Cross-talk in the A1-ATPase from Methanosarcina mazei Gö1 Due to Nucleotide Binding

Artigo Acesso aberto Revisado por pares

Cross-talk in the A1-ATPase from Methanosarcina mazei Gö1 Due to Nucleotide Binding

2002; Elsevier BV; Volume: 277; Issue: 19 Linguagem: Inglês

10.1074/jbc.m110407200

ISSN

1083-351X

Autores

Ünal Coskun, Gerhard Grüber, Michel H. J. Koch, Jasminka Godovac‐Zimmermann, Thorsten Lemker, Volker Müller,

Tópico(s)

Photosynthetic Processes and Mechanisms

Resumo

Changes in the A3B3CDF-complex of theMethanosarcina mazei Gö1 A1-ATPase in response to ligand binding have been studied by small-angle x-ray scattering, protease digestion, fluorescence spectroscopy, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, and CuCl2-induced disulfide formation. The value of the radius of gyration, R g, increases slightly when MgATP, MgADP, or MgADP + Pi (but not MgAMP-PNP) is present. The nucleotide-binding subunits A and B were reacted withN-4[4-[7-(dimethylamino)-4-methyl]coumarin-3-yl]maleimide, and spectral shifts and changes in fluorescence intensity were detected upon addition of MgAMP-PNP, MgATP, MgADP + Pi, or MgADP. Trypsin treatment of A1 resulted in cleavage of the stalk subunits C and F, which was rapid in the presence of MgAMP-PNP but slow when MgATP or MgADP were added to the enzyme. When A1 was supplemented with CuCl2 a clear nucleotide dependence of an A-A-D cross-linking product was generated in the presence of MgADP and MgATP but not when MgAMP-PNP or MgADP + Pi was added. The site of cross-link formation was located in the region of the N and C termini of subunit D. The data suggest that the stalk subunits C, D, and F in A1 undergo conformational changes during ATP hydrolysis. Changes in the A3B3CDF-complex of theMethanosarcina mazei Gö1 A1-ATPase in response to ligand binding have been studied by small-angle x-ray scattering, protease digestion, fluorescence spectroscopy, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, and CuCl2-induced disulfide formation. The value of the radius of gyration, R g, increases slightly when MgATP, MgADP, or MgADP + Pi (but not MgAMP-PNP) is present. The nucleotide-binding subunits A and B were reacted withN-4[4-[7-(dimethylamino)-4-methyl]coumarin-3-yl]maleimide, and spectral shifts and changes in fluorescence intensity were detected upon addition of MgAMP-PNP, MgATP, MgADP + Pi, or MgADP. Trypsin treatment of A1 resulted in cleavage of the stalk subunits C and F, which was rapid in the presence of MgAMP-PNP but slow when MgATP or MgADP were added to the enzyme. When A1 was supplemented with CuCl2 a clear nucleotide dependence of an A-A-D cross-linking product was generated in the presence of MgADP and MgATP but not when MgAMP-PNP or MgADP + Pi was added. The site of cross-link formation was located in the region of the N and C termini of subunit D. The data suggest that the stalk subunits C, D, and F in A1 undergo conformational changes during ATP hydrolysis. The membrane-integrated archaeal A1AO-ATPase (A3B3CDEFGHIKx, the exact stoichiometry of the subunits is unknown), like the bacterial F1FO-ATPase (α3β3γδεabcx) and the eucaryotic V1VO-ATPase (A3B3CDEFGxHyaczde), possesses an extrinsic domain (A1), containing the catalytic sites, and an intrinsic domain (AO), involved in ion translocation (1.Perzov N. Padler-Karavani V. Nelson H. Nelson N. FEBS Lett. 2001; 504: 223-228Crossref PubMed Scopus (25) Google Scholar, 2.Schäfer G. Engelhard M. Müller V. Mol. Biol. Rev. 1999; 63: 570-620Crossref PubMed Google Scholar, 3.Grüber G. Wieczorek H. Harvey W.R. Müller V. J. Exp. Biol. 2001; 204: 2597-2605Crossref PubMed Google Scholar). The primary structure of the archaeal ATPase is very similar to that of the V-ATPase, but its function, as an ATP-synthase, is more similar to that of F-ATPases (2.Schäfer G. Engelhard M. Müller V. Mol. Biol. Rev. 1999; 63: 570-620Crossref PubMed Google Scholar, 4.Gogarten J.P. Trends Ecol. Evol. 1995; 10: 147-151Abstract Full Text PDF PubMed Scopus (50) Google Scholar, 5.Hilario E. Gogarten J.P. J. Mol. Evol. 1998; 46: 703-715Crossref PubMed Scopus (68) Google Scholar, 6.Marin I. Fares M.A. Gonzáles-Candelas F. Barrio E. Moya A. J. Mol. Evol. 2001; 52: 17-28Crossref PubMed Scopus (15) Google Scholar). Electron microscopy has shown that the major nucleotide-binding subunits A and B of the A1/V1 and the corresponding β and α subunits of the F1 form an alternating hexagonal arrangement (7.Lübben M. Lünsdorf H. Schäfer G. Biol. Chem. Hoppe-Seyler. 1988; 369: 1259-1266Crossref PubMed Scopus (32) Google Scholar, 8.Radermacher M. Ruiz T. Harvey W.R. Wieczorek H. Grüber G. FEBS Lett. 1999; 453: 383-386Crossref PubMed Scopus (25) Google Scholar, 9.Gogol E.P. Lücken U. Bork T. Capaldi R.A. Biochemistry. 