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Role of Cysteine Residues in Structural Stability and Function of a Transmembrane Helix Bundle

2001; Elsevier BV; Volume: 276; Issue: 42 Linguagem: Inglês

10.1074/jbc.m104006200

ISSN

1083-351X

Autores

Christine B. Karim, Marta Paterlini, Laxma G. Reddy, Gregory W. Hunter, George Bárány, David D. Thomas,

Tópico(s)

Advanced biosensing and bioanalysis techniques

Resumo

To study the structural and functional roles of the cysteine residues at positions 36, 41, and 46 in the transmembrane domain of phospholamban (PLB), we have used Fmoc (N-(9-fluorenyl)methoxycarbonyl) solid-phase peptide synthesis to prepare α-amino-n-butyric acid (Abu)-PLB, the analogue in which all three cysteine residues are replaced by Abu. Whereas previous studies have shown that replacement of the three Cys residues by Ala (producing Ala-PLB) greatly destabilizes the pentameric structure, we hypothesized that replacement of Cys with Abu, which is isosteric to Cys, might preserve the pentameric stability. Therefore, we compared the oligomeric structure (from SDS-polyacrylamide gel electrophoresis) and function (inhibition of the Ca-ATPase in reconstituted membranes) of Abu-PLB with those of synthetic wild-type PLB and Ala-PLB. Molecular modeling provides structural and energetic insight into the different oligomeric stabilities of these molecules. We conclude that 1) the Cys residues of PLB are not necessary for pentamer formation or inhibitory function; 2) the steric properties of cysteine residues in the PLB transmembrane domain contribute substantially to pentameric stability, whereas the polar or chemical properties of the sulfhydryl group play only a minor role; 3) the functional potency of these PLB variants does not correlate with oligomeric stability; and 4) acetylation of the N-terminal methionine has neither a functional nor a structural effect in full-length PLB. To study the structural and functional roles of the cysteine residues at positions 36, 41, and 46 in the transmembrane domain of phospholamban (PLB), we have used Fmoc (N-(9-fluorenyl)methoxycarbonyl) solid-phase peptide synthesis to prepare α-amino-n-butyric acid (Abu)-PLB, the analogue in which all three cysteine residues are replaced by Abu. Whereas previous studies have shown that replacement of the three Cys residues by Ala (producing Ala-PLB) greatly destabilizes the pentameric structure, we hypothesized that replacement of Cys with Abu, which is isosteric to Cys, might preserve the pentameric stability. Therefore, we compared the oligomeric structure (from SDS-polyacrylamide gel electrophoresis) and function (inhibition of the Ca-ATPase in reconstituted membranes) of Abu-PLB with those of synthetic wild-type PLB and Ala-PLB. Molecular modeling provides structural and energetic insight into the different oligomeric stabilities of these molecules. We conclude that 1) the Cys residues of PLB are not necessary for pentamer formation or inhibitory function; 2) the steric properties of cysteine residues in the PLB transmembrane domain contribute substantially to pentameric stability, whereas the polar or chemical properties of the sulfhydryl group play only a minor role; 3) the functional potency of these PLB variants does not correlate with oligomeric stability; and 4) acetylation of the N-terminal methionine has neither a functional nor a structural effect in full-length PLB. phospholamban wild-type replacement of Cys-36, -41, -46 in PLB with alanine α-amino-n-butyric acid dioleoylphosphatidylcholine dioleoylphosphatidylethanolamine 9-fluorenylmethyloxycarbonyl β-nicotinamide adenine dinucleotide polyacrylamide gel electrophoresis sarcoplasmic reticulum Phospholamban (PLB)1 is a 52-residue integral membrane