Unique Dimeric Structure of BNip3 Transmembrane Domain Suggests Membrane Permeabilization as a Cell Death Trigger
2007; Elsevier BV; Volume: 282; Issue: 22 Linguagem: Inglês
10.1074/jbc.m701745200
ISSN1083-351X
AutoresEduard V. Bocharov, Yulia Pustovalova, Konstantin V. Pavlov, Pavel E. Volynsky, Marina V. Goncharuk, Yaroslav S. Ermolyuk, Dmitry V. Karpunin, Alexey Schulga, М. П. Кирпичников, Roman G. Efremov, Innokentiy Maslennikov, Alexander S. Arseniev,
Tópico(s)RNA Interference and Gene Delivery
ResumoBNip3 is a prominent representative of apoptotic Bcl-2 proteins with rather unique properties initiating an atypical programmed cell death pathway resembling both necrosis and apoptosis. Many Bcl-2 family proteins modulate the permeability state of the outer mitochondrial membrane by forming homo- and hetero-oligomers. The structure and dynamics of the homodimeric transmembrane domain of BNip3 were investigated with the aid of solution NMR in lipid bicelles and molecular dynamics energy relaxation in an explicit lipid bilayer. The right-handed parallel helix-helix structure of the domain with a hydrogen bond-rich His-Ser node in the middle of the membrane, accessibility of the node for water, and continuous hydrophilic track across the membrane suggest that the domain can provide an ion-conducting pathway through the membrane. Incorporation of the BNip3 transmembrane domain into an artificial lipid bilayer resulted in pH-dependent conductivity increase. A possible biological implication of the findings in relation to triggering necrosis-like cell death by BNip3 is discussed. BNip3 is a prominent representative of apoptotic Bcl-2 proteins with rather unique properties initiating an atypical programmed cell death pathway resembling both necrosis and apoptosis. Many Bcl-2 family proteins modulate the permeability state of the outer mitochondrial membrane by forming homo- and hetero-oligomers. The structure and dynamics of the homodimeric transmembrane domain of BNip3 were investigated with the aid of solution NMR in lipid bicelles and molecular dynamics energy relaxation in an explicit lipid bilayer. The right-handed parallel helix-helix structure of the domain with a hydrogen bond-rich His-Ser node in the middle of the membrane, accessibility of the node for water, and continuous hydrophilic track across the membrane suggest that the domain can provide an ion-conducting pathway through the membrane. Incorporation of the BNip3 transmembrane domain into an artificial lipid bilayer resulted in pH-dependent conductivity increase. A possible biological implication of the findings in relation to triggering necrosis-like cell death by BNip3 is discussed. Mitochondria hold a crucial role in programmed cell death required to control cell development and to maintain homeostasis in multicellular organisms (1.Green D.R. Reed J.S. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar). Mitochondria-mediated cell death is both promoted and suppressed by apoptotic proteins of the Bcl-2 family, most of which contain a C-terminal hydrophobic domain essential for membrane targeting (2.Aouacheria A. Brunet F. Gouy M. Mol. Biol. Evol. 2005; 22: 2395-2416Crossref PubMed Scopus (96) Google Scholar). A major function of Bcl-2 family proteins is to regulate the permeability state of the outer mitochondrial membrane by forming homo- and hetero-oligomers inside the membrane that determine cell fate (3.Harris M.H. Thompson C.B. Cell Death Differ. 2000; 7: 1182-1191Crossref PubMed Scopus (432) Google Scholar, 4.Tsujimoto Y. J. Cell. Physiol. 