1989; 28: 4709-4716Crossref PubMed Scopus (72) Google Scholar) around a central mass (10.Grüber G. Radermacher M. Ruiz T. Godovac-Zimmermann J. Canas B. Kleine-Kohlbrecher D. Huss M. Harvey W.R. Wieczorek H. Biochemistry. 2000; 39: 8609-8616Crossref PubMed Scopus (69) Google Scholar, 11.Gogol E.P. Aggeler R. Sagermann M. Capaldi R.A. Biochemistry. 1989; 28: 4717-4724Crossref PubMed Scopus (68) Google Scholar). The hexameric headpiece is attached to the AO/FO/VO part by at least one stalk (3.Grüber G. Wieczorek H. Harvey W.R. Müller V. J. Exp. Biol. 2001; 204: 2597-2605Crossref PubMed Google Scholar). Recent three-dimensional structures of F1 (12.Gibbons C. Montgomery M.G. Leslie A.G.W. Walker J.E. Nat. Struct. Biol. 2000; 7: 1055-1061Crossref PubMed Scopus (437) Google Scholar) and V1 (13.Radermacher M. Ruiz T. Wieczorek H. Grüber G. J. Struct. Biol. 2001; 135: 26-37Crossref PubMed Scopus (94) Google Scholar) confirm these features and show the stalk part extending from and therefore partly composed of F1(γ-ε; mitochondrial subunit nomenclature (12.Gibbons C. Montgomery M.G. Leslie A.G.W. Walker J.E. Nat. Struct. Biol. 2000; 7: 1055-1061Crossref PubMed Scopus (437) Google Scholar)) and V1subunits (C–H). The A3B3CDF complex of the Methanosarcina mazei Gö1 A1-ATPase, which is investigated here, consists of an ∼96-Å long headpiece and an 84-Å high and 60-Å diameter stalk as shown by small-angle x-ray scattering (14.Grüber G. Svergun D.I. Coskun Ü. Lemker T. Koch M.H.J. Schägger H. Müller V. Biochemistry. 2000; 40: 1890-1896Crossref Scopus (42) Google Scholar). A comparison of the central stalk of this A1 complex with the F1- and V1-ATPase indicates different shapes and lengths of these domains (3.Grüber G. Wieczorek H. Harvey W.R. Müller V. J. Exp. Biol. 2001; 204: 2597-2605Crossref PubMed Google Scholar, 14.Grüber G. Svergun D.I. Coskun Ü. Lemker T. Koch M.H.J. Schägger H. Müller V. Biochemistry. 2000; 40: 1890-1896Crossref Scopus (42) Google Scholar) which account for linking catalytic events in the headpiece with ion pumping through the membrane portion. In particular, the F1-ATPase has a significantly shorter stalk than A1, ∼40–45 Å long and 50–53 Å wide (12.Gibbons C. Montgomery M.G. Leslie A.G.W. Walker J.E. Nat. Struct. Biol. 2000; 7: 1055-1061Crossref PubMed Scopus (437) Google Scholar, 15.Grüber G. J. Bioenerg. Biomembr. 2000; 32: 341-346Crossref PubMed Scopus (16) Google Scholar). The prevailing view is, however, that ATP hydrolysis in the A1 headpiece is coupled to ion flow in AOthrough movements of the central stalk subunit(s) (2.Schäfer G. Engelhard M. Müller V. Mol. Biol. Rev. 1999; 63: 570-620Crossref PubMed Google Scholar) as visualized by optical microscopy for F1 by fixing this enzyme on a surface and attaching either an actin filament or a bead to the γ subunit to mark its orientation (reviewed in Ref. 16.Yoshida M. Muneyuki E. Hisabori T. Nat. Rev. Mol. Cell. Biol. 2001; 2: 669-677Crossref PubMed Scopus (723) Google Scholar). Although the energy-transducing mechanism of A-ATPase is thought to be similar to that of the F-ATPase, evidence for structural alterations during coupling in the A-ATPase has been lacking. Here we describe this phenomenon in the A1-ATPase by a variety of biophysical and biochemical methods. Altered overall dimensions of the A1complex, fluorescence changes, trypsin susceptibility, and CuCl2-induced cross-link formation between various subunits of A1 are discussed in the light of different ligand-dependent states. All chemicals were at least of analytical grade and were obtained from Biomol (Hamburg, Germany), Merck, Promega (Madison, WI), Sigma, or Serva (Heidelberg, Germany). The A1-ATPase from M. mazei Gö1 was obtained from Escherichia coli strain DK8 expressing the A1-ATPase genes A–G on a multicopy vector pTL2 (17.Lemker T. Ruppert C. Stöger H. Wimmers S. Müller V. Eur. J. Biochem. 2001; 268: 3744-3750Crossref PubMed Scopus (23) Google Scholar). The enzyme was isolated by gel permeation chromatography followed by ion exchange chromatography, as will be described elsewhere. 1T. Lemker and V. Müller, manuscript in preparation. For solution x-ray scattering experiments (see below) the enzyme was subsequently applied onto a Sephacryl S-300 HR column (10/30, Amersham Biosciences) equilibrated in 50 mm Tris-HCl (pH 6.9), 150 mmNaCl and subjected to gel permeation chromatography (FPLC) 2The abbreviations used are: FPLCfast protein liquid chromatographyCMN-[4-[7-(dimethylamino)-4-methyl]coumarin-3-yl)]maleimideAMP-PNPadenosine 5′-(β,γ-imino)triphosphateMALDI-TOFmatrix-assisted laser desorption ionization time-of-flightDTTdithiothreitolSAXSby small-angle x-ray scattering in order to isolate a homogeneous and nucleotide-depleted A1 complex (14.Grüber G. Svergun D.I. Coskun Ü. Lemker T. Koch M.H.J. Schägger H. Müller V. Biochemistry. 