Interactions between HIV-1 gp41 Core and Detergents and Their Implications for Membrane Fusion
2000; Elsevier BV; Volume: 275; Issue: 3 Linguagem: Inglês
10.1074/jbc.275.3.1839
ISSN1083-351X
Autores Tópico(s)Hepatitis C virus research
ResumoThe gp41 envelope protein mediates entry of human immunodeficiency virus type 1 (HIV-1) into the cell by promoting membrane fusion. The crystal structure of a gp41 ectodomain core in its fusion-active state is a six-helix bundle in which a N-terminal trimeric coiled coil is surrounded by three C-terminal outer helices in an antiparallel orientation. Here we demonstrate that the N34(L6)C28 model of the gp41 core is stabilized by interaction with the ionic detergent sodium dodecyl sulfate (SDS) or the nonionic detergentn-octyl-β-d-glucopyranoside (βOG). The high resolution x-ray structures of N34(L6)C28 crystallized from two different detergent micellar media reveal a six-helix bundle conformation very similar to that of the molecule in water. Moreover, N34(L6)C28 adopts a highly α-helical conformation in lipid vesicles. Taken together, these results suggest that the six-helix bundle of the gp41 core displays substantial affinity for lipid bilayers rather than unfolding in the membrane environment. This characteristic may be important for formation of the fusion-active gp41 core structure and close apposition of the viral and cellular membranes for fusion. The gp41 envelope protein mediates entry of human immunodeficiency virus type 1 (HIV-1) into the cell by promoting membrane fusion. The crystal structure of a gp41 ectodomain core in its fusion-active state is a six-helix bundle in which a N-terminal trimeric coiled coil is surrounded by three C-terminal outer helices in an antiparallel orientation. Here we demonstrate that the N34(L6)C28 model of the gp41 core is stabilized by interaction with the ionic detergent sodium dodecyl sulfate (SDS) or the nonionic detergentn-octyl-β-d-glucopyranoside (βOG). The high resolution x-ray structures of N34(L6)C28 crystallized from two different detergent micellar media reveal a six-helix bundle conformation very similar to that of the molecule in water. Moreover, N34(L6)C28 adopts a highly α-helical conformation in lipid vesicles. Taken together, these results suggest that the six-helix bundle of the gp41 core displays substantial affinity for lipid bilayers rather than unfolding in the membrane environment. This characteristic may be important for formation of the fusion-active gp41 core structure and close apposition of the viral and cellular membranes for fusion. hemagglutinin human immunodeficiency virus type 1 sodium dodecyl sulfate n-octyl-β-d-glucopyranoside molar ellipticity at 222 nm circular dichroism high performance liquid chromatography midpoint of thermal denaturation neutral pH phosphate-buffered saline dimyristoylphosphatidylglycerol 1-palmitoyl, 1,2-oleyl phosphatidylcholine Enveloped viruses enter cells by a viral envelope protein-promoted membrane fusion process that mediates penetration of the viral genome into host cells. The mechanism of viral membrane fusion is best understood for the hemagglutinin (HA)1 protein of influenza virus. The labile native (nonfusogenic) structure of HA is transformed, in a "spring-loaded" manner, by acidic pH to an energetically more stable, fusion-active (fusogenic) conformation (1.Carr C.M. Kim P.S. 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Protein dissection studies revealed that two 4,3 hydrophobic (heptad) repeat regions within the gp41 ectodomain form a soluble, α-helical complex consisting of a trimer of antiparallel heterodimers (Fig. 1) (16.Lu M. Blacklow S.C. Kim P.S. Nat. Struct. Biol. 1995; 2: 1075-1082Crossref PubMed Scopus (668) Google Scholar, 17.Lu M. Kim P.S. J. Biomol. Struct. Dyn. 1997; 15: 465-471Crossref PubMed Scopus (157) Google Scholar, 18.Lu M. Ji H. Shen S. J. Virol. 