R.B. Johnson J.E. Glover N. Tracey A. Biochemistry. 1996; 35: 11975-11984Crossref PubMed Scopus (91) Google Scholar). It consists of a continuous α-helix between residues 242–268, linked via a ∼50o bend to a loosely coiled N terminus (25Dunne S.J. Cornell R.B. Johnson J.E. Glover N. Tracey A. Biochemistry. 1996; 35: 11975-11984Crossref PubMed Scopus (91) Google Scholar). Fig. 1 B shows the positioning of amino acids that were targeted for mutation. In the mutant peptide 5KQ, five basic amino acids on the right-hand interfacial zone were changed to glutamines: Arg-245, Lys-248, Lys-252, Lys-259, and Lys-266. These residues are generally conserved in animal CTs. Assuming protonation of His-241, the substitutions changed the net charge from +4 to −1. In the mutant peptide 8KQ, an additional three lysines on the opposite face, Lys-250, -254, and -261, were changed to glutamine, to generate a peptide with a net charge of −4. In the wild-type and 8KQ peptide F263 was substituted with tryptophan to facilitate monitoring of fluorescence. The three interfacial glutamates (Glu-257, Glu-268, Glu-279) were mutated to glutamine using the VEEKS subdomain peptide (residues 256–288; Fig. 1 C). The membrane binding behavior and secondary structure of the wild-type version of this peptide have been extensively studied (26Johnson J.E. Cornell R.B. Biochemistry. 1994; 33: 4327-4335Crossref PubMed Scopus (67) Google Scholar, 27Johnson J.E. Rao N.M. Hui S.W. Cornell R.B. Biochemistry. 1998; 37: 9509-9519Crossref PubMed Scopus (52) Google Scholar). The NMR-derived structure of a 22-mer peptide containing the second and third VEEKS repeats in complex with SDS micelles is a continuous α-helix (25Dunne S.J. Cornell R.B. Johnson J.E. Glover N. Tracey A. Biochemistry. 1996; 35: 11975-11984Crossref PubMed Scopus (91) Google Scholar). The 3E to 3Q mutation changes the net charge from −2 to +1. This peptide also contains 6 interfacial basic residues that likely contribute to electrostatic binding. The substitutions did not alter the propensity of the peptides for helix formation, as determined by CD in 50% trifluoroethanol, a solvent that promotes internal H-bonding (data not shown). The helical contents estimated from the molar ellipticity at 222 nm were similar: 60% for Pep-8K, 73% for Pep-5KQ and 56% for Pep-8KQ; 69% for Pep-3E and 62% for Pep-3EQ. All peptides were predominantly random coil in water, except for Pep-8KQ, which adopted mostly β-structure in water (data not shown). The interactions of subdomain N peptides with SUVs were monitored by a direct vesicle-filtration binding assay, by CD, and by fluorescence changes. Thus, the binding event can be correlated with conformational changes and bilayer insertion processes. The progressively weakened response to PG/PC (1:1) lipid vesicles at 22 mm ionic strength upon removal of 5 or 8 interfacial lysines is evidenced by all three methods in the sets of parallel binding curves shown in Fig. 3. Weakened membrane binding (Fig. 3 A) is accompanied by reduced α-helix formation (Fig. 3 B) and by weaker insertion (Fig. 3 C). The bilayer insertion of wild-type peptide and Pep-8KQ was monitored via the blue shift in the fluorescence (330:350 nm) of a tryptophan located in the nonpolar face of the amphipathic helix. This change reached a plateau at a lipid/peptide ratio between 30–50 for the wild-type peptide. By contrast, no change in the fluorescence of Pep-8KQ was observed below a lipid/peptide ratio of 100. For Pep-5KQ, which was not engineered with a tryptophan, we monitored an increase in fluorescence at 304 nm, indicative of the movement of Tyr-240, the lone fluorophore in this peptide, into a more hydrophobic environment. A lipid/peptide ratio of ≥100 was required for maximum change of the F304for Pep-5KQ. These data suggest that both strips of interfacial lysines contribute to peptide binding. In addition they provide the first evidence for membrane insertion of the lone aromatic residue in the bend at the N terminus of domain M. We next probed the electrostatic nature of the interaction of WT, 5KQ, and 8KQ peptides by measuring their partitioning between aqueous and lipid phases as a function of mole percent anionic lipid and medium ionic strength. The effect of vesicle anionic lipid content is shown in Fig. 4 A. The binding of the peptides to pure PC vesicles was too weak to obtain a reliable estimate of their molar partition coefficients, Kx(sensitivity limit of the assay was Kx ≈ 1 × 104). The Kx of the wild-type peptide increased at least 2 orders of magnitude when the mol% PG was increased from 10 to 100% (Fig. 4 A). The response to increasing anionic lipid content was progressively muted upon substitution of the 5 or 8 lysines. This is in keeping with an elimination of the electrostatic component of the binding. The effect of ionic strength is shown in Fig. 4 B. Using 50% PG vesicles, the Kx for the wild-type peptide was reduced ∼50-fold as the ionic strength was raised from 11 mm to 0.75 m. At the lowest ionic strength, su" @default.
• W2000896070 created "2016-06-24" @default.
• W2000896070 creator A5010469481 @default.
• W2000896070 creator A5033402032 @default.
• W2000896070 creator A5042368566 @default.
• W2000896070 creator A5081558769 @default.
• W2000896070 creator A5082299606 @default.
• W2000896070 date "2003-01-01" @default.
• W2000896070 modified "2023-10-06" @default.
• W2000896070 title "Both Acidic and Basic Amino Acids in an Amphitropic Enzyme, CTP:Phosphocholine Cytidylyltransferase, Dictate Its Selectivity for Anionic Membranes" @default.
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