modestum in the organic solvent mixture with that in dodecylsulfate micelles several deviations became apparent: in the organic solvent, the interruption of the a helical structure withi
Trang 1NMR investigations of subunit c of the ATP synthase from
Ulrich Matthey1, Daniel Braun2and Peter Dimroth1
1
Institut fu¨r Mikrobiologie, and2Institut fu¨r Molekularbiologie und Biophysik, Eidgeno¨ssische Technische Hochschule, Zu¨rich, Switzerland
The subunit c from the ATP synthase of Propionigenium
modestumwas studied by NMR in chloroform/methanol/
water (4 : 4 : 1) In this solvent, subunit c consists of two
helical segments, comprised of residues L5 to I26 and G29 to
N82, respectively On comparing the secondary structure of
subunit c from P modestum in the organic solvent mixture
with that in dodecylsulfate micelles several deviations
became apparent: in the organic solvent, the interruption of
the a helical structure within the conserved GXGXGXGX
motif was shortened from five to two residues, the prominent
interruption of the a helical structure in the cystoplasmic
loop region was not apparent, and neither was there a break
in the a helix after the sodium ion-binding Glu65 residue The folding of subunit c of P modestum in the organic solvent also deviated from that of Escherichia coli in the same environment, the most important difference being that sub-unit c of P modestum did not adopt a stable hairpin struc-ture like subunit c of E coli
Keywords: ATP synthase; stable isotope labeling, NMR spectroscopy; Propionigenium modestum; subunit c
F1F0ATP synthases catalyse the formation of ATP from
ADP and inorganic phosphate that is driven by an
electrochemical gradient of protons or in some cases Na+
ions Similar enzymes are found in chloroplast,
mitochon-dria and bacteria They consist of a cytoplasmic F1part with
the subunit composition a3b3cde and a membrane intrinsic
F0moiety, which in bacteria has the subunit composition
ab2cx The mechanism for ATP synthesis, proposed to
involve binding changes of the three catalytic binding sites
on the b sunbunits [1], was in remarkable agreement with
the atomic resolution X-ray structure of F1 [2] Based on
these data rotation of subunit c within the cylinder made of
alternating a and b subunits was suggested and confirmed
[3–5] More recent structural data have shown that the c and
e subunits forming the central stalk are permanently fixed to
the ring of c subunits [6], and consequently, all three
subunits were demonstrated to rotate as a unit [7–9]
A high-resolution structure of the ion-translocating F0
part remains to be determined Electron and atomic force
microscopy of F0 indicated that subunits a and b are
attached to the periphery of an oligomeric ring of c subunits
[10,11] The subunits a and c are directly involved in ion
translocation [12–15], whereas subunit b is presumed to
form a peripheral stalk, which connects the F0part to F1via
association with the d subunit [16–18] Based on structural
data, the number of c subunits forming the ring is c10for the yeast ATP synthase [6], c14for the chloroplast enzyme [19], and c11 for the Ilyobacter tartaricus ATP synthase [20] According to cross-linking studies, c10 appears to be the preferred stoichiometry for the ATP synthase of Escherichia coli[21]
The NMR structure of the monomeric E coli subunit c was determined in chloroform/methanol/water (4 : 4 : 1),
in which the protein folds like a hairpin [22,23] Two extended a helices are connected by a hydrophilic loop, and the proton-binding residue D61 is located in the centre of the C-terminal helix The monomeric P modestum sub-unit c was studied by NMR in SDS micelles [24] In this biphasic system the protein consists of four a helical segments, that are connected by short linker peptides with nonregular secondary structures The Na+-binding residues Q32, E65 and S66 [12] are located in the I–II and III–IV helix connections No long-range NOEs could be identified that would indicate the presence of a three-dimensional fold with close packing of the helices
In order to enable a direct comparison of the P mode-stumsubunit c with its E coli homologue, we now prepared
an NMR sample in chloroform/methanol/water (4 : 4 : 1) The key questions to be investigated were how the secondary structure in the organic solvent mixture compares with that in dodecylsulfate micelles, and whether the
P modestumsubunit c forms similar interhelix contacts as the E coli protein in the same solvent
E X P E R I M E N T A L P R O C E D U R E S
Overproduction and purification of subunit c Uniformly 13C,15N-labelled subunit c was overproduced
in E coli PEF42(DE3)pT7c on Martek 9-CN medium
as described previously [24] Subunit c was purified by chloroform/methanol extraction and anion-exchange