1999; 73: 4433-4438Crossref PubMed Google Scholar). X-ray crystallographic analyses confirmed that this gp41 core is a six-helix bundle (19.Chan D.C. Fass D. Berger J.M. Kim P.S. Cell. 1997; 89: 263-273Abstract Full Text Full Text PDF PubMed Scopus (1833) Google Scholar, 20.Weissenhorn W. Dessen A. Harrison S.C. Skehel J.J. Wiley D.C. Nature. 1997; 387: 426-430Crossref PubMed Scopus (1459) Google Scholar, 21.Tan K. Liu J. Wang J. Shen S. Lu M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12303-12308Crossref PubMed Scopus (518) Google Scholar). Three N-terminal helices form a central, three-stranded coiled coil, while three C-terminal helices pack in the antiparallel manner into conserved hydrophobic grooves on the surface of the coiled-coil trimer. On the basis of these findings and a number of recent studies, it was proposed that this six-helix bundle structure represents the core of fusion-active gp41 (16.Lu M. Blacklow S.C. Kim P.S. Nat. Struct. Biol. 1995; 2: 1075-1082Crossref PubMed Scopus (668) Google Scholar, 19.Chan D.C. Fass D. Berger J.M. Kim P.S. Cell. 1997; 89: 263-273Abstract Full Text Full Text PDF PubMed Scopus (1833) Google Scholar, 20.Weissenhorn W. Dessen A. Harrison S.C. Skehel J.J. Wiley D.C. Nature. 1997; 387: 426-430Crossref PubMed Scopus (1459) Google Scholar, 21.Tan K. Liu J. Wang J. Shen S. Lu M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12303-12308Crossref PubMed Scopus (518) Google Scholar, 22.Judice J.K. Tom J.Y.K. Huang W. Wrin T. Vennari J. Petropoulos C.J. McDowell R.S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13426-13430Crossref PubMed Scopus (142) Google Scholar, 23.Furuta R.A. Wild C.T. Weng Y. Weiss C.D. Nat. Struct. Biol. 1998; 5: 276-279Crossref PubMed Scopus (466) Google Scholar, 24.Munoz-Barroso I. Durell S. Sakaguchi K. Appella E. Blumenthal R. J. Cell Biol. 1988; 140: 315-323Crossref Scopus (269) Google Scholar). Consistent with this view, a monoclonal antibody specifically recognizing the gp41 core binds to the surface of HIV-1 infected cells only after interaction of the envelope protein complex with soluble CD4 (25.Jiang S. Lin K. Lu M. J. Virol. 1998; 72: 10213-10217Crossref PubMed Google Scholar). The structure of the gp41 core resembles the proposed fusion-active conformations of the transmembrane envelope proteins from influenza virus and Moloney murine leukemia virus (2.Bullough P.A. Hughson F.M. Skehel J.J. Wiley D.C. Nature. 1994; 371: 37-43Crossref PubMed Scopus (1370) Google Scholar, 19.Chan D.C. Fass D. Berger J.M. Kim P.S. Cell. 1997; 89: 263-273Abstract Full Text Full Text PDF PubMed Scopus (1833) Google Scholar, 20.Weissenhorn W. Dessen A. Harrison S.C. Skehel J.J. Wiley D.C. Nature. 1997; 387: 426-430Crossref PubMed Scopus (1459) Google Scholar, 21.Tan K. Liu J. Wang J. Shen S. Lu M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12303-12308Crossref PubMed Scopus (518) Google Scholar, 26.Fass D. Harrison S.C. Kim P.S. Nat. Struct. Biol. 1997; 3: 465-469Crossref Scopus (302) Google Scholar). The core of each of the three structures consists of a trimeric coiled coil adjacent to the N-terminal fusion peptide, with three α-helices packed in an antiparallel orientation against the coiled coil. This conserved structure suggests a common theme for viral membrane fusion, notably that formation of the helical-hairpin structure leads to juxtaposition of the virus and cell membranes for fusion (20.Weissenhorn W. Dessen A. Harrison S.C. Skehel J.J. Wiley D.C. Nature. 