A pH-dependent Molten Globule Transition Is Required for Activity of the Steroidogenic Acute Regulatory Protein, StAR
2005; Elsevier BV; Volume: 280; Issue: 50 Linguagem: Inglês
10.1074/jbc.m510241200
ISSN1083-351X
AutoresBo Y. Baker, Dustin C. Yaworsky, Walter L. Miller,
Tópico(s)Bacterial Genetics and Biotechnology
ResumoThe steroidogenic acute regulatory protein (StAR) simulates steroid biosynthesis by increasing the flow of cholesterol from the outer mitochondrial membrane (OMM) to the inner membrane. StAR acts exclusively on the OMM, and only StAR's carboxyl-terminal α-helix (C-helix) interacts with membranes. Biophysical studies have shown that StAR becomes a molten globule at acidic pH, but a physiologic role for this structural transition has been controversial. Molecular modeling shows that the C-helix, which forms the floor of the sterol-binding pocket, is stabilized by hydrogen bonding to adjacent loops. Molecular dynamics simulations show that protonation of the C-helix and adjacent loops facilitates opening and closing the sterol-binding pocket. Two disulfide mutants, S100C/S261C (SS) and D106C/A268C (DA), designed to limit the mobility of the C-helix but not disrupt overall conformation, were prepared in bacteria, and their correct folding and positioning of the disulfide bonds was confirmed. The SS mutant lost half, and the DA mutant lost all cholesterol binding capacity and steroidogenic activity with isolated mitochondria in vitro, but full binding and activity was restored to each mutant by disrupting the disulfide bonds with dithiothreitol. These data strongly support the model that StAR activity requires a pH-dependent molten globule transition on the OMM. The steroidogenic acute regulatory protein (StAR) simulates steroid biosynthesis by increasing the flow of cholesterol from the outer mitochondrial membrane (OMM) to the inner membrane. StAR acts exclusively on the OMM, and only StAR's carboxyl-terminal α-helix (C-helix) interacts with membranes. Biophysical studies have shown that StAR becomes a molten globule at acidic pH, but a physiologic role for this structural transition has been controversial. Molecular modeling shows that the C-helix, which forms the floor of the sterol-binding pocket, is stabilized by hydrogen bonding to adjacent loops. Molecular dynamics simulations show that protonation of the C-helix and adjacent loops facilitates opening and closing the sterol-binding pocket. Two disulfide mutants, S100C/S261C (SS) and D106C/A268C (DA), designed to limit the mobility of the C-helix but not disrupt overall conformation, were prepared in bacteria, and their correct folding and positioning of the disulfide bonds was confirmed. The SS mutant lost half, and the DA mutant lost all cholesterol binding capacity and steroidogenic activity with isolated mitochondria in vitro, but full binding and activity was restored to each mutant by disrupting the disulfide bonds with dithiothreitol. These data strongly support the model that StAR activity requires a pH-dependent molten globule transition on the OMM. Molten globules are compact, partially unfolded proteins that retain their secondary structure but have lost some tertiary structure (1Ptitsyn O.B. Adv. Protein. Chem. 1995; 47: 83-229Crossref PubMed Google Scholar); such partially unfolded structures are typically inactive but may be intermediates in membrane insertion (2van der Goot F.G. Gonzalez-Manas J.M. Lakey J.H. Pattus F.A. Nature. 