Correspondence to U Matthey, Institut fu¨r Mikrobiologie,
Eidgeno¨ssische Technische Hochschule Zu¨rich, ETH-Zentrum, CH
8092 Zu¨rich, Switzerland.
Fax: + 41 1632 13 78, Tel.: + 41 1632 55 23,
E-mail: matthey@micro.biol.ethz.ch
Enzymes: H+-transporting ATP synthase (EC 3.6.1.34);
Na+-transporting ATP synthase (EC 3.6.1.37).
Note: web page available at http://www.micro.biol.ethz.ch
(Received 3 December 2001, revised 18 February 2001, accepted 20
February 2001)
Trang 2chromatography [25] Aliquots of 4 mL were applied to a
Sephadex LH20 column (160 mm· 20 mm) and eluted
with chloroform/methanol (2 : 1) Fractions of 3 mL were
collected and the protein was monitored by A280
NMR sample preparation
Subunit c was transferred into C2HCl3/C2H3OH/H2O
(4 : 4 : 1), applying the same conditions as for the E coli
protein (M Girvin, Biochemistry Department, Albert
Einstein College of Medicine, New York, USA, personal
communication) Ten milliliters of C2HCl3 were added to
11 mL of subunit c [1 mgÆmL)1 in chloroform/methanol
(2 : 1)] and the mixture was concentrated to less than
0.1 mL by a gentle stream of Argon with periodical swirling
of the solution to keep the solvent composition uniform
After addition of 0.5 mL C2HCl3/C2H3OH (2 : 1), the
sample was incubated for 15 min at room temperature, and
then 0.4 mL C2HCl3was added The sample was
concen-trated to 0.05–0.1 mL and the same cycle of solvent
addition and subsequent volume reduction was repeated
The sample was then brought to complete dryness with a
stream of Argon Whenever the sample became cloudy
during the preparation, C2HCl3was added to redissolve the
material The dried sample was covered with 0.6 mL of
C2HCl3/C2H3OH/H2O containing 25 mMD11-Tris/HCl at
pH 7.5, and incubated for 1 h at 30°C with periodical,
gentle swirling After adjusting the pH to 7.5, the sample
was centrifuged and transferred to an NMR tube, which
was flame-sealed The final concentration of the protein was
2 mM
NMR spectroscopy
Spectra were recorded at 27°C on Bruker DRX500,
DRX600, DRX750 and DRX800 spectrometers For the
resonance assignments and the collection of conformational
constraints, the following experiments were recorded: 3D
CBCA(CO)NH [26], 3D HNCACB [27], 3D15N-resolved
[1H,1H]-TOCSY (mixing time sm¼ 60 ms) [28], 3D
13C-resolved HCCH-TOCSY (sm¼ 14 ms) [29], 3D
15N-resolved [1H,1H]-NOESY (sm¼ 60 ms) [30], 3D13
C-resolved [1H,1H]-NOESY (sm¼ 60 ms) [31], and 3D13
C-resolved [1H,1H]-NOESY (sm¼ 150 ms) Spectra were
processed and analysed with the programsPROSA[32] and
XEASY [33] Chemical shifts were calibrated with sodium
3-(trimethylsilyl)propane-1-sulfonate
R E S U L T S
Stability ofP modestum subunit c in
chloroform/methanol/water (4 : 4 : 1)
Samples of 2 mM unlabelled subunit c of Propionigenium
modestum in C2HCl3/C2H3OH/H2O (4 : 4 : 1), 25 mM
D11-Tris/HCl were prepared to test the stability of the
protein Two-dimensional homonuclear NMR spectra
were recorded to monitor structural changes over 4 weeks
During this period, no spectral differences were observed
with samples at pH 5.8, pH 7.0 and pH 7.5 that were kept
at 20°C These observations indicated that P modestum
subunit c was stable in the solvent mixture at the pH values
indicated
Resonance assignment Sequence-specific backbone assignments for subunit c were obtained from 3D HNCA, 3D CBCA(CO)NH and HNCACB experiments using a 2-mM 15N/13C-labelled sample in chloroform/methanol/water (4 : 4 : 1) With the exception of the N-terminal dipeptide segment H-Met-Asp-all amide protons and nitrogen resonances could be assigned Proton resonances of aliphatic side chains were achieved by 3D15N-resolved [1H,1H]-TOCSY and 3D15 N-resolved [1H,1H]-NOESY Furthermore, homonuclear 2D [1H,1H]-TOCSY and 2D [1H,1H]-NOESY spectra were used to assign the aromatic spin systems While all aromatic