1997; 387: 426-430Crossref PubMed Scopus (1459) Google Scholar, 23.Furuta R.A. Wild C.T. Weng Y. Weiss C.D. Nat. Struct. Biol. 1998; 5: 276-279Crossref PubMed Scopus (466) Google Scholar, 27.Hughson F.M. Curr. Biol. 1997; 7: R565-R569Abstract Full Text Full Text PDF PubMed Google Scholar). According to this theory, the helical-bundle molecule, a protein, is required to break the energy barrier for fusion of two membranes, which is energetically unfavorable. This model implies that the helical structure interacts with lipid membranes. This notion is supported by electron paramagnetic resonance spectroscopic experiments that indicate that the coiled-coil region adjacent to the fusion peptide region of HA interacts with lipid bilayers only in the fusion-active state (28.Yu Y.G. King D.S. Shin Y.-K. Science. 1994; 266: 274-276Crossref PubMed Scopus (105) Google Scholar). Again, using electron paramagnetic resonance, a peptide corresponding to the N-terminal heptad-repeat region within the gp41 ectodomain of HIV-1 shows membrane binding (29.Rabenstein M. Shin Y.K. Biochemistry. 1995; 34: 13390-13397Crossref PubMed Scopus (89) Google Scholar). These lipid-binding phenomena are postulated to facilitate membrane fusion (28.Yu Y.G. King D.S. Shin Y.-K. Science. 1994; 266: 274-276Crossref PubMed Scopus (105) Google Scholar). The surface of the gp41 core is highly grooved and possesses distinct hydrophilic and hydrophobic regions, features that may be essential for the binding of lipid membranes. Here we show that the N34(L6)C28 model of the gp41 core is stabilized by interaction with the anionic detergent sodium dodecyl sulfate (SDS) or the nonionic detergentn-octyl-β-d-glucopyranoside (βOG). The x-ray structures of N34(L6)C28 crystals grown from these detergent micellar media at resolutions of 2.7 (SDS) and 1.45 Å (βOG) reveal a six-helix bundle conformation that is very similar to that of the molecule in water. Moreover, N34(L6)C28 folds into a fully helical conformation in lipid vesicles. Our results suggest that the six-helix bundle structure can interact with lipid bilayers. This membrane binding may play a role in facilitating both the gp41 conformational change during fusion activation and local apposition of the viral and cellular membranes for fusion. The recombinant N34(L6)C28 model was expressed in Escherichia coli BL21(DE3)/pLysS using the T7 expression system (30.Studier F.W. Rosenberg A.H. Dunn J.J. Dubendorff J.W. Methods Enzymol. 1990; 185: 60-89Crossref PubMed Scopus (5998) Google Scholar) and purified to homogeneity by reverse-phase high performance liquid chromatography (HPLC) as described previously (17.Lu M. Kim P.S. J. Biomol. Struct. Dyn. 1997; 15: 465-471Crossref PubMed Scopus (157) Google Scholar). Protein identity was confirmed by mass spectrometry. Protein concentrations were determined by absorbance at 280 nm in 6m guanidinium hydrochloride, with an extinction coefficient calculated based on tryptophan and tyrosine (31.Edelhoch H. Biochemistry. 1967; 6: 1948-1954Crossref PubMed Scopus (2999) Google Scholar). CD spectra were acquired in 50 mm sodium phosphate, pH 7.0, and 150 mmsodium chloride (PBS) with an Aviv 62 DS spectrometer as described previously (32.Shu W. Ji H. Lu M. Biochemistry. 1999; 38: 5378-5385Crossref PubMed Scopus (53) Google Scholar). The same buffer was also used to prepare micellar solutions of βOG and SDS. The wavelength dependence of molar ellipticity, [θ], was monitored at 20 °C as the average of five scans, using a five-second integration time at 1.0-nm wavelength increments. Spectra were baseline-corrected against the cuvette with buffer alone. Helix content was estimated from the CD signal by dividing the mean residue ellipticity at 222 nm by the value expected for 100% helix formation by helices of comparable size, −33,000 degrees cm2 dmol−1 (33.Chen Y.H. Yang J.T. Chau K.H. Biochemistry. 