2000; 40: 1890-1896Crossref Scopus (42) Google Scholar). The purity and homogeneity of the protein sample was analyzed by Native-PAGE (18.Schägger H. von Jagow G. Anal. Biochem. 1991; 199: 223-231Crossref PubMed Scopus (1918) Google Scholar) and SDS-PAGE (19.Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar). SDS gels were stained with Coomassie Brilliant Blue G-250. Protein concentrations were determined according to Lowry et al. (20.Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). ATPase activity was measured as described previously (14.Grüber G. Svergun D.I. Coskun Ü. Lemker T. Koch M.H.J. Schägger H. Müller V. Biochemistry. 2000; 40: 1890-1896Crossref Scopus (42) Google Scholar, 21.Heinonen J.K. Lahti R.J. Anal. Biochem. 1981; 113: 313-317Crossref PubMed Scopus (789) Google Scholar). fast protein liquid chromatography N-[4-[7-(dimethylamino)-4-methyl]coumarin-3-yl)]maleimide adenosine 5′-(β,γ-imino)triphosphate matrix-assisted laser desorption ionization time-of-flight dithiothreitol by small-angle x-ray scattering Bound nucleotides were removed by passing the A1 complex through a size exclusion column (Superdex 200 HR (10/30), Amersham Biosciences) equilibrated in 20 mm Tris-HCl (pH 7.4) and 150 mm NaCl. After preincubation of the enzyme with 5 mm nucleotide for 5 min, cross-linking was induced by supplementation with 2 mm CuCl2 in a buffer containing 20 mm Tris-HCl (pH 7.4) and 150 mmNaCl on a rotary shaker (450 rpm) at 4 °C for 30 min. The cross-linking reaction was stopped by addition of 10 mmEDTA, subsequently dissolved in DTT-free dissociation buffer, and applied to an SDS-polyacrylamide gel as described above. The subunits involved in cross-linking were identified by Western blotting (22.Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44939) Google Scholar) using antisera against subunit A and B as described previously (17.Lemker T. Ruppert C. Stöger H. Wimmers S. Müller V. Eur. J. Biochem. 2001; 268: 3744-3750Crossref PubMed Scopus (23) Google Scholar) and mass spectrometric analysis. For the latter the cross-linked bands were cut out from the SDS-polyacrylamide gel and destained with a solution of 25 mm ammonium bicarbonate and 50% acetonitrile for 12 h. The gel band was cut into pieces of 1 mm3, which were washed three times with acetonitrile, dried for 30 min in a speed-vacuum concentrator, and digested according to a procedure modified from Hellmann et al. (23.Hellman U. Wernstedt C. Gonez J. Heldin C.-H. Anal. Biochem. 1995; 224: 451-455Crossref PubMed Scopus (687) Google Scholar) and Roos et al. (24.Roos M. Soskic V. Poznanovic S. Godovac-Zimmermann J. J. Biol. Chem. 1998; 273: 924-931Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). For MALDI mass spectrometry, aliquots of 0.5 μl of the digested solution were applied to a target disc and allowed to dry in the air. Subsequently, 0.5 μl of matrix solution (1% w/v α-cyano-4-hydroxycinnamic acid in 50% acetonitrile, 0.1% (v/v) trifluoroacetic acid) was applied to the dried sample and also allowed to dry. Spectra were obtained using a Bruker Biflex III MALDI-TOF mass spectrometer. The protein fragments were identified using programs from the University of California, San Francisco (rafael.ucsf.edu/cgi-bin/msfit), the ProFound program of Rockefeller University (prowl.rockefeller.edu/cgi-bin/ProFound), the PepSearch program of the EMBL in Heidelberg (www.mann.embl-heidelberg.de/Services/PeptideSearch/FR_peptideSearchForm.html), and TagIdent available on the ExPASy WWW server. Before labeling withN-[4-[7-(dimethylamino)-4-methyl]coumarin-3-yl)]maleimide (CM), A1 was depleted of nucleotides as described above. The enzyme was labeled with 50 μm CM for 10 min in 20 mm Tris-HCl (pH 6.9) and 150 mm NaCl (buffer A). Excess label was removed by one pass through a Sephadex G-25 spin column, equilibrated in buffer A. Fluorescence emission spectra of CM-bound A1 in the presence and absence of 5 mmof different nucleotides were recorded at 10 °C using an SLM-Aminco 8100 spectrofluorimeter. Protein samples were excited at 365 nm, and the emission was recorded from 410 to 560 nm with excitation and emission bandpasses set to 4 nm. In order to determine the amount of hydrolyzed MgATP after the fluorescent measurement, a 200-μl portion of the sample was denatured with 20 μl of 14% perchloric