1H side chain resonances of Y34, Y70 and Y80 could be determined, only one aromatic side chain resonance was found for F84 due to signal overlap
The13C chemical shifts were obtained by 3D13C-resolved HCCH-COSY, 3D13C-resolved HCCH-TOCSY and 3D
13C-resolved [1H,1H]-NOESY experiments However, chemical shift dispersion of the methyl groups was limited, complete assignment of all CHngroups was received except those of the N-terminal methionine
Conformational constraints Overall 3035 NOESY cross-peaks were assigned We found
515 intraresidual, 283 sequential and 331 medium-range NOEs Observed dadNOEs showed that the peptide bonds G27–P28, Q46–P47 and N82–P83 are all in trans-confor-mation No long-range NOEs could be identified in the spectra recorded with a mixing time of 60 ms that would indicate the occurrence of close interhelix contacts There-fore, additional 2D [1H,1H]-NOESY and 3D13C-resolved [1H,1H]-NOESY spectra with 150 ms mixing time were recorded on a Bruker DRX800 spectrometer and analysed Possible long-range NOEs were collected using the chemical shift comparison function of CANDID Evaluation of these signals did not indicate the presence of any long-range NOEs In particular, cross-peaks of aromatic protons could
be completely assigned as short-range and medium-range NOEs (Fig 1)
The secondary structure characteristic connectivities were collected from NOESY spectra recorded with 60 ms Surprisingly, the proposed hydrophilic loop between Q46 and D52 showed significant a helix connectivities (Fig 2)
In contrast to previous NMR studies of P modestum subunit c in SDS micelles [24], subunit c in chloroform/ methanol/water (4 : 4 : 1) exhibited no a helix interruption
at the C-terminal part (T67, G68) of the protein Similar to the SDS sample, subunit c in chloroform/methanol/water (4 : 4 : 1) contains a nonhelical linker peptide near P28, which is shorter than in the SDS structure Overall, typical
a helix connectivities were found from L5 to I26, from G29
to Q46 and from P47 to N82 As indicated by the
daN(i,i + 2) connectivities the two helices possibly end with
310helix turns comprising residues I23 to I26 and N82 to L87, respectively
Chemical shift deviations The deviations from the random coil chemical shifts were calculated as the difference between the measured chemical shifts and the corresponding random coil values in aqueous
Trang 3solution [34] Continuous 13Ca downfield chemical shift
deviations were found from V4 to G23, V30 to A44 and
I53 to Y80 (Fig 2) Significantly smaller deviations were
observed for the peptide segments G25 to G29, R45 to
D52 and A81 to G89 The shape of the chemical shift
deviations did not change when calculated with
corre-sponding random coil values in chloroform/methanol
(1 : 1), which were referenced with tetramethylsilane
(H Kessler, Institute fu¨r Organische Chemie and
Bio-chemie, Tu Mu¨nchen, Garching, Germany, personal
communication) However, the chemical shift deviations
were about 1.9 p.p.m higher, which can most probably
be attributed to the usage of different reference
stan-dards
Secondary structure and global fold
As indicated by helix-characteristic NOE connectivities [35],
subunit c in chloroform/methanol/water (4 : 4 : 1) consists
of two helices Helix I comprises residues L5 to I26, and
helix II comprises the segment G29 to N82 with a short