2003; 195: 158-167Crossref PubMed Scopus (444) Google Scholar, 5.Sharpe J.C. Arnoult D. Youle R.J. Biochim. Biophys. Acta. 2004; 1644: 107-113Crossref PubMed Scopus (338) Google Scholar). The pro-apoptotic protein BNip3 (Bcl-2 Nineteen-kDa interacting protein 3) with a single Bcl-2 homology 3 (BH3) domain is one of the most intensively studied members of the family (6.Chen G. Cizeau J. Vande Velde C. Park J.H. Bozek G. Bolton J. Shi L. Dubik D. Greenberg A. J. Biol. Chem. 1999; 274: 7-10Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). BNip3 and its homologues belong to an independent monophyletic branch with individual evolutionary history (2.Aouacheria A. Brunet F. Gouy M. Mol. Biol. Evol. 2005; 22: 2395-2416Crossref PubMed Scopus (96) Google Scholar) and are essentially different from other BH3-only proteins such as Bid/Bik not only in that they do not require BH3 domain for their function but also because they directly cause changes of mitochondrial potential (7.Shimizu S. Tsujimoto Y. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 577-582Crossref PubMed Scopus (263) Google Scholar). BNip3-induced cell death is independent of caspases and cytochrome c release; it is believed to represent a novel form of programmed cell death, resembling necrosis rather than classical apoptosis (8.Vande Velde C. Cizeau J. Dubik D. Alimonti J. Brown T. Israels S. Hakem R. Greenberg A.H. Mol. Cell. Biol. 2000; 20: 5454-5468Crossref PubMed Scopus (534) Google Scholar).For all cells, loss of nutrient supply represents a potent signal for programmed death. BNip3 plays an important role in hypoxic cell death of normal and malignant cells (9.Lee H. Paik S.G. Mol. Cells. 2006; 28: 1-6Google Scholar). Hypoxia induces expression and accumulation of cytoplasmic or loosely membrane-bound BNip3; however, in order to activate cell death pathway acidosis is required (10.Webster K.A. Graham R.M. Bishopric N.H. J. Mol. Cell. Cardiol. 2005; 38: 35-45Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Transition from respiratory to glycolytic metabolism with increased glucose consumption, lactic acid production, and decrease of cytosolic pH causes redistribution of BNip3 to the outer mitochondrial membrane and integration of homodimeric BNip3 into it, triggering a cell death cascade, which ultimately leads to opening of the mitochondrial permeability transition pore (8.Vande Velde C. Cizeau J. Dubik D. Alimonti J. Brown T. Israels S. Hakem R. Greenberg A.H. Mol. Cell. Biol. 2000; 20: 5454-5468Crossref PubMed Scopus (534) Google Scholar, 10.Webster K.A. Graham R.M. Bishopric N.H. J. Mol. Cell. Cardiol. 2005; 38: 35-45Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). BNip3 inserts into the outer mitochondrial membrane with its N terminus in the cytoplasm and C terminus inside the mitochondria. It was demonstrated that the BNip3 transmembrane domain (TM) 3The abbreviations used are: TM, transmembrane; BNip3tm, TM fragment 146–190 of human pro-apoptotic protein BNip3; GpAtm, TM fragment 61–98 of human protein glycophorin A; BLM, bilayer lipid membrane; MHP, molecular hydrophobic potential; DMPC, dimyristoyl-phosphatidylcholine; DHPC, dihexanoyl-phosphatidylcholine; DPhPC, diphytanoyl-phosphatidylcholine; NOE, nuclear Overhauser effect; MD, molecular dynamics; MES, 4-morpholineethanesulfonic acid; VDAC, voltage-dependent anionic channel.3The abbreviations used are: TM, transmembrane; BNip3tm, TM fragment 146–190 of human pro-apoptotic protein BNip3; GpAtm, TM fragment 61–98 of human protein glycophorin A; BLM, bilayer lipid membrane; MHP, molecular hydrophobic potential; DMPC, dimyristoyl-phosphatidylcholine; DHPC, dihexanoyl-phosphatidylcholine; DPhPC, diphytanoyl-phosphatidylcholine; NOE, nuclear Overhauser effect; MD, molecular dynamics; MES, 4-morpholineethanesulfonic acid; VDAC, voltage-dependent anionic channel. is crucial for pro-apoptotic activity, mitochondrial