acid and cooled on ice for 15 min. The denatured protein was then pelleted by centrifugation at 5000 rpm for 2 min (25.Bullough D.A. Brown E.L. Saario J.D. Allison W.S. J. Biol. Chem. 1988; 263: 14053-14060Abstract Full Text PDF PubMed Google Scholar, 26.Grüber G. Hausrath A. Sagermann M. Capaldi R.A. FEBS Lett. 1997; 410: 165-168Crossref PubMed Scopus (12) Google Scholar). The supernatant was transferred to a microcentrifuge tube containing 3 μl of 5m K2CO3 for neutralization. The nucleotide content of 100-μl samples of this supernatant was determined by FPLC (Econo-System, Bio-Rad) using a DEAE-MEMSEP (10/10) column eluted by a linear gradient of 0–1 m triethylamine, pH 7.5, at a flow rate of 5 ml/min at room temperature. The eluents were monitored by absorption at 254 nm, and the amounts were determined by integration of absorption peaks, calibrated with ATP and ADP standards. A1-ATPase was incubated at a concentration of 8 μg with trypsin in a ratio of 900:1 (w/w) in 20 mm Tris-HCl (pH 7.5) and 150 mmNaCl in the absence or presence of 5 mm nucleotide at 30 °C. Trypsin cleavage was stopped by addition of the protease inhibitor Pefabloc SC (8 mm). Peptides were separated by SDS-PAGE (14.Grüber G. Svergun D.I. Coskun Ü. Lemker T. Koch M.H.J. Schägger H. Müller V. Biochemistry. 2000; 40: 1890-1896Crossref Scopus (42) Google Scholar). The synchrotron radiation x-ray scattering data were collected following standard procedures on the X33 camera (27.Koch M.H.J. Bordas J. Nucl. Instrum. Methods. 1983; 208: 461-469Crossref Scopus (287) Google Scholar, 28.Boulin C. Kempf R. Koch M.H.J. McLaughlin S.M. Nucl. Instrum. Methods. 1986; 249: 399-407Crossref Scopus (296) Google Scholar, 29.Boulin C.J. Kempf R. Gabriel A. Koch M.H.J. Nucl. Instrum. Methods. 1988; 269: 312-320Crossref Scopus (232) Google Scholar) of the EMBL on the storage ring DORIS III of the Deutsches Elektronen Synchrotron (DESY) using multiwire proportional chambers with delay line readout (30.Gabriel A. Dauvergne F. Nucl. Instrum. Methods. 1982; 201: 223-224Crossref Scopus (139) Google Scholar). Solutions with protein concentrations of 4.3 and 7.9 mg/ml were measured. At sample detector distances of 3.9 m and 1.4 m and a wavelength λ = 0.15 nm, the ranges of momentum transfer 0.14 < s < 2.1 nm−1 were covered (s = 4π sinθ/λ, where 2θ is the scattering angle). The data were normalized to the intensity of the incident beam and corrected for the detector response; the scattering of the buffer was subtracted, and the difference curves were scaled for concentration using the program SAPOKO. 3D. I. Svergun and M. H. J. Koch, unpublished data.The maximum dimension, D max, of the A1-ATPase samples, their distance distribution functionp(r) and radii of gyration,R g, were computed by the indirect Fourier transform program GNOM (31.Svergun D.I. J. Appl. Crystallogr. 1993; 26: 258-267Crossref Scopus (106) Google Scholar, 32.Svergun D.I. Semenyuk A.V. Feigin L.A. Acta Crystallogr. Sect. A. 1988; 44: 244-250Crossref Scopus (277) Google Scholar). The molecular masses of the solutes were estimated by comparison with the forward scattering of a reference solution of bovine serum albumin. Previously, we have characterized the A1-ATPase from M. mazei Gö1 by small-angle x-ray scattering (SAXS) and determined the maximum dimension (18.0 ± 0.1 nm) and the radius of gyration (R g, 5.03 ± 0.1 nm (14.Grüber G. Svergun D.I. Coskun Ü. Lemker T. Koch M.H.J. Schägger H. Müller V. Biochemistry. 2000; 40: 1890-1896Crossref Scopus (42) Google Scholar)). Here SAXS was used to investigate possible changes of the quaternary structure of A1 due to substrate binding. Fig. 1 displays a native gel and the scattering profile of A1, free of loosely bound nucleotides and an ATPase activity of ∼8.0 μmol of ATP hydrolysis per mg of enzyme per min. The radius of gyration (5.02 ± 0.1 nm) is in agreement with previous results (14.Grüber G. Svergun D.I. Coskun Ü. Lemker T. Koch M.H.J. Schägger H. Müller V. Biochemistry. 