1991; 354: 408-410Crossref PubMed Scopus (416) Google Scholar). Steroidogenic acute regulatory protein (StAR) 3The abbreviations used are: StARsteroidogenic acute regulatorySBPsteroid-binding pocketMDmolecular dynamicsOMMouter mitochondrial membraneIMMinner mitochondrial membraneNBD-cholesterol22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino-23,24-bisnor-5-cholen-3β-olC-helixcarboxyl-terminal α-helixDTTdithiothreitolPBRperipheral benzodiazepine receptorMS/MStandem mass spectrometryLC/MSliquid chromatography/mass spectrometry.3The abbreviations used are: StARsteroidogenic acute regulatorySBPsteroid-binding pocketMDmolecular dynamicsOMMouter mitochondrial membraneIMMinner mitochondrial membraneNBD-cholesterol22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino-23,24-bisnor-5-cholen-3β-olC-helixcarboxyl-terminal α-helixDTTdithiothreitolPBRperipheral benzodiazepine receptorMS/MStandem mass spectrometryLC/MSliquid chromatography/mass spectrometry., which is essential for normal adrenal and gonadal steroidogenesis, facilitates the flow of cholesterol from the outer mitochondrial membrane (OMM) to the inner mitochondrial membrane (IMM), where cholesterol is converted to pregnenolone by the cholesterol side chain cleavage enzyme, P450scc (3Stocco D.M. Clark B.J. Endocr. Rev. 1996; 17: 221-244Crossref PubMed Scopus (926) Google Scholar, 4Miller W.L. Strauss J.F. II I J. Steroid Biochem. Mol. Biol. 1999; 69: 131-141Crossref PubMed Scopus (142) Google Scholar). Mutation of human StAR causes potentially lethal congenital lipoid adrenal hyperplasia (5Lin D. Sugawara T. Strauss J.F. II I Clark B.J. Stocco D.M. Saenger P. Rogol A. Miller W.L. Science. 1995; 267: 1828-1831Crossref PubMed Scopus (852) Google Scholar, 6Tee M.K. Lin D. Sugawara T. Holt J.A. Guiguen Y. Buckingham B. Strauss J.F. II I Miller W.L. Hum. Mol. Genet. 1995; 4: 2299-2305Crossref PubMed Scopus (117) Google Scholar); all missense mutations that cause this disease are found in the carboxyl-terminal 50% of the protein, indicating that these sequences are required for StAR activity (7Bose H.S. Sugawara T. Strauss J.F. II I Miller W.L. N. Engl. J. Med. 1996; 335: 1870-1878Crossref PubMed Scopus (512) Google Scholar, 8Miller W.L. J. Mol. Endocrinol. 1997; 19: 227-240Crossref PubMed Scopus (121) Google Scholar, 9Chen X. Baker B.Y. Abduljabbar M.A. Miller W.L. J. Clin. Endocrinol. Metab. 2005; 90: 835-840Crossref PubMed Scopus (60) Google Scholar). StAR is synthesized as a 37-kDa phosphoprotein with an amino-terminal mitochondrial leader sequence that is cleaved during mitochondrial entry (3Stocco D.M. Clark B.J. Endocr. Rev. 1996; 17: 221-244Crossref PubMed Scopus (926) Google Scholar, 10Granot Z. Geiss-Friedlander R. Melamed-Book N. Eimerl S. Timberg R. Weiss A.M. Hales K.H. Hales D.B. Stocco D.M. Orly J. Mol. Endocrinol. 2003; 17: 2461-2476Crossref PubMed Scopus (75) Google Scholar, 11Bose H.S. Lingappa V.R. Miller W.L. Nature. 2002; 417: 87-91Crossref PubMed Scopus (284) Google Scholar).The mechanism by which StAR moves cholesterol from the OMM to IMM remains unclear. The x-ray crystal structures of two closely related proteins, N-216 MLN64 (12Tsujishita Y. Hurley J.H. Nat. Struct. Biol. 2000; 7: 408-414Crossref PubMed Scopus (436) Google Scholar) and StarD4 (13Romanowski M.J. Soccio R.E. Breslow J.L. Burley S.K. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6949-6954Crossref PubMed Scopus (137) Google Scholar), reveal a β-barrel structure with a hydrophobic sterol-binding pocket (SBP) that will accommodate one cholesterol molecule, although the apparent access channel to the SBP is too small to accommodate a cholesterol molecule. Models of StAR show the same fold (12Tsujishita Y. Hurley J.H. Nat. Struct. Biol. 2000; 7: 408-414Crossref PubMed Scopus (436) Google Scholar, 14Mathieu A.P. Fleury A. Ducharme L. Lavigne P. LeHoux J.G. J. Mol. Endocrinol. 