localization, and homodimerization of the protein, whereas its N-terminal region can be involved in interaction with Bcl-2 proteins (11.Ray R. Chen G. Vande Velde C. Cizeau J. Park J.H. Reed J.C. Gietz R.D. Greenberg A.H. J. Biol. Chem. 2000; 275: 1439-1448Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar, 12.Lamy L. Ticchioni M. Rouquette-Jazdanian A.K. Samson M. Deckert M. Greenberg A.H. Bernard A. J. Biol. Chem. 2003; 278: 23915-23921Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar).As was demonstrated recently, the BNip3 TM segment exists in the form of a tightly associated dimer, in both the detergent and lipid environments (13.Solistijo E.S. Jaszewski T.M. Mackenzie K.R. J. Biol. Chem. 2003; 278: 51950-51956Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). So far, the structure of the transmembrane domain of a representative of the Bcl-2 family in the lipid environment has not been reported. We describe the spatial structure and internal dynamics of the homodimeric TM domain of human pro-apoptotic protein BNip3 in a membrane-mimicking lipid environment obtained with the aid of a combination of NMR spectroscopic technique and molecular dynamics (MD) energy relaxation. The structure suggests a possibility that the BNip3 TM domain alone can form an ion-conducting pathway in the membrane, as supported by direct measurements on the bilayer lipid membranes (BLMs). This property of the TM domain can help to explain the mechanism of BNip3 action, in particular in hypoxia acidosis-induced cell death.EXPERIMENTAL PROCEDURESConstruct Design, Cloning, and Expression−The DNA sequence encoding human BNip3 fragment 146–190 (BNip3tm) was synthesized from six oligonucleotides via PCR. The TrxA-BNip3tm fusion protein was constructed by fusing the gene for thioredoxin of Escherichia coli with an N terminus His tag extension to the gene for BNip3tm in the pGEMEX1 (Promega) vector. To facilitate purification, a His tag and enterokinase cleavage site were placed between the genes for the thioredoxin and BNip3tm fragment. The fusion protein was expressed in E. coli BL21(DE3)pLysS and grown in 2-liter flasks at 37 °C in M9 minimal medium containing (15NH4)2SO4 (CIL) or (15NH4)2SO4/[U-13C]glucose (CIL) for the production of uniformly 15N- or 15N/13C-labeled protein samples. After induction with 50 μm isopropyl-1-thio-β-d-galactopyranoside and an additional 40 h of growth at 13 °C, the cells were harvested and stored at –20 °C.Protein Purification−Cell pellets (1-liter equivalents) were resuspended in 50 ml of lysis buffer (50 mm Tris, pH 8.0, 150 mm NaCl, 10 mm 2-mercaptoethanol, 10 mm imidazole, 15 mm Triton X-100, 0.2 mm phenylmethylsulfonyl fluoride) and lysed by ultrasonication. Centrifugally clarified lysate was applied to chelating Sepharose FF beads (Amersham Biosciences) pre-treated with NiSO4 and eluted with 200 mm imidazole. After overnight incubation with recombinant light chain of human enterokinase, the cleaved BNip3tm fragment was passed through chelating Sepharose FF, loaded onto an SP-Sepharose FF column (Amersham Biosciences), and eluted by gradient of NaCl. Yields of ∼10 mg of protein/liter of cell culture could be obtained by this procedure. Protein concentration was determined by A280 values. Protein identity and purity were confirmed by gel electrophoreses, mass spectrometry, and NMR spectroscopy in a methanol/chloroform (1/1) mixture containing 5–10% of water. The unlabeled human glycophorin A fragment 61–98 (GpAtm), including a GpA TM segment with adjacent N- and C-terminal regions, was obtained in a similar way as the BNip3tm fragment.NMR Spectroscopy and Structure Calculations−Four BNip3tm samples were prepared: uniformly 15N/13C-labeled; 15N-labeled; unlabeled; and a 