2000; 40: 1890-1896Crossref Scopus (42) Google Scholar). However, when 5 mmMgADP, MgADP + Pi, or MgATP was added to the protein solution, the radii of gyration of the complexes increased to 5.14 ± 0.1, 5.21 ± 0.1, and 5.23 ± 0.1 nm, respectively (see Table I). In the presence of the unhydrolyzable ATP analogue, AMP-PNP, the enzyme had a slightly lowerR g = 4.92 ± 0.1 nm. The maximum dimension of the unligated or ligated A1-ATPase remained the same (18.0 ± 0.1 nm).Table IExperimentally determined radii of gyration, R g, of the A1-ATPase from M. mazei Gö1 dependent on nucleotide conditionsAdded nucleotidesR g(nm)None5.02 ± 0.15 mm MgATP5.23 ± 0.15 mmMgADP5.14 ± 0.15 mm MgADP + Pi5.21 ± 0.15 mm MgAMP PNP4.92 ± 0.1 Open table in a new tab To characterize further the structural changes described above, the nucleotide-binding subunits A and B were specifically labeled using the fluorescent label CM, as visualized on the polyacrylamide gel in Fig. 2 A. The higher intensity of CM bound at subunit A may be due to the fact that subunit A includes five Cys residues (Cys28, Cys65, Cys173, Cys255, and Cys372 (33.Wilms R. Freiberg C. Wegerle E. Meier I. Mayer F. Müller V. J. Biol. Chem. 1996; 271: 18843-18852Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar)) and thereby more possible sulfhydryl groups reacting with the maleimide. The activity of the CM-bound enzyme was not altered by the chemical modification. Fig. 3 shows the fluorescence spectrum of the CM labeled A1-ATPase, which was freed of bound nucleotides (Fig. 3, curve ▴) as described under “Experimental Procedures.” For comparison, addition of 5 mm MgATP to the protein (in a 1:1 ratio) causes the signal to increase and to shift to shorter wavelength (Fig. 3,curve ▵). Addition of MgADP (Fig. 3, curve ■) gave a spectrum similar to that obtained with MgATP but with a lower fluorescence maximum. In contrast, A1-ATPase in the presence of MgADP + Pi (Fig. 3, curve ○) displayed a lower fluorescence intensity and a small blue shift. Interestingly, addition of the non-cleavable nucleotide analogue AMP-PNP (Fig. 3, curve ⋄) caused an increase of the fluorescence signal, indicating that the MgATP-bound enzyme is catalyzing ATP hydrolysis during the measurements. This is also confirmed by the fact that 85% of the added MgATP is still present after the fluorescence measurement, as determined by FPLC. The results presented indicate that the fluorescence spectrum of CM bound to subunits A and B is sensitive to nucleotide binding.Figure 3Nucleotide-induced fluorescence changes of the CM-bound A1-ATPase. The fluorescence emission spectra of the A1-ATPase was measured with a protein concentration of 200 nm and a 1:1 ratio of Mg2+to nucleotide at 10 °C. The enzyme was diluted in 20 mmTris-HCl (pH 6.9) and 150 mm NaCl and preincubated with 5 mm MgAMP-PNP (curve ⋄), MgATP (curve ▵), MgADP (curve ■), and MgADP + Pi (curve ○) on ice. Curve ▴, A1-ATPase in the absence of nucleotides. The spectra were recorded at λex of 365.1 nm over a range of 410–560 nm with the emission and excitation slits at 4 nm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Intersubunit cross-linking is a useful method for establishing the relative position of subunits (34.Aggeler R. Haughton M.A. Capaldi R.A. J. Biol. Chem. 1995; 270: 9185-9191Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 35.Tsunoda S.P. Muneyuki E. Amano T. Yoshida M. Noji H. J. Biol. Chem. 1999; 274: 5701-5706Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 36.Hutcheon M.L. Duncan T.M. Ngai H. Cross R.L. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8519-8524Crossref PubMed Scopus (64) Google Scholar). Disulfide bond formation was mediated by Cu2+. Fig. 4 A illustrates the results of cross-linking of the A1-ATPase with CuCl2 under different nucleotide conditions. When the enzyme was incubated with 5 mm MgAMP-PNP at 4 °C before Cu2+ treatment, two new bands (I and V) involving subunit(s) B (I) and A-B (V) were generated, as indicated by Western blotting with antibodies to subunits A and B (Fig. 4 B). MALDI mass spectrometry confirmed that band I derived from subunit B by identifying the peptides Ala102–Arg120, Gly271–Arg286, and Ala366–Arg379. When A1 was suspended in MgADP + Pi, the cross-link product I and a low amount of band V are obtained. In the presence of MgATP the bands I and V increased, and the new bands III, IV, and VI-VIII appeared, including the subunits A-A (III) and A-A-D (IV), respectively, and several forms of A-B oligomers (V-VIII). The A-A-D (IV) formation, which cannot be cleaved by DTT, was analyzed more precisely by MALDI mass spectrometry (Fig. 5). Ten peptides were unequivocally identified as bands deriving from either the N or C terminus of subunit D, together with five peptides of subunit A (see Table II). However, we were not able to monitor unequivocally the specific peptide of each subunit forming the linkage. This might be due to the incompleteness of in-gel tryptic digestion of the polypeptides (37.Svoboda M. Meister W. Vetter W. J. Mass. Spectrom. 