2002; 29: 327-345Crossref PubMed Scopus (68) Google Scholar, 15Yaworsky D.C. Baker B.Y. Bose H.S. Best K.B. Jensen L.B. Bell J.D. Baldwin M.A. Miller W.L. J. Biol. Chem. 2005; 280: 2045-2054Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), suggesting action as a transport protein. Recent data suggest that members of the StarD4 family of related proteins, which lack mitochondrial targeting sequences, serve as cytosolic transporters for insoluble lipid molecules (16Soccio R.E. Adams R.M. Romanowski M.J. Sehayek E. Burley S.K. Breslow J.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6943-6948Crossref PubMed Scopus (151) Google Scholar, 17Soccio R.E. Breslow J.L. J. Biol. Chem. 2003; 278: 22183-22186Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). Furthermore, one molecule of StAR apparently facilitates the mitochondrial import of about 400 molecules of cholesterol (18Artemenko I.P. Zhao D. Hales D.B. Hales K.H. Jefcoate C.R. J. Biol. Chem. 2001; 276: 46583-46596Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar), suggesting a similar mechanism for StAR action. This would suggest that StAR acts in the intramembranous space (12Tsujishita Y. Hurley J.H. Nat. Struct. Biol. 2000; 7: 408-414Crossref PubMed Scopus (436) Google Scholar), but multiple lines of evidence indicate that StAR exerts its action on or in the OMM. First, deletion of 62 amino-terminal residues, including the leader peptide, results in a protein (N-62 StAR) that is confined to the cytoplasm but retains full activity, both in transfected cell systems and in isolated mitochondria in vitro (19Arakane F. Sugawara T. Nishino H. Liu Z. Holt J.A. Pain D. Stocco D.M. Miller W.L. Strauss J.F. II I Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13731-13736Crossref PubMed Scopus (251) Google Scholar, 20Arakane F. Kallen C.B. Watari H. Foster J.A. Sepuri N.B. Pain D. Stayrook S.E. Lewis M. Gerton G.L. Strauss J.F. II I J. Biol. Chem. 1998; 273: 16339-16345Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 21Wang X. Liu Z. Eimerl S. Timberg R. Weiss A.M. Orly J. Stocco D.M. Endocrinology. 1998; 139: 3903-3912Crossref PubMed Scopus (0) Google Scholar). Second, fluorescence resonance energy transfer experiments indicate that StAR directly interacts with synthetic membranes (22Christensen K. Bose H.S. Harris F.M. Miller W.L. Bell J.D. J. Biol. Chem. 2001; 276: 17044-17051Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Third, N-62 StAR promotes cholesterol transfer from synthetic vesicles lacking P450scc to those containing P450scc (23Tuckey R.C. Headlam M.J. Bose H.S. Miller W.L. J. Biol. Chem. 2002; 277: 47123-47128Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Fourth, affixing N-62 StAR to the cytoplasmic aspect of the OMM constitutively activates StAR, whereas localizing N-62 StAR to the intramembranous space or matrix side of IMM ablated all activity (11Bose H.S. Lingappa V.R. Miller W.L. Nature. 2002; 417: 87-91Crossref PubMed Scopus (284) Google Scholar). Fifth, in vitro mitochondrial protein import assays show that constructs that slow mitochondrial entry increase StAR activity and constructs that speed its entry decrease activity (11Bose H.S. Lingappa V.R. Miller W.L. Nature. 2002; 417: 87-91Crossref PubMed Scopus (284) Google Scholar). Thus, all direct experimental evidence indicates that StAR's site of action is the OMM. Finally, synthetic unilamellar vesicles having a lipid composition that models the OMM only protect StAR's carboxyl-terminal α-helix (C-helix) from proteolysis, implying that when StAR interacts with the OMM, most of the protein remains exposed to the cytoplasm (15Yaworsky D.C. Baker B.Y. Bose H.S. Best K.B. Jensen L.B. Bell J.D. Baldwin M.A. Miller W.L. J. Biol. Chem. 2005; 280: 2045-2054Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar).Spectral data and partial proteolysis indicate that StAR undergoes a structural transition to a molten globule at low pH in solution (24Bose H.S. Whittal R.M. Baldwin M.A. Miller W.