1:1 mixture of uniformly 15N/13C-labeled and unlabeled protein (“heterodimer” sample). The lyophilized protein was solubilized in the form of an aqueous suspension of dimyristoyl-phosphatidylcholine (DMPC)/dihexanoyl-phosphatidylcholine (DHPC) lipid (Avanti Polar Lipids) bicelles prepared with a lipid molar ratio of 0.25, and then the samples were subjected to several freeze/thaw cycles resulting in uniform protein distribution among the lipid bicelles. To verify the validity of NMR experimental conditions, circular dichroism spectra of BNip3tm in the DMPC/DHPC bicelles and in DMPC monolamellar liposomes (phospholipid bilayer) were recorded. After base-line substraction, the spectra proved virtually identical (for results and experimental procedures, see the supplemental data).NMR experiments were performed on a 600-MHz (1H) Varian Unity spectrometer equipped with a pulsed field gradient unit and triple resonance probe. NMR spectra were acquired at 40 °C using 1-mm samples of BNip3tm incorporated into lipid bicelles (with a lipid/protein molar ratio of 40) dissolved in buffer solution (pH 5.0) containing 20 mm deuterated sodium acetate, 0.15 μm sodium azide, 1 mm EDTA, and either 5 or 99.9% D2O unless otherwise specified.The backbone and side chain 1H, 13C, and 15N resonances of BNip3tm were assigned using standard triple resonance techniques (14.Sattler M. Schleucher J. Griesinger C. Prog. Nucl. Magn. Reson. Spectrosc. 1999; 34: 93-158Abstract Full Text Full Text PDF Scopus (1369) Google Scholar, 15.Cavanagh J. Fairbrother W.J. Palmer A.G. Skelton N.J. Protein NMR Spectroscopy: Principles and Practice.2nd Ed. Academic Press, San Diego, CA2006Google Scholar). Two- and three-dimensional heteronuclear 1H-15N HSQC, 1H-13C HSQC, 15N-edited TOCSY (40-ms mixing time), HNCA, HN(CO)CA, HNCACB, and CBCA(CO)NH spectra in H2O provided backbone and partial side chain assignments, while HCCH-TOCSY (15.6- and 23.4-ms mixing times) and homonuclear 1H NOESY (60-ms mixing time) experiments in D2O facilitated side chain assignments. Resonance assignments were performed with CARA (www.nmr.ch).The values of heteronuclear 15N{1H} steady state NOE, 15N longitudinal (T1), and transverse (T2) relaxation times were obtained for the 15N-labeled sample as described (16.Bocharov E.V. Sobol A.G. Pavlov K.V. Korzhnev D.M. Jaravine V.A. Gudkov A.T. Arseniev A.S. J. Biol. Chem. 2004; 279: 17697-17706Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). The effective rotation correlation times, τR, for the individual 15N nuclei were calculated from a T1/T2 ratio using DASHA (17.Orekhov V.Y. Nolde D.E. Golovanov A.P. Korzhnev D.M. Arseniev A.S. Appl. Magn. Reson. 1995; 9: 581-588Crossref Scopus (80) Google Scholar).For the study of hydrogen bonding and water accessibility to protein groups, slowly exchanging amide protons were identified by reconstituting lyophilized 15N-labeled sample in D2O and recording a series of 1H-15N HSQC spectra over 1 week at 30 °C. In addition, two-dimensional 1H ROESY (50-ms mixing time) and 1H NOESY (80-ms mixing time) spectra were acquired for the unlabeled sample in H2O using the watergate scheme. The water-protein cross-peaks in these spectra were narrowed by suppressing radiation dumping in evolution and mixing periods with the aid of weak (50 mG·cm–1) magnetic field gradients (18.Sklenar V. J. Magn. Res. A. 1995; 114: 132-135Crossref Scopus (143) Google Scholar).NMR spatial structures of the BNip3tm dimer were calculated using CYANA (19.Güntert P. Prog. Nucl. Magn. Res. Spectrosc. 