protein that regulates the enzymatic activity of the Ca-ATPase in cardiac sarcoplasmic reticulum (1Lindemann J.P. Jones L.R. Hathaway D.R. Henry B.G. Watanabe A.M. J. Biol. Chem. 1983; 258: 464-471Abstract Full Text PDF PubMed Google Scholar). PLB is predominantly a homopentamer, with a small fraction of monomer, as assayed by SDS-PAGE (2Simmerman H.K.B. Collins J.H. Theibert J.L. Wegener A.D. Jones L.R. J. Biol. Chem. 1986; 261: 13333-13341Abstract Full Text PDF PubMed Google Scholar), electron paramagnetic resonance spectroscopy (3Cornea R.L. Jones L.R. Autry J.M. Thomas D.D. Biochemistry. 1997; 36: 2960-2967Crossref PubMed Scopus (158) Google Scholar, 4Thomas D.D. Reddy L.G. Karim C.B. Li M. Cornea R. Stamm J.D. Ann. N. Y. Acad. Sci. 1998; 853: 186-195Crossref PubMed Scopus (40) Google Scholar, 5Karim C.B. Stamm J.D. Karim J. Jones L.R. Thomas D.D. Biochemistry. 1998; 37: 12074-12081Crossref PubMed Scopus (53) Google Scholar), and fluorescence energy transfer (6Li M. Reddy L.G. Bennett R. Silva N.D. Jones Jr., L.R. Thomas D.D. Biophys. J. 1999; 76: 2587-2599Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 7Reddy L.G. Jones L.R. Thomas D.D. Biochemistry. 1999; 38: 3954-3962Crossref PubMed Scopus (86) Google Scholar). It has been suggested, however, that the less predominant monomeric form of PLB is primarily responsible for inhibition of the Ca-ATPase (3Cornea R.L. Jones L.R. Autry J.M. Thomas D.D. Biochemistry. 1997; 36: 2960-2967Crossref PubMed Scopus (158) Google Scholar, 4Thomas D.D. Reddy L.G. Karim C.B. Li M. Cornea R. Stamm J.D. Ann. N. Y. Acad. Sci. 1998; 853: 186-195Crossref PubMed Scopus (40) Google Scholar,7Reddy L.G. Jones L.R. Thomas D.D. Biochemistry. 1999; 38: 3954-3962Crossref PubMed Scopus (86) Google Scholar, 8Autry J.M. Jones L.R. J. Biol. Chem. 1997; 272: 15872-15880Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 9Kimura Y. Kurzydlowski K. Tada M. MacLennan D.H. J. Biol. Chem. 1997; 272: 15061-15064Abstract Full Text Full Text PDF PubMed Scopus (245) Google Scholar). The residues presumed responsible for stabilizing the pentameric structure of PLB are located in the hydrophobic transmembrane domain (10Wegener A.D. Jones L.R. J. Biol. Chem. 1984; 259: 1834-1841Abstract Full Text PDF PubMed Google Scholar, 11Arkin I.T. Adams P.D. MacKenzie K.R. Lemmon M.A. Brunger A.T. Engelman D.M. EMBO J. 1994; 13: 4757-4764Crossref PubMed Scopus (173) Google Scholar, 12Simmerman H.K.B. Kobayashi Y.M. Autry J.M. Jones L.R. J. Biol. Chem. 1996; 271: 5941-5946Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). These transmembrane domain residues are largely composed of Leu and Ile, but this arrangement is punctuated with three cysteines in a five-residue repeat (Fig. 1). Mutation of these cysteines (at positions 36, 41, and 46 to Ser, Ala, or Phe, respectively) induces changes in the oligomeric stability of PLB (13Fujii J. Maruyamma K. Tada M. MacLennan D.H. J. Biol. Chem. 1989; 264: 12950-12955Abstract Full Text PDF PubMed Google Scholar). The mutation Cys-41 to Phe shows the strongest effect, decreasing the apparent pentameric stability. We have found that Cys-41 is unreactive and is located at a crucial site for the maintenance of the pentameric structure (5Karim C.B. Stamm J.D. Karim J. Jones L.R. Thomas D.D. Biochemistry. 1998; 37: 12074-12081Crossref PubMed Scopus (53) Google Scholar). Based on these results, a structural model for the PLB pentamer has been proposed, in which each pair of subunits is stabilized by interhelical interactions between leucines 37, 44, and 51 with isoleucines 40 and 47 to form a Leu/Ile zipper (5Karim C.B. Stamm J.D. Karim J. Jones L.R. Thomas D.D. Biochemistry. 