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7250-7255Crossref PubMed Scopus (195) Google Scholar) and in association with synthetic membranes (22Christensen K. Bose H.S. Harris F.M. Miller W.L. Bell J.D. J. Biol. Chem. 2001; 276: 17044-17051Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 25Song M. Shao H. Mujeeb A. James T.L. Miller W.L. Biochem. J. 2001; 356: 151-158Crossref PubMed Scopus (40) Google Scholar). We have proposed that this conformational change is induced in vivo by StAR's interaction with protonated phospholipid head groups of the OMM and is required for StAR activity (11Bose H.S. Lingappa V.R. Miller W.L. Nature. 2002; 417: 87-91Crossref PubMed Scopus (284) Google Scholar, 22Christensen K. Bose H.S. Harris F.M. Miller W.L. Bell J.D. J. Biol. Chem. 2001; 276: 17044-17051Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 24Bose H.S. Whittal R.M. Baldwin M.A. Miller W.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7250-7255Crossref PubMed Scopus (195) Google Scholar). Consistent with this model, disrupting the mitochondrial proton pump with mCCCP (26King S.R. Liu Z.M. Soh J. Eimerl S. Orly J. Stocco D.M. J. Steroid Biochem. Mol. Biol. 1999; 69: 143-154Crossref PubMed Scopus (53) Google Scholar) or with lipopolysaccharide (27Allen J.A. Diemer T. Janus P. Hales K.H. Hales D.B. Endocrine. 2004; 25: 265-275Crossref PubMed Scopus (111) Google Scholar) abolishes StAR activity, and liposome protection of the C-helix is more complete at pH 4.0 than at pH 6.5 (15Yaworsky D.C. Baker B.Y. Bose H.S. Best K.B. Jensen L.B. Bell J.D. Baldwin M.A. Miller W.L. J. Biol. Chem. 2005; 280: 2045-2054Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). As the available structural (12Tsujishita Y. Hurley J.H. Nat. Struct. Biol. 2000; 7: 408-414Crossref PubMed Scopus (436) Google Scholar, 13Romanowski M.J. Soccio R.E. Breslow J.L. Burley S.K. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6949-6954Crossref PubMed Scopus (137) Google Scholar) and modeling (12Tsujishita Y. Hurley J.H. Nat. Struct. Biol. 2000; 7: 408-414Crossref PubMed Scopus (436) Google Scholar, 14Mathieu A.P. Fleury A. Ducharme L. Lavigne P. LeHoux J.G. J. Mol. Endocrinol. 2002; 29: 327-345Crossref PubMed Scopus (68) Google Scholar, 15Yaworsky D.C. Baker B.Y. Bose H.S. Best K.B. Jensen L.B. Bell J.D. Baldwin M.A. Miller W.L. J. Biol. Chem. 2005; 280: 2045-2054Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) data indicate that no access route to the SBP of StAR is large enough to accommodate a cholesterol molecule, it is apparent that StAR must undergo a conformational change to permit cholesterol to reach the SBP. This conformational change is apparently associated with the spectroscopic changes interpreted as a molten globule (24Bose H.S. Whittal R.M. Baldwin M.A. Miller W.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7250-7255Crossref PubMed Scopus (195) Google Scholar). Nevertheless, the molten globule model of StAR's action has been controversial. Therefore, we have sought direct evidence for the potential involvement of the molten globule structural transition in StAR activity. We now show that the conformational changes that have previously been characterized as a molten globule transition (24Bose H.S. Whittal R.M. Baldwin M.A. Miller W.