1974; 13: 3350-3359Crossref PubMed Scopus (1958) Google Scholar). Thermal stability was determined by monitoring the change in CD signal at 222 nm as a function of temperature, and thermal melts were performed in 2-degree intervals with a 2-min equilibration at the desired temperature and an integration time of 30 s. The thermal melts were not reversible, and the protein precipitated after thermal denaturation. The midpoint of the thermal unfolding transition (apparent melting temperature,T m) was determined from the maximum of the first derivative, with respect to the reciprocal of the temperature, of the [θ]222 values (34.Cantor C. Schimmel P. Biophysical Chemistry, Part III. W. H. Freeman Co., New York1980: 1131-1132Google Scholar). The error in estimation ofT m is ± 1 °C. Sedimentation equilibrium analysis was performed on a Beckman XL-A analytical ultracentrifuge as described previously (32.Shu W. Ji H. Lu M. Biochemistry. 1999; 38: 5378-5385Crossref PubMed Scopus (53) Google Scholar). Protein solutions were dialyzed overnight against PBS containing SDS or βOG, loaded at initial concentrations of 10, 40, and 150 μm, and analyzed at rotor speeds of 22 and 25 krpm at 20 °C. Data sets were fitted to a single-species model. Protein partial specific volume and solvent density were calculated with constants from Laue et al. (35.Laue T.M. Shah B.D. Ridgeway T.M. Pelletier S.L. Harding S.E. Rowe A.J. Horton J.C. Analytical Ultracentrifugation in Biochemistry and Polymer Science. Royal Society of Chemistry, Cambridge, United Kingdom1992: 90-125Google Scholar). Molecular weights were all within 10% of those calculated for an ideal trimer, with no systematic deviation of the residuals. Small unilamellar vesicles of dimyristoylphosphatidylglycerol (DMPG) (Sigma) and 1-palmitoyl, 1,2-oleyl phosphatidylcholine (POPE) (Avanti Polar Lipids, Birmingham, AL) were prepared by nitrogen stream evaporation of a 5 mg/ml chloroform solution of the lipid followed by redispersion in PBS (pH 7.0) and sonication in a bath sonicator at 4 °C for 45 min. N34(L6)C28 was crystallized at room temperature by vapor diffusion. A stock of HPLC-purified N34(L6)C28 was dissolved in water, and its final protein concentration was adjusted to 10 mg/ml. Initial crystallization conditions were found by using the hanging-droplet method with sparse matrix crystallization kits (Crystal Screen I and II, Hampton Research, Riverside, CA) and then optimized. Centered cubic crystals with the symmetry of space group I213 were obtained from 0.1m sodium HEPES, pH 7.5, 0.8 m potassium sodium tartrate, and 10 mm SDS. Data to 2.7 Å resolution on the cubic crystals were collected at room temperature at the X-ray Crystallography Facility at the Weill Medical College of Cornell University using an R-axis IV image plate detector mounted on a Rigaku RU200 rotating anode x-ray generator. Primitive rhombohedral crystals with the symmetry of space group R3 were obtained from 0.1 m sodium citrate, pH 5.6, 1.0 mammonium dihydrogen phosphate, and 35 mm βOG. The crystals were transferred to a cryoprotectant solution containing 25% (v/v) glycerol in the corresponding mother liquor. Cryoprotected crystals were frozen in propane before data collection. Data to 1.45 Å resolution were collected at 95 K using a Mar research 300 image plate scanner at the X12B beamline of the National Synchrotron Light Source. All diffraction intensities were integrated and scaled with the HKL suite (36.Otwinowski Z. Minor W. Methods Enzymol. 1996; 276: 307-326Crossref Scopus (38445) Google Scholar). The structures of N34(L6)C28 crystallized in the presence of detergent were determined by molecular replacement by using the program AMoRe (37.Navaza J. Acta Crystallogr. Sect. A. 1994; 50: 157-163Crossref Scopus (5027) Google Scholar). The 2.4 Å structure of N34(L6)C28 (Protein Data Bank code 1STZ) was used in a combined rotation-translation search (using 8.0–3.5 Å data) to yield a solution for N34(L6)C28/SDS (correlation coefficient, 60.9%;R-factor, 45.1%). Density interpretation and model building were done with the program O (38.Jones T.A. Zou J.