1998; 37: 12074-12081Crossref PubMed Scopus (53) Google Scholar, 11Arkin I.T. Adams P.D. MacKenzie K.R. Lemmon M.A. Brunger A.T. Engelman D.M. EMBO J. 1994; 13: 4757-4764Crossref PubMed Scopus (173) Google Scholar, 12Simmerman H.K.B. Kobayashi Y.M. Autry J.M. Jones L.R. J. Biol. Chem. 1996; 271: 5941-5946Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). The transmembrane Cys residues do not appear to be involved in intermolecular disulfide bonding (2Simmerman H.K.B. Collins J.H. Theibert J.L. Wegener A.D. Jones L.R. J. Biol. Chem. 1986; 261: 13333-13341Abstract Full Text PDF PubMed Google Scholar, 15Karim C.B. Marquardt C.G. Stamm J.D. Barany G. Thomas D.D. Biochemistry. 2000; 39: 10892-10897Crossref PubMed Scopus (67) Google Scholar). To evaluate the role of the chemical and steric packing properties of the cysteines in the pentameric structure of PLB as well as their function, we have used Fmoc solid-phase peptide synthesis to design a sterically identical PLB derivative. In the present study, we replaced all cysteine residues in PLB with α-amino-n-butyric acid (Abu), which is isosteric to cysteine (16Ferrer M. Woodward C. Barany G. Int. J. Peptide Protein Res. 1992; 40: 194-207Crossref PubMed Scopus (50) Google Scholar). Like many eukaryotic proteins, PLB is “capped” at the N-terminal methionine by posttranslational acetylation (17Polevoda B. Norbeck J. Takakura H. Blomberg A. Sherman F. EMBO J. 1999; 18: 6155-6168Crossref PubMed Scopus (171) Google Scholar). The N-terminal cytoplasmic portion of WT-PLB has a net charge of +3 with the posttranslational acetylation, but this charge increases to +4 in the absence of the acetyl group. It is not known whether this acetyl group in the full-length PLB is necessary for pentameric stabilization and interaction with the Ca-ATPase. No effect of nonacetylated PLB1–25 was reported, and only acetylated peptide Ac-PLB1–25 showed inhibition of the Ca-ATPase (18Starling A.P. Sharma R.P. East J.M. Lee A.G. Biochem. Biophys. Res. Commun. 1996; 226: 352-355Crossref PubMed Scopus (13) Google Scholar). To clarify this subject, we determined the oligomeric states of acetylated and nonacetylated Abu-PLB in detergent solution by SDS-PAGE. The synthetic peptides were then co-reconstituted with the Ca-ATPase in lipid vesicles, and Ca-ATPase inhibition assays were performed in comparison to WT-PLB. Materials, solvents, instrumentation, and general methods of solid-phase peptide synthesis were essentially as described in our previous publications (15Karim C.B. Marquardt C.G. Stamm J.D. Barany G. Thomas D.D. Biochemistry. 2000; 39: 10892-10897Crossref PubMed Scopus (67) Google Scholar, 16Ferrer M. Woodward C. Barany G. Int. J. Peptide Protein Res. 1992; 40: 194-207Crossref PubMed Scopus (50) Google Scholar, 20Barany G. Gross C.M. Ferrer M. Barbar E. Pan H. Woodward C. Marshak D. Techniques in Protein Chemistry VII. Academic Press, San Diego1996: 503-514Google Scholar, 21Kates S.A. Solé N.A. Beyermann M. Barany G. Albericio F. Peptide Res. 1996; 9: 106-113PubMed Google Scholar, 22Han Y. Bontems S.L. Hegyes P. Munson M.C. Minor C.A. Kates S.A. Albericio F. Barany G. J. Org. Chem. 1996; 61: 6326-6339Crossref PubMed Scopus (46) Google Scholar). We used acetic anhydride for the acetylation of the N-terminal amino group (23Tarr G.E. Black S.D. Fujita V.S. Coon M.J. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 6552-6556Crossref PubMed Scopus (140) Google Scholar). First, an Fmoc removal step was carried out on 200 mg of peptide resin (15Karim C.B. Marquardt C.G. Stamm J.D. Barany G. Thomas D.D. Biochemistry. 2000; 39: 10892-10897Crossref PubMed Scopus (67) Google Scholar), followed by treatment with 0.5 m acetic anhydride in 10 ml ofN,N-dimethylformamide. After 2 h, the acetylated peptide resin was filtered and then used for cleavage and purification (15Karim C.B. Marquardt C.G. Stamm J.D. Barany G. Thomas D.D. Biochemistry. 