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7250-7255Crossref PubMed Scopus (195) Google Scholar) are required for StAR activity.MATERIALS AND METHODSMolecular Dynamics (MD)—The parallel MD program AMBER7 using the amber force field ff94 (28Cornell W.D. Cieplak P. Bayly C.I. Gould K.M. Merz D.M. Ferguson D.C. Spellmeyer T. Fox J.W.C. Kollman P.A. J. Am. Chem. Soc. 1995; 117 (Jr.): 5179-5197Crossref Scopus (11437) Google Scholar) plus the modified frcmod. mod_phipsi.2 file (AMBER 8) were used at the University of California San Francisco Computer Graphics Laboratory. Programs were run on the socrates host of a Hewlett-Packard AlphaSever mainframe that includes a 32-processor GS1280 and four 4-processor ES45s. The model of human N-62 StAR (15Yaworsky D.C. Baker B.Y. Bose H.S. Best K.B. Jensen L.B. Bell J.D. Baldwin M.A. Miller W.L. J. Biol. Chem. 2005; 280: 2045-2054Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) was solvated using the pre-equilibrated TIP3P water model in a rectangular box with a minimum solute-wall distance of 10 Å. Images of the model were generated with the program Chimera (45Pettersen E.F. Goddard T.D. Huang C.C. Couch G.S. Greenblatt D.M. Meng E.C. Ferrin T.E. J. Comput. Chem. 2004; 25: 1605-1612Crossref PubMed Scopus (26714) Google Scholar). We used periodic boundary simulations based on the particle mesh Ewald method (AMBER 7), and the nonbonded cutoff, which is used to limit the direct space sum for particle mesh Ewald, was set at default value (8 Å). The system was subjected to a steepest descent energy minimization (1000 steps) with positional restraints on the protein and second minimization (1000 steps) on the whole system. At minimization convergence, the whole system was heated to 300 K over 100 ps, and another 100 ps equilibration was performed under constant pressure with the restraints switched off to allow the water box to relax. The whole system was then subjected to a 3-ns production MD run, (1.5 × 108 steps at 2 fs/step). The system energy, temperature, and pressure were checked before and after each MD run.Protein Preparation—Wild-type full-length StAR DNA cloned into pTWIN1 (New England Biolabs Inc.) (a gift from X. Chen) was modified by deleting the region encoding the first 62 residues and adding an amino-terminal His6 tag fused to the Ssp DnaB intein domain at StAR's amino terminus and then cloned into the SapI and BamHI sites. The carboxyl-terminal Cys-285 residue was mutated to Phe, and the SapI site in the cDNA was mutated to facilitate cloning, yielding pTWIN-N-His6-N-62 StAR. Transformed Escherichia coli BL21(DE3) (New England Biolabs) grown in Luria broth were induced with 0.5 mm isopropyl-β-d-thiogalactopyranoside at 30 °C for 4 h and stored at -80 °C in 20 mm Tris, pH 8.5, 500 mm NaCl. Bacteria were lysed by sonication and cleared of debris, and the supernatant was filtered (0.45 μm) before loading onto chitin-binding columns (New England Biolabs). The columns were prepared, loaded, washed, and eluted according to the manufacturer's suggestions. Protein was collected, dialyzed against 20 mm Tris, pH 7.5, 300 mm NaCl, and concentrated using 15 ml of ultrafiltration cells with YM10MWC membrane (Amicon, Inc). Protein concentration was estimated at A280; the molar extinction coefficient of His6-N-62 StAR is 1.034 mg-1 cm-1 calculated from the amino acid sequence using the ProtParam program, ExPASy. Free sulfhydryl groups were titrated at 412 nm with 5,5′-dithiobis (2-nitrobenzoate) at pH 7.5 in the absence and presence of 2% SDS (Ellman's reagent instructions, Pierce). The N-62 StAR mutants S100C/S261C (SS) and D106C/A268C (DA) were constructed by site-directed mutagenesis, sequenced, and expressed as above.Mass Spectrometry—Bands of purified wild-type, SS, and DA mutant N-62 StAR were excised from SDS-PAGE gels and diced into ∼1 mm3 pieces. Samples were destained twice with 100 μl of 25 mm NH4HCO3 in 50% acetonitrile to eliminate formation of spurious disulfides (29Sechi S. Chait B.T. Anal. Chem. 1998; 70: 5150-5158Crossref PubMed Scopus (355) Google Scholar), evaporated to dryness in a SpeedVac centrifuge, and reacted