1995; 30: 1562-1566Crossref Scopus (18) Google Scholar), which covers only 47.8 and 23.9% of the D and A sequence, respectively. The presence of MgADP leads to a slight decrease of the A-A-D (IV) formation and additionally to several closely spaced bands running just above band VIII, including A-B and A-A formations as shown by antibody blotting (Fig. 4 B). Consistently, the staining intensity of the A and B bands decreased in parallel with the occurrence of these A-B and A-A oligomer bands. These results rule out that MgATP was completely converted into MgADP or MgADP + Pi before cross-link formation under the conditions used, and it can be concluded that the differences in the fluorescence spectra seen above depend on whether MgATP or MgADP is bound in the nucleotide-binding site of the A1-ATPase. As shown by MALDI mass spectrometry, addition of MgADP leads to an intrinsic cross-link of the contaminant DnaK (band II). A cross-linked B product (I) and an A-B oligomer formation (V) were also formed in the presence of the A1 inhibitor dienestrol (17.Lemker T. Ruppert C. Stöger H. Wimmers S. Müller V. Eur. J. Biochem. 2001; 268: 3744-3750Crossref PubMed Scopus (23) Google Scholar) and under atmospheric oxygen without CuCl2 as shown by two-dimensional SDS-PAGE (Fig. 4 C). Addition of DTT after CuCl2 treatment reversed cross-linking of the oxidized A1 complex.Figure 5Detail of a MALDI-TOF mass spectrum of peptides from subunit D involved in the cross-link product IV.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IIMALDI-mass spectrometry analysis of peptides from subunits A and D involved in the cross-linking product IVSubunitStart residueEnd residueExpected massMeasured massDelta massSequenceA1992101338.71338.70.0LTPEKPLVTGQR3413571911.81911.80.0LEEMPGEEGYPAYLSAR3413571927.81927.80.0LEEMPGEEGYPAYLSAR2-aOxidized Met.4334511940.91940.8−0.1IMKAIMKWGDAAMDALK2-aOxidized Met.3764012571.22571.3+0.1GSITAIGAVSPPGGDESEPVTQNTLRD11191073.31072.5−0.8SELINLKKK19281127.31126.5−0.8KIKLSESGHK1841941517.81517.6−0.2YIRFMLEEMER2-aOxidized Met.20331629.01628.7−0.3IKLSESGHKLLKMK2-aOxidized Met.1871991748.91748.8−0.1FMLEEMERENTFR2-aOxidized Met.32492210.62211.0+0.4MKRDGLILEFFKILNEAR2-aOxidized Met.1431642564.02564.1+0.1IITAAELETTMKRLLDEIEKTK2-aOxidized Met.1431652720.22720.1−0.1IITAAELETTMKRLLDEIEKTKR2-aOxidized Met.35613095.63095.5−0.1DGLILEFFKILNEARNVRTELDAAFAK1741993337.93338.5+0.6VIPELIDTMKYIRFMLEEMERENTFR2-aOxidized Met.2-a Oxidized Met. Open table in a new tab Recently, limited tryptic digestion of A1 has shown that the subunits A–C are cleaved most rapidly, leading to the fragments A1–4, B1, C1, followed by F, which becomes cleaved into fragment F1, whereby subunit D remains shielded by the complex (14.Grüber G. Svergun D.I. Coskun Ü. Lemker T. Koch M.H.J. Schägger H. Müller V. Biochemistry. 2000; 40: 1890-1896Crossref Scopus (42) Google Scholar). The cleavage products and their intensities were the same as observed upon trypsin treatment of A1 in the presence of 5 mm MgAMP-PNP (Fig. 6 A), with subunit F cleaved into fragment F1 after 20 min (data not shown). In contrast, there is slow cleavage of these subunits with MgATP or MgADP. Subunit C, which completely vanishes in the absence (14.Grüber G. Svergun D.I. Coskun Ü. Lemker T. Koch M.H.J. Schägger H. Müller V. Biochemistry. 2000; 40: 1890-1896Crossref Scopus (42) Google Scholar) or presence of MgAMP-PNP after 50 min, is slowly cleaved when MgATP or MgADP is added to the enzyme. There is, however, a slight difference in cleavage of this stalk subunit depending on whether MgATP or MgADP is bound. Quantitation of the scanned C-bands indicate that 43 and 59.4% of this subunit remained after 70 min when MgATP or MgADP, respectively, is bound to the enzyme (Fig. 6 B). Proteolysis of A1after addition of MgADP + Pi leads to a time-dependent cleavage pattern in which only 4.7% of subunit C remained after 70 min. Furthermore, under this nucleotide condition the subunits A, B, and F also become more accessible to trypsin than in the presence of MgATP or MgADP. Interestingly, the cleavage pattern of the MgADP + Pi-bound A1-complex is quite comparable with the feature seen when MgAMP-PNP is present. This is in agreement with the results of Cu2+-induced cross-link formations described above when MgADP + Pi or MgAMP-PNP are bound to the enzyme. X-ray solution scattering was used to investigate the influence of nucleotide binding to the quaternary structure of the A1-ATPase from M. mazei Gö1. Binding of the uncleaved MgATP (obtained by adding MgAMP-PNP) causes a slight decrease of the radius of gyration of this complex, consistent with the observation on the closely related F1FO-ATPase from E. coli, where the diameter of the F1complex decreases upon MgAMP-PNP binding as determined by three-dimensional reconstructions (38.Böttcher B. Bertsche I. Reuter R. Gräber P. J. Mol. Biol. 2000; 