2000; 39: 10892-10897Crossref PubMed Scopus (67) Google Scholar). Fractions containing peptides were lyophilized to yield 26 mg of Abu-PLB (Fig. 1) (12% yield based on starting resin). Edward McKenna (Merck Research Laboratories), who synthesized the protein using an Applied Biosystems 430A synthesizer, graciously provided WT-PLB. WT-PLB was expressed in Sf21/baculovirus insect cell system and purified by monoclonal antibody affinity chromatography as described previously (7Reddy L.G. Jones L.R. Thomas D.D. Biochemistry. 1999; 38: 3954-3962Crossref PubMed Scopus (86) Google Scholar, 12Simmerman H.K.B. Kobayashi Y.M. Autry J.M. Jones L.R. J. Biol. Chem. 1996; 271: 5941-5946Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). SR vesicles were prepared from the fast-twitch skeletal muscle of New Zealand White rabbits (24Fernandez J.L. Rosemblatt M. Hidalgo C. Biochim. Biophys. Acta. 1980; 599: 552-568Crossref PubMed Scopus (113) Google Scholar). The Ca-ATPase from the SR vesicles was purified using a reactive-red affinity column (25Stokes D.L. Green N.M. Biophys. J. 1990; 57: 1-14Abstract Full Text PDF PubMed Scopus (116) Google Scholar). SDS-PAGE was performed using 16.5% Tris/Tricine gel (Bio-Rad) (15Karim C.B. Marquardt C.G. Stamm J.D. Barany G. Thomas D.D. Biochemistry. 2000; 39: 10892-10897Crossref PubMed Scopus (67) Google Scholar). The peptide samples from the stock methanol/chloroform 2:1 solution were dried overnight. 20 µl of 1% SDS was added to the samples that contained 5 µg of Abu-PLB and WT-PLB. For SDS-PAGE, samples contained 20 µl of Tricine sample buffer (26Schagger H. Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10410) Google Scholar) with a final SDS concentration of 1.5%. The temperature was controlled during electrophoresis by using a recirculating water bath. For the quantitation of Abu-PLB and WT-PLB monomers, the gels were scanned by a densitometer using the transmittance mode, and then the bands were quantitated using the volume (area × density) analysis method (27Reddy L.G. Autry J.M. Jones L.R. Thomas D.D. J. Biol. Chem. 1999; 274: 7649-7655Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). Mass spectral data was acquired with a Bruker Biflex III matrix-assisted laser desorption/ionization time of flight system, which is equipped with an N2-laser (337 nm, 3-ns pulse length) and a microchannel plate detector. The data was collected in the linear mode, positive polarity, with an accelerating potential of 19 kV. Each spectrum is the accumulation of 100–400 laser shots. The samples were co-crystallized with the matrix 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid). The method used for the functional reconstitution of Ca-ATPase with PLB has been described (28Reddy L.G. Jones L.R. Cala S.E. O'Brian J.J. Tatulian S.A. Stokes D.L. J. Biol. Chem. 1995; 270: 9390-9397Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). In short, 33 µg of Abu-PLB or its analogues was dried and solubilized in 240 µl of chloroform containing 2.4 mg of lipids (DOPC/DOPE, 4:1). The dried film of lipid and PLB was hydrated with 120 µl of 25 mm imidazole, pH 7.0, by vortexing followed by a brief sonication. The resulting vesicles were diluted to 20 mm imidazole, pH 7.0, 0.1 m KCl, 5 mm MgCl2, 10% glycerol. Then, 4.8 mg of β-octyl glucoside was added, followed by 60 µg of purified Ca-ATPase. The final volume was adjusted to 300 µl with buffer. The detergent was then removed by incubation with 120 mg of hydrated Biobeads for 3 h at room temperature. The Ca-ATPase/PLB lipid vesicles were separated from Biobeads and assayed immediately. All Ca-ATPase/PLB co-reconstitution in the present study used a fixed molar ratio of 10 PLB/Ca-ATPase. As shown previously (27Reddy L.G. Autry J.M. Jones L.R. Thomas D.D. J. Biol. Chem. 1999; 274: 7649-7655Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), this ratio gives substantial effects, comparable to those in cardiac SR. Ca-ATPase activity was measured by an enzyme-linked assay performed in microtiter plates (200 µl total volume in each well) as described previously (15Karim C.B. Marquardt C.G. Stamm J.D. Barany G. Thomas D.D. Biochemistry. 