with 50 μl of 55 mm iodoacetamide in 25 mm NH4HCO3 for 1 h at 20° C in the dark. After removing the supernatant, the gel pieces were evaporated to dryness, digested with 10 μl of 12.5 ng/μl sequencing grade modified trypsin (Promega, Madison, WI), and extracted with 50 μl of 5% formic acid in 50% acetonitrile and then sonicated for 30 min. The supernatant was evaporated nearly to dryness and dissolved in 10 μl of 0.1% formic acid. A 1-μl aliquot was chromatographed on a 1100 nano-high pressure liquid chromatograph (Agilent Technologies, Santa Clara, CA) using a 75 μm × 15 cm, 5-μm particle size C-18 column (Phenomenex, Torrence, CA) and eluted in 0.1% formic acid with a 30-min linear gradient of 5-60% acetonitrile flowing at 5 μl/min. The effluent was routed into a QSTAR (o)-TOF MS (ABI, Foster City, CA) operated in electrospray positive mode. Data were acquired in IDA-mode scanning from 305 to 1400 m/z, low Q1 resolution, 25-V collision voltage, with a time-of-flight m/z range of 50-2000 for the MS/MS acquisition.Spectroscopy—Protein samples were diluted to 0.3 mg/ml in 20 mm Tris-HCl, pH 7.5, and CD spectroscopy was performed at room temperature in a 1-mm path length cuvette in a Jasco 720 spectropolarimeter equipped with a Peltier temperature controller. For pH studies, samples were dialyzed against 20 mm phosphate buffer at the desired pH and clarified at 10,000 × g for 10 min to eliminate aggregates, and the protein concentrations were determined following the CD spectroscopy. Each spectrum represents the average of at least five accumulations with subtraction of the appropriate background. Each experiment was repeated three times, and the spectra from each experiment were averaged and converted to mean residue ellipticity (Θ). Secondary structure compositions were estimated using the three computational tools in CDPro (30Sreerama N. Woody R.W. Anal. Biochem. 2000; 287: 252-260Crossref PubMed Scopus (2473) Google Scholar). Analysis with CONTIN (31Povencher S.W. Glockner J. Biochemistry. 1981; 20: 33-37Crossref PubMed Scopus (1866) Google Scholar) yielded the lowest normalized root mean square deviation score; thus, data are portrayed with this program. Protein samples (0.5 mg/ml) in phosphate-buffered saline were excited at 280 nm, and emission spectra were recorded from 360 to 500 nm on a Spectra Max M2 microplate reader (Molecular Devices). Each experiment was performed three times, and the fluorescence spectra were averaged.Cholesterol Binding—N-62 StAR (0.5 μm) in phosphate-buffered saline was mixed with various amounts of NBD-cholesterol 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino-23,24-bisnor-5-cholen-3β-ol) (Molecular Probes Inc.) in 96-well plates (final volume 200 μl) incubated 15 min at 37 °C, and steady state fluorescence was monitored on a SpectraMax M2 microplate reader. NBD-cholesterol was excited at 460 nm, and emission was recorded at 534 nm. Data were analyzed by non-linear regression using GraphPad Prism version 3.00 plotting NBD-cholesterol concentration versus the relative fluorescence.Steroidogenic Activity—MA-10 cell mitochondria were isolated (11Bose H.S. Lingappa V.R. Miller W.L. Nature. 2002; 417: 87-91Crossref PubMed Scopus (284) Google Scholar), suspended in 0.25 m sucrose, 10 mm HEPES, pH 7.4, and stored at -80 °C. About 1 μg of mitochondrial protein (Bradford method) was incubated with wild-type or mutant N-62 StAR at 37 °C for 60 min in 125 mm KCl, 5 mm MgCl2, 10 mm KH2PO4, 25 mm HEPES, 250 ng/ml trilostane, 100 μm GTP, 10 mm isocitrate with or without 1 mm DTT. Conversion of cholesterol to pregnenolone was determined by radioimmunoassay.RESULTSMolecular Dynamics Simulations—We previously reported a computational model of human N-62 StAR (15Yaworsky D.C. Baker B.Y. Bose H.S. Best K.B. Jensen L.B. Bell J.D. Baldwin M.A. Miller W.L. J. Biol. Chem. 2005; 280: 2045-2054Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) based largely on the x-ray crystal structure of N-216 MLN64 (12Tsujishita Y. Hurley J.H. Nat. Struct. Biol. 2000; 7: 408-414Crossref PubMed Scopus (436) Google Scholar). N-216 MLN64 has 36% sequence identity with StAR (32Moog-Lutz C. Tomasetto C. Regnier C.H. Wendling C. Lutz Y. Muller D. Chenard M.P. Basset P. Rio M.C. Int. J. Cancer. 