-Y. Cowan S.W. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13006) Google Scholar). Crystallographic refinement of the structure was done with the program X-PLOR (39.Brünger A.T. X-PLOR Version 3.1: A System for X-ray Crystallography and NMR. Yale University Press, New Haven, CT1992Google Scholar). Prior to refinement, 5% of the diffraction data were set aside for cross-validation (freeR-factor calculation). Noncrystallographic symmetry restraints were not used in the final refinement. Despite the relatively high mean B-factor 42 Å2, the model is generally well defined in the electron density (see Fig.5 A). The final refined model (R cryst= 18.3%; R free = 31.5%) includes 162 of 204 residues in the N34(L6)C28 trimer. No ordered density was observed for SDS. The following regions do not have clear electron density and are presumed to be disordered: residues 546–550 and 653–655 of gp41 and the linker region. All φ and ψ angles are in allowed regions of the Ramachandran plot. Cross-rotation and cross-translation functions (using 10.0–3.0 Å data) by using the 2.4 Å structure of N34(L6)C28 as a search model yielded a solution for N34(L6)C28/βOG (correlation coefficient, 58.2%; R-factor, 43.8%). The model was subjected to rigid body refinement (8.0–2.5 Å) of the whole molecule by using the program X-PLOR (39.Brünger A.T. X-PLOR Version 3.1: A System for X-ray Crystallography and NMR. Yale University Press, New Haven, CT1992Google Scholar). This procedure was followed by least squares minimization of the atomic positions with the program O (38.Jones T.A. Zou J.-Y. Cowan S.W. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13006) Google Scholar). The resulting map was subjected to rounds of automatic tracing and model building by using the program Arp-Warp 5.0 (40.Lamzin V.S. Wilson K.S. Acta Crystallogr. Sect. D. 1993; 49: 129-147Crossref PubMed Google Scholar) and the program REFMAC from the CCP4 program suite (41.CCP4Acta Crystallogr. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (19730) Google Scholar). The refinement was monitored by using the free R-factor. At this stage, the model yielded higher quality of the 2F o − F c andF o − F c electron density maps compared with the starting maps. Noncrystallograhic symmetry restraints were not used in the refinement. Crystallographic refinement of the structure was done with the program X-PLOR (39.Brünger A.T. X-PLOR Version 3.1: A System for X-ray Crystallography and NMR. Yale University Press, New Haven, CT1992Google Scholar). The final refined model (R cryst = 20.0%;R free = 24.5%) contains residues 548–578 and 628–651 of gp41, Gly-5 and Gly-6 of the linker, and 90 water molecules. No ordered density was observed for βOG. Residues 546, 547, 579, and 652–655 of gp41 and Ser-1, Gly-2, Gly-3, and Arg-4 of the linker are not seen in the electron density, and the side chains of Gln-550 and Glu-647, as well as Met-629, are disordered and were thus modeled as serine and alanine, respectively. All φ and ψ angles are within allowed regions. The atomic coordinates have been deposited in the Protein Data Bank (codes 1DF4 and 1DF5). We have previously shown that the recombinant N34(L6)C28 model of the HIV-1 gp41 core forms a six-helix-bundle structure in solution and in crystals and shows a cooperative thermal unfolding transition (18.Lu M. Ji H. Shen S. J. Virol. 