2000; 39: 10892-10897Crossref PubMed Scopus (67) Google Scholar, 29Madden T.D. Chapman D. Quinn P.J. Nature. 1979; 279: 538-541Crossref PubMed Scopus (111) Google Scholar). Each well contained 0.2–0.6 µg of Ca-ATPase (1–3 µl of vesicles) and was added to a buffer containing 50 mmimidazole, pH 7.0, 0.1 m KCl, 5 mmMgCl2, 0.5 mm EGTA, 0.5 mmphosphoenolpyruvate, 2.5 mm ATP, 0.2 mm NADH, 2 IU of pyruvate kinase, 2 IU of lactate dehydrogenase, and 1–2 µg of calcium ionophore (A23187). Each assay was done in triplicate at each of 12 different free calcium concentrations. The absorbance of NADH was monitored at 340 nm to determine the rate of ATP hydrolysis. The assays were performed at 15 and 25 °C in a Thermomax microplate reader (Molecular Devices). Each data point represents average ± S.E. (n ≥ 6). A t test was used to determine the statistical significance of the differences between peptides and the effects of temperature. We started with our previously constructed, experimentally verified model for the PLB transmembrane domain (residues 35–52) (5Karim C.B. Stamm J.D. Karim J. Jones L.R. Thomas D.D. Biochemistry. 1998; 37: 12074-12081Crossref PubMed Scopus (53) Google Scholar). This model was derived from that of Adamset al. (30Adams P.D. Arkin I.T. Engelman D.M. Brunger A.T. Nat. Struct. Biol. 1995; 2: 154-162Crossref PubMed Scopus (194) Google Scholar) (PDB entry 1PSL) by capping the N and C termini with N-acetyl and N′-methylamide groups, respectively, and then rotating each helix counter-clockwise around its axis by about 50° to produce a structure conforming to a leucine-zipper motif (12Simmerman H.K.B. Kobayashi Y.M. Autry J.M. Jones L.R. J. Biol. Chem. 1996; 271: 5941-5946Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar), as supported by cysteine reactivity and site-directed spin labeling (5Karim C.B. Stamm J.D. Karim J. Jones L.R. Thomas D.D. Biochemistry. 1998; 37: 12074-12081Crossref PubMed Scopus (53) Google Scholar). This structure was then used as the starting point for simulations carried out using AMBER 5.0 (31Pearlman D.A. Case D.A. Caldwell J.W. Ross W.S. Cheatham T.E. Debolt S. Ferguson D. Seibel G. Kollman P. Comp. Phys. Commun. 1995; 91: 1-41Crossref Scopus (2604) Google Scholar, 32Case D.A. Pearlman D.A. Caldwell T.E. Cheatham T.E. Ross W.S. Simmerling C.L. Darden T.A. Merz K.M. Stanton R.V. Cheng A.L. Vincent J.J. Crowley M. Ferguson D.M. Radmer R.J. Deibel G.L. Singh U.C. Weiner P.K. Kollman P.A. AMBER5. University of California, San Francisco1997Google Scholar), as recently updated for the peptide backbone parameters (parm96.dat). Before energy minimization, side chain rotamers were generated using the program SQWRL (33Dunbrack R.L. Karplus M. Nat. Struct. Biol. 1994; 1: 334-340Crossref PubMed Scopus (294) Google Scholar) to relieve unfavorable interactions between side chains. Side chains were found to be in the most favorable conformation for an α-helix (33Dunbrack R.L. Karplus M. Nat. Struct. Biol. 1994; 1: 334-340Crossref PubMed Scopus (294) Google Scholar). The template was energy-minimized using a 12-Å nonbonded cutoff, a distance-dependent dielectric constant of 4 r, and a converge criterion of 5 × 10−5 kcal/mol Å in the root mean square of the Cartesian elements of the energy gradient. Positional constraints were imposed on the backbone heavy atoms using a harmonic potential with a force constant of 5 kcal/mol Å2. Minimum energy conformations of the mutants were generated by replacing the side chains while maintaining the same backbone templates as in the wild-type protein. Interhelical energy differences were obtained as ΔEh = Eh –Eh0, where Eh is the nonbonded interchain energy per helix (34Némethy G. Scheraga H.A. Biopolymers. 