1997; 71: 183-191Crossref PubMed Scopus (114) Google Scholar), 50-60% of StAR's activity in vitro (33Watari H. Arakane F. Moog-Lutz C. Kallen C.B. Tomasetto C. Gerton G.L. Rio M.C. Baker M.E. Strauss J.F. II I Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8462-8467Crossref PubMed Scopus (204) Google Scholar, 34Bose H.S. Whittal R.M. Huang M.C. Baldwin M.A. Miller W.L. Biochemistry. 2000; 39: 11722-11731Crossref PubMed Scopus (81) Google Scholar), and a tightly folded amino-terminal domain and a more loosely folded carboxyl-terminal domain (34Bose H.S. Whittal R.M. Huang M.C. Baldwin M.A. Miller W.L. Biochemistry. 2000; 39: 11722-11731Crossref PubMed Scopus (81) Google Scholar), similar to StAR (24Bose H.S. Whittal R.M. Baldwin M.A. Miller W.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7250-7255Crossref PubMed Scopus (195) Google Scholar). The model closely resembles the structures of N-216 MLN64 (12Tsujishita Y. Hurley J.H. Nat. Struct. Biol. 2000; 7: 408-414Crossref PubMed Scopus (436) Google Scholar) and StarD4 (13Romanowski M.J. Soccio R.E. Breslow J.L. Burley S.K. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6949-6954Crossref PubMed Scopus (137) Google Scholar), scores favorably in the programs PROCHECK and WHATIF, and has a calculated free energy of -4.3 × 103 kcal/mol, which is similar to the value of -5.9 × 103 kcal/mol calculated based on the coordinates of the N-216 MLN64 crystal structure (Fig. 1A).Models and crystal structures yield rigid representations, analogous to visualizing the protein at 0 K. To visualize the movement of the protein in solution at 300 K, we "hydrated" the model of human N-62 StAR in silico by adding 9631 water molecules as a 73.6 × 71.8 × 77.1 Å box so that there is at least 10 Å between any atom in StAR and the edge of the box. Following energy minimization, the system was heated from 0 to 300 K over 100 ps and equilibrated for 100 ps, and then 3-ns MD runs were performed encompassing 1.5 × 108 molecular steps at 2 fs/step. The output files were written every 500 steps (1 ps) and the trajectory files were saved every 5000 steps, yielding 300 structures for each simulation.MD runs were done under three computational conditions. First, all residues were left in their charged states (the "default" settings of the program) to model pH 7 ("neutral" conditions). Second, all Asp, Glu, and His residues were protonated to model acidic conditions near pH 4 ("acidic" conditions). Third, only the Asp, Glu, and His residues in the C-helix and in the adjacent Ω1 and Ω3 loops were protonated to model a neutral StAR molecule interacting with an acidified membrane ("acidified C-helix" conditions). The total energy under each condition remained stable during the MD runs (Fig. 1B). At neutral pH, the model showed stable trajectories with conserved secondary and tertiary structures, but when acidic conditions were modeled, StAR had a partially opened tertiary structure. The distal end of the carboxyl-terminal helix became unfolded further, and the Ω1 loop and C-helix moved away from each other, opening and closing the SBP. Measurements of the Cα root mean square deviation showed that StAR is more labile under the two acidic conditions (Fig. 1C). The C-helix and Ω1 