1999; 73: 4433-4438Crossref PubMed Google Scholar, 21.Tan K. Liu J. Wang J. Shen S. Lu M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12303-12308Crossref PubMed Scopus (518) Google Scholar). To analyze the conformation of N34(L6)C28 in the hydrophobic environment of the lipid bilayer, we first characterized the physicochemical properties of N34(L6)C28 in the presence of the ionic detergent SDS. SDS micellar system (the critical micellar concentration is 8.2 mm) has been widely used to study peptide-lipid and protein-lipid interactions (42.van de Ven F.J.M. van Os J.W. Wymenga S.S. Remerowski M.L. Konings R.N. Hibers C.W. Biochemistry. 1993; 32: 8322-8328Crossref PubMed Scopus (60) Google Scholar, 43.Roth M. Lewit-Bentley A. Michel H. Deisenhofer J. Huber R. Oesterhelt D. Nature. 1989; 340: 659-662Crossref Scopus (186) Google Scholar, 44.Barber J. Nature. 1989; 340: 601Crossref PubMed Scopus (8) Google Scholar, 45.Hoyt D.W. Gierasch L.M. Biochemistry. 1991; 30: 10155-10163Crossref PubMed Scopus (61) Google Scholar, 46.Gordon L.M. Curtain C.C. Zhong Y.C. Kirkpatrick A. Mobley P.W. Waring A.J. Biochim. Biophys. Acta. 1992; 1139: 257-274Crossref PubMed Scopus (91) Google Scholar, 47.Li S.C. Deber C.M. Nat. Struct. Biol. 1994; 1: 368-373Crossref PubMed Scopus (139) Google Scholar, 48.Rotering B.V. Ohms J.P. Hagenmaier H. Eur. J. Biochem. 1976; 70: 601-610Crossref PubMed Scopus (31) Google Scholar). On the basis of CD measurements at a 10 μm protein concentration in PBS at 20 °C in the presence of 2 and 10 mm SDS, N34(L6)C28 displays a ∼75% α-helical structure, with characteristic double minima in CD spectra at 222 and 208 nm (Fig. 2 A). Under these conditions, N34(L6)C28 exhibits a cooperative melt with an apparent melting temperature (T m) of 88 °C in 2 mm SDS, in contrast to that of 70 °C in aqueous buffer (Fig. 2 B; Table I). Remarkably, N34(L6)C28 has a thermal stability that exceeds 100 °C in 10 mm SDS micelles (Fig. 2 B). Because N34(L6)C28 denatures irreversibly with temperature in all cases,T m values measured by CD spectroscopy are useful as only a qualitative guide to stability. Moreover, sedimentation equilibrium measurements indicate that N34(L6)C28 sediments as a clean trimer in 10 mm SDS micelles (Fig. 2 C; TableI).Table ISummary of circular dichroism and sedimentation equilibrium data for N34(L6)C28 in aqueous and membrane-mimetic mediaMediumConcentration−[θ]222aAll scans and melts were performed at a 10 μm protein concentration.TmaAll scans and melts were performed at a 10 μm protein concentration.Molecular massmmdeg cm 2 dmol −1°CkDaPBS29,6007024.4SDS223,4008826.5SDS1022,800>10025.9βOG3528,600>10023.6DMPG433,800POPE439,100a All scans and melts were performed at a 10 μm protein concentration. Open table in a new tab Taken together, these results indicate that N34(L6)C28 can form a highly stable six-helix bundle in SDS micelles. The lower helicity of N34(L6)C28 in the presence of SDS compared with PBS may be due to the partially unfolded region in the molecule (see below). It is extremely unusual for a small protein motif, in SDS solution that is in excess of its critical micellar concentration, to fold into a stable structure that is similar to that found under native conditions. By extension, the six-helix bundle of the gp41 core likely interacts preferentially with SDS micelles. To determine the general feature of the gp41 core-detergent interaction, we analyzed the conformation of N34(L6)C28 in βOG micelles. βOG is a nonionic detergent with a critical micellar concentration of 25.3 mm. In PBS at a 10 μmprotein concentration in the presence of 35 mm βOG, CD experiments indicate that the folded N34(L6)C28 molecule appears to be ∼90% helical at 20 °C and ∼45% helical at 90 °C (Fig.3 A; Table I). In addition, sedimentation equilibrium experiments indicate that N34(L6)C28 is trimeric in 35 mm βOG micelles (Fig. 3 B; TableI). Thus, N34(L6)C28 folds into a stable