1989; 28: 1573-1584Crossref PubMed Scopus (16) Google Scholar), andEh0 is the value for WT-PLB. The buried surface area was defined as ΔA = (Ai + Aj) –Aij, where Ai andAj are the surface areas of the individual helicesi and j, and Aij is the surface area of the dimer complex. Values were averaged over the five helix interfaces of the pentamer. Surface areas were generated using GRASP (35Nicholls A. Sharp K. Honig B. Proteins. 1991; 11: 281Crossref PubMed Scopus (5310) Google Scholar) with a probe size of 1.4 Å. Free energies of disassociation were estimated from the buried surface area by considering a penalty of 20 cal mol−1 Å–2 upon loss of interfacing surface (36Fleming K.G. Ackerman A.L. Engelman D.M. J. Mol. Biol. 1997; 272: 266-275Crossref PubMed Scopus (205) Google Scholar). Mass spectroscopy for acetylated Abu-PLB (Fig. 2) yielded anm/z value of 6066, [M + H], which is in agreement with the predicted value of 6066.44 Da. Mass spectral analysis of the free N-terminal Abu-PLB showed a peak at 6025 m/z, [M + H], which corresponded to the calculated molecular mass of 6023.40 Da. Amino acid analysis was also consistent with the expected composition (data not shown). We compared expressed WT-PLB (Fig. 3,lane 1) with synthetic acetylated (lane 2) and nonacetylated Abu-PLB (lane 3) on SDS-PAGE. WT-PLB and Abu-PLB each appears primarily as a 30-kDa pentamer, with a faint band at 6 kDa (monomer). This shows that in SDS solution at 25 °C, the pentamer is just as stable for Abu-PLB as for WT-PLB. Fig.4 shows the effects of synthetic WT-PLB and Abu-PLB on Ca-ATPase activity as a function of Ca2+concentration, measured in reconstituted membranes. Both WT-PLB and Abu-PLB decrease the activity of the Ca-ATPase at pCa below 5.5, resulting in an increase in pKCa (the calcium concentration, in pCa units, required for 50% calcium activation). Abu-PLB shifted pKCa by – 0.27 (control, 6.23 ± 0.02; Abu-PLB, 5.96 ± 0.02), whereas the shift by WT-PLB was –0.20 (6.03 ± 0.03) for this co-reconstitution system. There is a significant difference between the control and Abu-PLB (p = 2.5 E-06) but not between Abu-PLB and WT-PLB (p = 0.2). Thus, Abu-PLB shows similar inhibitory activity to WT-PLB, and Cys residues are not required for inhibition of the Ca-ATPase. To study the function of the acetyl group capping the N terminus, acetylated and nonacetylated Abu-PLB were reconstituted in membranes. Both peptides showed the same increase in pKCa (Fig. 5). There is no significant difference between acetylated and nonacetylated Abu-PLB (p = 0.60). This result shows that the acetyl group plays no role in the inhibition of the Ca-ATPase. Fig.6 shows the inhibitory effects of Ala-PLB and WT-PLB on Ca-ATPase activity. Ala-PLB decreases pKCa by 0.29 (control, 6.31 ± 0.02; Ala-PLB, 6.02 ± 0.02), whereas the decrease from WT-PLB is 0.20 (6.03 ± 0.03). There is a significant difference between the control and Ala-PLB (p = 1.7 E-09) but not between Ala-PLB and WT-PLB (p = 0.6). Table I summarizes the inhibitory potencies, measured as in Figs. 4 and 6, and oligomeric stabilities, measured from densitometry of SDS-PAGE, of the three PLB derivatives at three different temperatures. There was no significant difference in inhibitory potency among the three peptides, nor was there a significant temperature dependence. In contrast, there were