296: 449-457Crossref PubMed Scopus (52) Google Scholar). In contrast, addition of the hydrolyzable MgATP, MgADP + Pi, and MgADP increase the radii of gyration. Conformational changes of the quaternary and tertiary structure of the related F1 due to nucleotide binding are in line with the most recent crystallographic model of bovine F1-ATPase, indicating changes of the quaternary and tertiary structure of the complex (39.Menz R.I. Walker J.E. Leslie A.G. Cell. 2001; 106: 331-341Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar). Depending on the nucleotide bound to one of the catalytic β subunits significant changes of the lower part of the nucleotide binding domain and the C-terminal domain have been observed. Superposition of the so-called βDP, βTP, and βADP+Pisubunits of the bovine F1-ATPase (39.Menz R.I. Walker J.E. Leslie A.G. Cell. 2001; 106: 331-341Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar) indicate a 33° rotation of the C-terminal domain of βADP+Pi when compared with βDP and significant changes in backbone torsion angles in regions of the nucleotide binding domain. Incorporation of the fluorescent label CM into the nucleotide-binding site A and B of A1, as described above, provides strong evidence for rearrangements of these subunits, which also suggests that the small but systematic differences observed in SAXS are significant. Addition of MgADP causes a significant fluorescent enhancement and blue shift, whereas the binding of MgAMP-PNP only increases the signal. In contrast, the presence of MgADP + Pi results in a quenching of the signal, suggesting that structural changes in and around the bound CM occur in response to ATP binding and subsequent bond cleavage to ADP and Pi. The nucleotide-induced rearrangement of the major subunits A and B is also confirmed by the quantity of Cu2+-induced A-B dimers or the formation of A-B oligomers. The formation of A-B oligomers reflects the proximity of these subunits, which are proposed to alternate around a cavity in a hexameric fashion, thereby locating the nucleotide-binding sites at their interfaces (2.Schäfer G. Engelhard M. Müller V. Mol. Biol. Rev. 1999; 63: 570-620Crossref PubMed Google Scholar, 40.Schäfer G. Meyering-Vos M. Biochim. Biophys. Acta. 1992; 1101: 232-235Crossref PubMed Scopus (36) Google Scholar). Significantly, A-A dimers can be observed even though separated by an intervening B subunit. Close proximity of the related α and β subunits of the F1-ATPase has also been demonstrated by α-α or β-β products formed via disulfide bridges of the N or C terminus, respectively (35.Tsunoda S.P. Muneyuki E. Amano T. Yoshida M. Noji H. J. Biol. Chem. 1999; 274: 5701-5706Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 41.Ogilvie I. Aggeler R. Capaldi R.A. J. Biol. Chem. 1997; 272: 16652-16656Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Like in the closely related V-ATPases, the A1 subunit A contains a region of 80–90 additional amino acids near the N terminus (5.Hilario E. Gogarten J.P. J. Mol. Evol. 1998; 46: 703-715Crossref PubMed Scopus (68) Google Scholar, 6.Marin I. Fares M.A. Gonzáles-Candelas F. Barrio E. Moya A. J. Mol. Evol. 2001; 52: 17-28Crossref PubMed Scopus (15) Google Scholar, 33.Wilms R. Freiberg C. Wegerle E. Meier I. Mayer F. Müller V. J. Biol. Chem. 1996; 271: 18843-18852Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). This extension is assumed to be located at the top of the three A subunits, forming protuberances as described for the V-ATPase (10.Grüber G. Radermacher M. Ruiz T. Godovac-Zimmermann J. Canas B. Kleine-Kohlbrecher D. Huss M. Harvey W.R. Wieczorek H. Biochemistry. 2000; 39: 8609-8616Crossref PubMed Scopus (69) Google Scholar, 13.Radermacher M. Ruiz T. Wieczorek H. Grüber G. J. Struct. Biol. 2001; 135: 26-37Crossref PubMed Scopus (94) Google Scholar, 42.Wilkens S. Vasilyeva E. Forgac M. J. Biol. Chem. 1999; 274: 31804-31810Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Therefore, depending on nucleotide binding to the catalytic A subunits, these protuberances might come in close contact thereby facilitating an A-A formation. A key finding of the present study is that subunit D can be cross-linked to the catalytic A subunit depending on nucleotide binding. The A-D formation occurs after addition of MgADP and to a small amount in the presence of the hydrolyzable MgATP but not in the presence of MgADP + Pi, MgAMP-PNP, or the absence of nucleotides. The interaction between the subunits A and D involves the N and C termini of D, indicating their close proximity to the catalytic A subunit. It is of particular interest that neither of the termini