six-helix bundle structure in βOG micelles. These results strongly suggest that the fusion-active gp41 core displays substantial affinity for detergents. To further investigate the gp41 core-membrane interaction, we examined the conformation of N34(L6)C28 in DMPG and POPE bilayer vesicles, systems that imitate more closely the structural features of physiologically relevant bilayer membranes (e.g. 49, 50). CD measurements at 10 μm protein and 4 mm lipid concentrations in PBS (pH 7.0) at 20 °C indicate that N34(L6)C28 folds into α-helical structures in both DMPG and POPE vesicles (Fig.4). Because up to 2-fold reductions in ellipticity have been observed in the spectra of membrane-bound proteins (51.Glaeser R.M. Jap B.K. Biochemistry. 1985; 24: 6398-6401Crossref PubMed Scopus (28) Google Scholar, 52.Mao D. Wallace B.A. Anal. Biochem. 1984; 142: 317-328Crossref PubMed Scopus (95) Google Scholar), it is striking that N34(L6)C28 appears to be more helical in lipid environments than that in aqueous solution; the ellipticities of N34(L6)C28 at 222 nm are 30,000, 34,000, and 39,000 degrees cm2/dmol in water, DMPG, and POPE, respectively (Fig. 4). In the light of recent evidence that helix formation for amino acids in lipid micelles/vesicles is qualitatively different from that in water and that hydrophobic interactions between the side chain and lipid govern helix formation in membranes (47.Li S.C. Deber C.M. Nat. Struct. Biol. 1994; 1: 368-373Crossref PubMed Scopus (139) Google Scholar), the hydrophobic residues on the surface of the six-helix bundle appear to contribute to the net extent of helix formation in lipid environments. In addition, the CD spectra of N34(L6)C28 obtained in the DMPG and POPE vesicles differ slightly from that in aqueous solution, probably due to the stronger light-scattering effect of the phospholipid samples, which produce higher noise levels during CD measurements. To evaluate the high resolution structural features of the gp41 core in detergent micelles, we determined the x-ray structure of a new centered cubic crystal form of N34(L6)C28 grown in the presence of 10 mm SDS at 2.7 Å resolution (see under "Experimental Procedures"). This crystal structure, designated N34(L6)C28/SDS, was refined to a conventional R-factor of 18.3% with a freeR-factor of 31.5% and root mean square deviations from ideal bond lengths and bond angles of 0.004 Å and 0.763°, respectively (Table II). Moreover, we determined the 1.45 Å resolution x-ray structure of primitive rhombohedral crystals of N34(L6)C28 grown in 35 mm βOG micelles (see under "Experimental Procedures"). The rhombohedral crystals are isomorphous with those crystallized from aqueous solution. The N34(L6)C28/βOG structure was refined to a conventionalR-factor of 20.0% with a free R-factor of 24.5% and root mean square deviations from ideal bond lengths and bond angles of 0.017 Å and 2.0°, respectively (Table II). Representative portions of the final 2F o − F cmaps with the refined molecular models superimposed are shown in Fig.5. Details of the data collection and refinement statistics are presented in Table II.Table IIX-ray data collection and refinement statisticsDetergentSDSβOGProcessing statistics Space groupI213R3 a = b, c (Å)72.77, 72.7752.38, 60.98 Resolution (Å)30.0–2.720.0–1.45 Measured reflections10,35443,163 Unique reflections1,85911,818 Completeness (%)99.999.0 R merge (%)aRmerge = Σ‖I − ‖/ΣI, where I is the intensity of an individual measurement and is the mean recorded intensity over multiple recordings.4.62.8Refinement statistics Resolution (Å)8.0–2.716.5–1.45 Protein nonhydrogen atoms555465 Water molecules90 R free (%)bR = Σ‖Fobs −F calc‖/ΣFobs, where R free is calculated for a randomly chosen 5
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