substantial differences in oligomeric stability. At all three temperatures, WT-PLB was predominantly pentameric, and Ala-PLB was completely monomeric. At low temperature, Abu-PLB exhibited high pentameric stability, comparable to that of WT-PLB, but the Abu-PLB pentamer was much less stable at 37 °C. Fig.7 shows a comparison of the mobilities of the WT-PLB, Abu-PLB, and Ala-PLB on SDS-PAGE at room temperature. WT-PLB and Abu-PLB showed mobility characteristic of pentamers, whereas Ala-PLB was completely monomeric.Table IInhibitory effects and monomeric fractions of WT-PLB, Abu-PLB, and Ala-PLB, at 12, 25, and 37 °C12 °C25 °C37 °CInhibitionFraction monomerInhibitionFraction monomerInhibitionFraction monomerpKCashift%pKCa shift%pKCa shift%WT-PLB−0.23 ± .0712 ± 7−0.25 ± .0318 ± 5−0.25 ± .0522 ± 5Abu-PLB−0.34 ± .088 ± 5−0.27 ± .0232 ± 41-aSignificantly different from WT-PLB (p < 0.01).−0.30 ± .0478 ± 41-aSignificantly different from WT-PLB (p < 0.01).Ala-PLB−0.25 ± .07100 ± 21-aSignificantly different from WT-PLB (p < 0.01).−0.29 ± .02100 ± 21-aSignificantly different from WT-PLB (p < 0.01).−0.27 ± .04100 ± 21-aSignificantly different from WT-PLB (p < 0.01).1-a Significantly different from WT-PLB (p < 0.01). Open table in a new tab Previous studies have shown that mutations of the three cysteines in PLB to serine, alanine, or phenylalanine disrupts the pentameric structure of PLB, suggesting that the cysteine side chains are crucial for the oligomeric stability of PLB (12Simmerman H.K.B. Kobayashi Y.M. Autry J.M. Jones L.R. J. Biol. Chem. 1996; 271: 5941-5946Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 13Fujii J. Maruyamma K. Tada M. MacLennan D.H. J. Biol. Chem. 1989; 264: 12950-12955Abstract Full Text PDF PubMed Google Scholar). The principal goal of the present study was to determine whether the steric properties of Cys are sufficient for pentameric stability or whether the specific chemical properties of the thiol group are important. Another goal was to clarify the role of the N-terminal acetyl group for pentameric stability and interaction with the Ca-ATPase. We synthesized acetylated and nonacetylated Abu-PLB, an analogue of PLB in which the three Cys residues were replaced with Abu (Fig. 1), an amino acid analogue that is known to be isosteric with cysteine. SDS-PAGE of Abu-PLB indicated that it is primarily pentameric, as is WT-PLB (Fig. 3), indicating clearly that the apparent requirement of the three Cys residues for pentameric stability of PLB is based primarily on steric packing, not on the chemical properties of the thiol groups. N-terminal acetylation of Abu-PLB indicated no difference in the oligomeric stability and inhibitory function. Fig. 8 illustrates a structural model for helix packing in the transmembrane domain of the pentameric WT-PLB (left), Abu-PLB (middle), and Ala-PLB (right), focusing on the role of one of the three Cys residues, Cys-36. Note that the steric structure of the helix interface is indistinguishable for WT-PLB and Abu-PLB, showing excellent matching of the two helical surfaces. In this steric view, Abu-36 substitutes perfectly for Cys-36. However, in Ala-PLB, there is clearly a cavity created due to the smaller side chain of Ala-36, which should result in a loss of interhelical van der Waals stabilization energy. TableII summarizes calculations of predicted energetic properties of these three structures, focusing on the interhelical potential energy. Unlike total energies, interhelical potential energies can be compared between peptides with different sequences and can provide a direct comparison between mutants to estimate relative stability (34Némethy G. Scheraga H.A. Biopolymers. 