of D contain Cys residues and that the cross-linked formation cannot be disrupted by reducing agents, which rules out that the A-D product would be generated by disulfide bond formation. Inspection of the amino acid sequence of the peptides from subunit D reveals the presence of a His27 and a Tyr184 residue at the N and C termini of this subunit, respectively, both candidates to form a thioether bridge with a cysteinyl residue. Such covalent linkage of the sulfur of a Cys with an imidazole ring of a His residue or with a Tyr residue has been identified as an essential formation in the tyrosinase from Neurospora crassa (43.Lerch K. J. Biol. Chem. 1982; 257: 6414-6419Abstract Full Text PDF PubMed Google Scholar) and the galactose oxidase from Dactylium dendroides (44.Ito N. Phillips S.E.V. Stevens C. Ogel Z.B. McPherson M.J. Keen J.N. Yadav K.D.S. Kowles P.F. Nature. 1991; 350: 87-90Crossref PubMed Scopus (697) Google Scholar), respectively, and also occurred in cross-linking studies of the ATP synthase fromE. coli (45.Watts S.D. Zhang Y. Fillingame R.H. Capaldi R.A. FEBS Lett. 1995; 368: 235-238Crossref PubMed Scopus (84) Google Scholar). This close proximity of subunits A and D would allow coupling between the catalytic site events in A via D into the central stalk, which provides the physical and structural linkage between the A3B3 headpiece and the ion-conducting complex (2.Schäfer G. Engelhard M. Müller V. Mol. Biol. Rev. 1999; 63: 570-620Crossref PubMed Google Scholar, 3.Grüber G. Wieczorek H. Harvey W.R. Müller V. J. Exp. Biol. 2001; 204: 2597-2605Crossref PubMed Google Scholar). Secondary structure analysis of D predicted α-helical N and C termini (33.Wilms R. Freiberg C. Wegerle E. Meier I. Mayer F. Müller V. J. Biol. Chem. 1996; 271: 18843-18852Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 45.Watts S.D. Zhang Y. Fillingame R.H. Capaldi R.A. FEBS Lett. 1995; 368: 235-238Crossref PubMed Scopus (84) Google Scholar), as shown for both termini of subunit γ of F-ATPase (12.Gibbons C. Montgomery M.G. Leslie A.G.W. Walker J.E. Nat. Struct. Biol. 2000; 7: 1055-1061Crossref PubMed Scopus (437) Google Scholar, 47.Rodgers A.J.W. Wilce M.C.J. Nat. Struct. Biol. 2000; 7: 1051-1054Crossref PubMed Scopus (153) Google Scholar, 48.Hausrath A.C. Grüber G. Matthews B.W. Capaldi R.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13697-13702Crossref PubMed Scopus (82) Google Scholar), which has been proposed as a structural and functional homologue of subunit D (3.Grüber G. Wieczorek H. Harvey W.R. Müller V. J. Exp. Biol. 2001; 204: 2597-2605Crossref PubMed Google Scholar, 14.Grüber G. Svergun D.I. Coskun Ü. Lemker T. Koch M.H.J. Schägger H. Müller V. Biochemistry. 2000; 40: 1890-1896Crossref Scopus (42) Google Scholar,33.Wilms R. Freiberg C. Wegerle E. Meier I. Mayer F. Müller V. J. Biol. Chem. 1996; 271: 18843-18852Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 46.Müller V. Ruppert C. Lemker T. J. Bioenerg. Biomembr. 1999; 31: 15-27Crossref PubMed Scopus (53) Google Scholar). The x-ray structure revealed that the α-helical N and C termini of γ intercalate into the cavity of the α3β3 assembly of F1 (12.Gibbons C. Montgomery M.G. Leslie A.G.W. Walker J.E. Nat. Struct. Biol. 2000; 7: 1055-1061Crossref PubMed Scopus (437) Google Scholar, 48.Hausrath A.C. Grüber G. Matthews B.W. Capaldi R.A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 13697-13702Crossref PubMed Scopus (82) Google Scholar,49.Hausrath A.C. Capaldi R.A. Matthews B.W. J. Biol. Chem. 2001; 276: 47227-47232Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), thereby linking two differently occupied catalytic subunits, βTP (triphosphate-containing) and βDP(diphosphate-containing) (39.Menz R.I. Walker J.E. Leslie A.G. Cell. 2001; 106: 331-341Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar). Taken together, the results above provide several lines of evidence suggesting that, depending on the nucleotides bound to them, subunits A and B of the A1-ATPase change conformation and/or their interactions with structural alterations in the stalk subunits C, D, and F. The nucleotide dependence suggests also that there is a tight interaction of the D subunit at its catch region with the A subunit upon MgADP binding, which is broken when MgAMP-PNP is bound. Such binding followed by release may play a part in coupling the catalytic sites with the stalk region and the membrane-bound ion channel. We thank R. Genswein, Universität Mainz, for excellent technical assistance.

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Cross-talk in the A1-ATPase from Methanosarcina mazei Gö1 Due to Nucleotide Binding