2003; 43: 105-125Abstract Full Text Full Text PDF Scopus (220) Google Scholar). Intramonomeric nuclear Overhauser effect (NOE) distance restraints were identified with CARA through the analysis of three-dimensional 15N- and 13C-edited NOESY experiments (60-ms mixing time) (15.Cavanagh J. Fairbrother W.J. Palmer A.G. Skelton N.J. Protein NMR Spectroscopy: Principles and Practice.2nd Ed. Academic Press, San Diego, CA2006Google Scholar) performed for 15N- and 15N/13C-labeled samples in H2O and D2O, respectively. Two-dimensional 1H NOESY (60-ms mixing time) spectrum acquired for the unlabeled sample was used as an additional source of the structural information concerning aromatic ring protons. Intermonomeric distance restraints were derived from two-dimensional 15N,13C F1-filtered/F3-edited-NOESY and three-dimensional 13C F1-filtered/F3-edited-NOESY spectra (20.Zwahlen C. Legault P. Vincent S.J.F. Greenblatt J. Konrat R. Kay L.E. J. Am. Chem. Soc. 1997; 119: 6711-6721Crossref Scopus (536) Google Scholar) acquired with an 80-ms mixing time for the “heterodimer” sample in H2O and D2O, respectively. Protein-lipid NOE contacts were identified from two-dimensional 15N, 13C F1-filtered/F3-edited-NOESY and three-dimensional 13C F1-filtered/F3-edited-NOESY spectra acquired with an 80-ms mixing time for the 15N/13C-labeled sample. Stereospecific assignments and torsion angle restraints for ϕ, ψ, and χ1 were obtained by the analysis of local conformation in CYANA using sequential NOE data and the available 3JHNα and 3JNβ coupling constants evaluated quantitatively from three-dimensional 1H-15N HNHA and qualitatively from three-dimensional 1H-15N HNHB experiments (15.Cavanagh J. Fairbrother W.J. Palmer A.G. Skelton N.J. Protein NMR Spectroscopy: Principles and Practice.2nd Ed. Academic Press, San Diego, CA2006Google Scholar). Backbone dihedral angle restraints were also estimated based on the assigned chemical shifts using TALOS (21.Cornilescu G. Delaglio F. Bax A. J. Biomol. NMR. 1999; 13: 289-302Crossref PubMed Scopus (2729) Google Scholar). The slowly exchanging amide protons were assigned as hydrogen bond donors with related hydrogen-acceptor partners on the basis of preliminary structure calculations. Corresponding hydrogen bond restraints were employed in subsequent calculations for d(O,N), d(O,HN), d(C,HN) distances according to Ref. (22.Baker E.N. Hubbard R.E. Prog. Biophys. Mol. Biol. 1984; 44: 97-179Crossref PubMed Scopus (1636) Google Scholar). The standard CYANA simulated annealing protocol was applied to 200 random structures, and the resulting 16 structures with the lowest target function were selected. Constrained energy relaxation of the 16 best CYANA structures of the BNip3tm dimer was performed using available distance restraints by MD in the explicit DMPC bilayer.Structure Refinement via MD Energy Relaxation−MD energy relaxation calculations were performed using the GRO-MACS package (23.Lindahl E. Hess B. van der Spoel D. J. Mol. Mod. 2001; 7: 306-317Crossref Google Scholar) at a temperature of 315 K, with a time step of 2 fs, imposed three-dimensional periodic boundary conditions, in the NPT ensemble with an isotropic pressure of 1 bar. A twin range (12/20 Å) spherical cutoff and PME algorithm were used to treat van der Waals and electrostatic interactions, respectively. In total, 16 NMR structures of the BNip3tm dimer with different tautomeric forms of His173 were refined: 12 εHis173-BNip3tm and 4 δHis173-BNip3tm dimers with a protonated Nε or Nδ group of the imidazole ring, respectively. In each case, a 5-ns collection MD run with applied intra- and intermonomeric NOE distance restraints was carried out. Furthermore, two additional MD simulations were performed starting from coordinates of the resulting systems. In the former one, a 5-ns continuation MD run without restraints was carried out in order to check the stability of the dimer. The latter simulation (1-ns MD with NMR