1989; 28: 1573-1584Crossref PubMed Scopus (16) Google Scholar). We calculated interhelical energies,Eh, as described under “Experimental Procedures,” for the structural models of WT-PLB, Abu-PLB, and Ala-PLB (Fig. 8) by placing the side chains of residues at positions 36, 41, and 46 in the gauche(+) (χ1 = –60°) conformation.Table IIMolecular modeling of oligomeric stabilityWT-PLBAbu-PLBAla-PLBMonomers (%)2-aFrom SDS PAGE (25 °C).1532100ΔEh(kcal/mol)2-bTotal interchain nonbonded interaction energy per helix, relative to WT-PLB (Eh0 = −45.1 ± 1.0 kcal/mol). Calculations were performed with the side chain of Cys-36 and Abu-36 in either the gauche(+) (χ1 = −60 °) ortrans (χ1 = 180 °) conformation. All other side-chain conformations were the same in the two calculations.0.0+0.1 ± 1.0+3.9 ± 1.0Gauche(+) Trans+0.3 ± 1.0+1.3 ± 1.0ΔΔGA(kcal/mol)2-cFree energy differences due to burial of interhelical surface areas. Values are relative to WT-PLB.0.00.0+0.72 ± 0.32-a From SDS PAGE (25 °C).2-b Total interchain nonbonded interaction energy per helix, relative to WT-PLB (Eh0 = −45.1 ± 1.0 kcal/mol). Calculations were performed with the side chain of Cys-36 and Abu-36 in either the gauche(+) (χ1 = −60 °) ortrans (χ1 = 180 °) conformation. All other side-chain conformations were the same in the two calculations.2-c Free energy differences due to burial of interhelical surface areas. Values are relative to WT-PLB. Open table in a new tab As shown in Table II, the decreased pentameric stability (increased percentage of monomer) of Ala-PLB correlates with a calculated loss of energetically favorable interhelical van der Waals interactions, due to the smaller Ala side chain compared with Cys (ΔEh). In contrast, the isosteric Abu and Cys side chains result in isoenergetic interactions. These results mirror the calculated loss in free energy of association (ΔΔG in Table II), as obtained from a loss in the buried interhelical surface area, which is significantly less for Ala-PLB than for either WT-PLB or Abu-PLB. The estimated ΔΔG = 0.72 kcal/mol for an Ala-PLB dimer interface is about 50% of that calculated for the glycophorin mutants L75A and I76A (36Fleming K.G. Ackerman A.L. Engelman D.M. J. Mol. Biol. 1997; 272: 266-275Crossref PubMed Scopus (205) Google Scholar). The smaller values for Ala-PLB reflect the smaller volume change for a Cys to Ala mutation, as compared with a Leu (Ile) to Ala mutation. Whereas the PLB-Ala peptide is mostly monomeric, as measured by SDS-PAGE, the truncated peptide (residues 26–52) is mostly oligomeric (15Karim C.B. Marquardt C.G. Stamm J.D. Barany G. Thomas D.D. Biochemistry. 2000; 39: 10892-10897Crossref PubMed Scopus (67) Google Scholar). The different behavior between full-length and truncated peptide suggest that a decrease in van der Waals interactions upon Cys-to-Ala mutation is not sufficient for depolymerization but that the N-terminal region also contributes to the energetics of the PLB pentamer (15Karim C.B. Marquardt C.G. Stamm J.D. Barany G. Thomas D.D. Biochemistry. 2000; 39: 10892-10897Crossref PubMed Scopus (67) Google Scholar). Although the pentameric stabilities of WT-PLB and Abu-PLB are far more similar than that of Ala-PLB, SDS-PAGE at higher temperatures showed clearly that the WT-PLB pentamer is significantly more stable than Abu-PLB (Table I). This difference must be explained by interactions other than van der Waals, probably involving the specific chemical (polar) properties of the thiol group. Examination of the model structure reveals a possible hydrogen bond between Cys-36 and the backbone oxygen of Leu-37 and between Cys-36 and Cys-41 (Fig.9). These two interactions are absent in the model previously proposed by Arkin et al. (37Arkin I.T. Adams P.D. Bruenger A.T. Aimoto S. Engelman D.M. Smith S.O. J.

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