loop are closely associated; the distances between either of the resonating guanidino nitrogens of Arg-272 and either of the carboxylic oxygens of Asp-106 are 2.8 ± 0.1 Å under neutral conditions, favoring the formation of two hydrogen bonds (Fig. 1D).To visualize the movement of the C-helix, we superimposed images of the molecule obtained every 100 ps, and we plotted the distances between key pairs of atoms during MD (Fig. 2). The distance between the carboxyl carbon (Cγ) of Asp-106 in the Ω1 loop to the guanidino carbon (Cζ) of Arg-272 remained stable at 4 Å under neutral conditions, whereas under either acidic or acidified C-helix conditions, this distance fluctuated dramatically, reaching >10 Å (Fig. 2C, top). This indicates that the C-helix and Ω1 loop move dramatically with respect to one another, opening and closing the SBP. Similar hydrogen bonding was seen between Glu-136 and Arg-274 in the static model at neutral pH. This distance varied from 4 to 6 Å during MD at neutral pH but was relatively more stable under acidic conditions, particularly when only the C-helix was acidified (Fig. 2C, bottom). Thus, acidic pH had little effect (or even a stabilizing effect) on the distance between Arg-274 in the C-helix and Glu-136 in the short α2 helix that contributes to the roof of the SBP. Therefore, cholesterol is more likely to reach the SBP via a space between the Ω1 loop and the C-helix than between the Ω3 loop and the C-helix.FIGURE 2Movement of the StAR molecule during molecular dynamics. A, Cα superposition of conformations from MD snapshots every 100 ps over the 3-ns simulations at neutral (left panel), acidic (middle panel), and acidified C-helix (right panel) conditions. The protein is dynamic, particularly in the distal ends and the loop regions, but the overall structure is well maintained at neutral and acidic pHs. At low pH, conformation transitions are particularly prominent at the Ω1 region. B, interatomic distances (in Å) between Arg-272 and Asp-106 as a function of time during molecular dynamics simulations. The carboxyl carbon (Cγ) of Arg-106 in the Ω1 loop is bonded to two oxygen atoms, termed Oδ1 and Oδ2, and the guanidino carbon (Cζ) of Arg-272 in the C-helix is covalently bonded to two nitrogen atoms, termed Nω1 and Nω2. The distances are plotted between these atoms and shown as follows: in red,Oδ1/Nω2; green,Oδ1/Nω1; black,Oδ2/Nω1; and yellow,Oδ2/Nω2. Under neutral conditions, the Oδ1/Nω2 and Oδ2/Nω1 distances are 2.8 ± 0.1 Å, favoring hydrogen bonding; pairings of Oδ1/Nω1 and Oδ2/Nω2 are less likely. Upper panel, neutral; middle panel, acidic; lower panel, acidified C-helix. C, interatomic distances during MD. Top, distance (in Å) between Cζ in Arg-272 and Cγ in Arg-106. Bottom, distance (in Å) between Cζ in Arg-274 and Cδ in Glu-136. Black, neutral; red, acidic; green, acidified C-helix.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Design of Immobilizing Mutants—MD simulations based on different mathematical parameters might have yielded somewhat different results; therefore, we sought experimental confirmation of our computational results. To test the hypothesis that movement of the C-helix is required for StAR's activity, we sought to immobilize this helix in the position identified by the modeling and by the MD under neutral conditions. We systematically modeled the effects of changing residues in the C-helix and adjacent loops to Cys, seeking pairs in which the side chains were ∼ 4 Å apart, and then mutated them to Cys in silico. We identified two pairs of residues, S100C/S261C (SS) and D106C/A268C (DA), which, when changed to Cys, positioned their sulfhydryl groups to form disulfide bonds without altering the
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