distance restraints followed by 5-ns free MD) was done with protonated His173 in one subunit of the δεHis173/εHis173-BNip3tm dimer in order to assess the effect of the ionization state of His173 on the dimeric structure. Equilibrium parts of MD trajectories (last 2 ns) were analyzed using original software developed by the authors and utilities supplied with the GROMACS package. Hydrophobic properties of α-helices were calculated using the molecular hydrophobicity potential approach as described elsewhere (24.Efremov R.G. Vergoten G. J. Phys. Chem. 1995; 99: 10658-10666Crossref Scopus (46) Google Scholar). The resulting structures of the BNip3tm dimer were visualized with MOLMOL (25.Koradi R. Billeter M. Wüthrich K. J. Mol. Graphics. 1996; 14: 51-55Crossref PubMed Scopus (6469) Google Scholar) and PYMOL (www.pymol.org). Other details of MD calculations are described in the supplemental data.Electrochemical Measurements−Bilayers were formed either on 1–1.2-mm aperture in a vertical or on 200-μm aperture in a horizontal teflon partition with the aid of the Mueller-Rudin method. The membranes were formed from a 20-mg/ml solution of diphytanoyl-phosphatidylcholine (DPhPC; Avanti Polar Lipids) in decane. The measurements were performed at 23 °C in 100- or 10-mm KCl solutions containing 1 mm EDTA and buffered with 10 mm Hepes at pH 7.0 or 30 mm MES at pH 4.0. BNip3tm and GpAtm were added either into water solution in the form of solution in Me2SO (1 μg/μl) or directly into the solution of the lipid in decane used for formation of the membranes (to the final concentration of 0.5 μg/μl).For electrical measurements, silver chloride electrodes with standard agar-salt bridges with the total resistance not exceeding 50 kOm were used. The capacitance of BLM was determined from the electric current under applied alternating voltage with the aid of homemade software. The voltage was applied from analog output of a ADC-DAC board (Lcard L780). Conductivity to direct current was determined by applying constant voltage to the membrane for a period significantly exceeding the RC constant of the membrane and recording the current.RESULTSStructure Determination of the BNip3tm Dimer−The 45-residue BNip3 fragment 146–190 (BNip3tm), including the BNip3 TM segment with adjacent N- and C-terminal regions, was solubilized in an aqueous suspension of DMPC/DHPC lipid bicelles, and the standard heteronuclear NMR techniques were applied to determine the spatial structure and to describe the internal dynamics. Directly identified intra- and intermonomeric NOE contacts (a representative strip is shown on Fig. 1B) confirmed the dimeric helical structure of the BNip3 TM domain in the bicelles and demonstrated that the dimer subunits are in a parallel mutual arrangement. The presence of the single cross-peak set in the 1H-15N HSQC spectrum (Fig. 1A) implies that the BNip3tm dimer is symmetrical on the NMR time scale. Therefore, the dihedral angle restraints and both intra- and intermonomeric distance restraints were symmetrically doubled for each dimer subunit that resulted in a dimer with 2-fold symmetry averaged over the ensemble of calculated NMR structures. The full set of input data for NMR structure calculation included 608/28 intra/intermonomeric unambiguous NOE distance restraints, distance restraints for 38 hydrogen bonds, and 162 backbone (ϕ, ψ) and side chain (χ1) dihedral angle restraints. From the observed protein-lipid NOE contacts (Fig. 1C, supplemental Fig. S1) with lipid polar heads and hydrophobic tails we can conclude, similarly to Ref. (26.Fernandez C. Hilty C. Wider H. Wüthrich K. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 13533-13537Crossref PubMed Scopus (114) Google Scholar) where analogous contacts have been analyzed, that the DMPC/DHPC bicelles mimic the embedding of the BNip3 TM domain into a double layer lipid membrane reasonably well. As for the protein-lipid interaction, around the central part of the molecular surface of the BNip3tm dimer we identified many protein-lipid NOE contacts with lipid hydrophobic tails, whereas the protein-lipid NOE contacts with lipid polar heads were found only in the N- and C-terminal parts of the dimer helices. Thus, the dimer spans the hydrophobic phase of the bicelle.The resulting NMR structures of the BNip3tm dimers were subjected to energy relaxation using MD in an explicit hydrated lipid bilayer with the imposed experimentally derived distance restraints. The MD energy relaxation allowed adapting the structure to the lipid environment, with improvement of the quality (distribution of the torsion angles of the side chains, backbone conformation), while the geometry of interaction of the TM fragments remained practically unchanged. Moreover, a 5-ns continuation of MD without NMR restraints did not cause any change of the dimer structure or violations of the restraints. Hence, the obtained structure of the BNip3tm dimer is stable in the membrane. A survey of the structural statistics for the final ensemble of the 16 structures of the BNip3tm dimer (Fig. 2A) is provided in Table 1.FIGURE 2Spatial structure of the BNip3tm dimer. A, ensemble of 16 BNip3tm dimer structures (front and side views) after alignment of the backbone atoms of residues-(166–184)2. Side chain and backbone heavy atom bonds of folded regions-(159–187)2 are shown in magenta and black, respectively. B, ribbon diagram with side chains of the BNip3tm dimer colored according to half-exchange times of backbone amid protons for solvent deuterium. The structural elements are indicated. The lipid head phosphorus atoms and water molecules are presented as green balls and blue V bars, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE 1Structural statistics for the ensemble of 16 best NMR structures of the BNip3tm dimer after MD energy relaxationNMR distance and dihedral restraintsTotal unambiguous NOE restraints608Intra-residue202Inter-residue406Sequential (|i−j| = 1)162Medium range (1 〈|i − j| 〉4)240Long range (| i − j| > 4)4Inter-monomeric NOE28Hydrogen bond restraints (upper/lower)114/114Total torsion angle restraints162Backbone ϕ82Backbone ψ50Side chain χ130Structure calculation statisticsCYANA target function (Å2)0.37 ± 0.06Restraint violationsDistance (>0.2 Å)0Dihedral (>5°)0Average pairwise root mean square deviation (Å)All folded regions-(159-187)2Backbone atoms2.03 ± 0.53All heavy atoms2.83 ± 0.52Stable α-helical region-(166-184)2Backbone atoms0.95 ± 0.30All heavy atoms1.54 ± 0.29Ramachandran analysisaRamachandran statistics were determined using PROCHECK_NMR (48).% Residues in most favored regions85.8% Residues in additional allowed regions12.7% Residues in generously allowed regions0.8bResidues from unfolded and flexible regions.% Residues in disallowed regions0.7bResidues from unfolded and flexible regions.Helix-helix packingLennard-Jones contact energy (kJ/mol)Total-510 ± 100Stable α-helical region-(166-184)2-255 ± 30Contact surface area (Å2)Total650 ± 90Stable α-helical region-(166-184)2375 ± 25Angle θ (°) between the TM helix axes-45 ± 5Distance d (Å) between the TM helix axes5.7 ± 0.5a Ramachandran statistics were determined using PROCHECK_NMR (48.Laskowski R.A. Rullman J.A.C. MacArthur M.W. Kaptein R. Thornton J.M. J. Biomol. NMR. 1996; 8: 477-486Crossref PubMed Scopus (4329) Google Scholar).b Residues from unfolded and flexible regions. Open table in a new tab Tertiary Fold of the BNip3tm Dimer in Lipid Bicelles−Analysis of the calculated spatial structure revealed that the BNip3tm integrated in the lipid bicelles mostly adopted an α-helical structure. The membrane-spanning α-helices cross at the angle θ of –45 ± 5° with the distance d = 5.7 ± 0.5 Å between helix axes and form a right-handed parallel symmetric dimer (Fig. 2) typical for membrane-soluble helical pairs (27.Walters R.F.S. DeGrado W.F. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 13658-13663Crossref PubMed Scopus (223) Google Scholar). For the BNip3tm dimer, the overall rotation correlation time τR, estimated from a T1/T2 ratio and averaged over 15N nuclei with 1H{15N} NOE higher than 0.6, is ∼18 ns (Fig. 3). Based on the empirical dependence (28.Daragan V.A. Mayo K.H. Prog. Nucl. Magn. Reson. Spectrosc. 1997; 31: 63-105Abstract Full Text Full Text PDF Scopus (221) Google Scholar), this τR value corresponds to the effective molecular mass of ∼50 kDa that almost exactly coincides with the size of the DMPC/DHPC bicelle (29.Andersson A. Maler L. Langmuir. 2005; 21: 7702-7709Crossref PubMed Scopus (52) Google Scholar) composed of the BNip3tm dimer surrounded by 80 lipid molecules.FIGURE 3BNip3tm backbone dynamics. A–C, experimental steady-state 15N{1H} NOE, 15N longitudinal T1, and transverse T2 relaxation times for the backbone 15N nuclei of the BNip3tm dimer, respectively. D, effective rotation correlation times, τR, calculated from the T1/T2 ratio. Uncertainties are shown by bars. The BNip3tm sequence is presented below. Broken lines separate the unfolded regions 146–158 and 188–190 from the α-helical regions 159–165, 166–184, and 185–187 regions that have different flexibility.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The 15N{1H} NOE, 15N T1, and T2 values (Fig. 3) measured for the backbone 15N nuclei exhibit significant variations along the protein sequence and are indicative of a stable TM α-helical region (residues 166–184) adjacent to more flexible N- and C-terminal α-helices (residues 159–165 and 185–187, respectively) located in the membrane/water interface. The TM helix has the highest positive 15N{1H} NOEs, which suggests restricted internal mobility for the NH vectors in a pico-nano-second time scale. In contrast, the residues 146–158 and 188–190 from the N- and C termini, respectively, have nearly unrestricted mobility, resulting in low and negative 15N{1H} NOEs and decreased local rotation correlation times τR, estimated from the T1/T2 ratio (Fig. 3). For more detailed discussion of the stable and transitory structures of the BNip3tm dimer see the supplemental data.BNip3tm Dimerization Interface−The TM domain, though generally hydrophobic, contains certain relatively hydrophilic regions. The dimerization interface includes both polar and non-polar residues. A convenient parameter for visualization of spatial distribution of hydrophilic/hydrophobic properties is molecular hydrophobicity potential (MHP) (24.Efremov R.G. Vergoten G. J. Phys. Chem. 1995; 99: 10658-10666Crossref Scopus (46) Google Scholar). On Fig. 4A, contour isolines on a two-dimensional hydrophobicity map for the BNip3tm helices encircle hydrophobic regions with high values of MHP, and the red-hatched area (∼650 Å2) indicates the helix-packing interface of the symmetrical BNip3 dimer. It can be seen that the N-terminal helices interact exclusively through hydrophobic regions (Fig. 4). The dimerization interface on the TM helices is mostly polar with some hydrophobic segments on the outer surface. All the stable intermonomeric contacts (preserved over the MD energy relaxation) in the dimer are observed along the TM helices, whereas the less stable ones are along the mobile N-terminal helices, where the intermonomeric stacking in
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