interruption between Q46 and P47 In particular no signal
intensities were obtained for the a helix characteristic
connectivities daN(23,26), dab(23,27), daN(24,27), dad(24,28),
daN(25,29), dab(25,28), dad (25,28) and daN(26,30) The
interruption between helix I and helix II is further confirmed
by the small13Cadownfield chemical shift deviation of the segment A24 to G29
The proposed hydrophilic loop Q46 to D52, is a helical according to the medium-range NOEs However, the13Ca chemical shift deviation of this peptide segment is signifi-cantly smaller than that of the other helical segments Therefore, it is likely that the a helix conformation of this segment is poorly populated
D I S C U S S I O N
Structural studies of membrane proteins by NMR in solution require the usage of either organic solvents or detergents The structure of the E coli subunit c was studied in organic solvent by NMR [23] Addition of water
to E coli subunit c in chloroform:methanol (1 : 1) was found to stabilize interhelix contacts in a concentration depending manner (M Girvin, Biochemistry Department, Albert Einstein College of Medicine, New York, USA, personal communication) In chloroform/methanol/water (4 : 4 : 1) the protein consisted of two elongated helices, which were connected by a hydrophilic loop Two different structures at pH 5 and pH 8 were found and a conforma-tional change of subunit c during ion translocation was proposed [22]
The secondary structure of subunit c of P modestum in SDS micelles [24] deviates significantly from that of E coli
in chloroform/methanol/water (4 : 4 : 1): P modestum sub-unit c folds into four helices, that are connected by small linker peptides with nonregular secondary structure The
Na+-binding ligands (Q32, E65, S66) [12] are located in the peptides connecting helices I and II, and III and IV, respectively
As described above, the two NMR structures were determined in different solvent systems and for the c subunits from two different bacteria It was therefore intriguing to investigate whether the structure of the c subunit is dependent on the solvent conditions For this purpose, we determined the structure of subunit c from
P modestum in chloroform/methanol/water (4 : 4 : 1) Under these conditions, the protein folds significantly dif-ferent from subunit c in dodecylsulfate micelles (Fig 3B,B¢) and it also folds significantly different than subunit c of
E coli in the organic solvent mixtures (Fig 3A) The secondary structure of P modestum subunit c in the organic solvent is mainly helical with only a short linker peptide consisting of residues G27 and P 28, but without interrup-tion of the helical folding in the hydrophilic loop region as indicated by helix-characteristic NOEs Subunit c of
P modestum therefore does not fold into a stable helical hairpin in chloroform/methanol/water (4 : 4 : 1) The hair-pin structure of subunit c is indicated, however, by a wealth
of experiments [12,36–40] and was unequivocally demon-strated by the F1c crystal structure from yeast mitochondria [6] Moreover, the inconsistency between chemical shift deviations and NOE connectivities at the peptide seg-ment R45 to D52 implies structural polymorphism The
P modestumsubunit c showed structural variety in differ-ent solvdiffer-ent systems It is likely that small changes in the organic solvent composition can cause structural polymor-phism Further, homologue proteins (P modestum and
E colisubunit c) can form different structures in the same solvent, depending on their individual sequences
Fig 1 Part of a contour plot of a two-dimensional [ 1 H, 1 H]-NOESY
spectrum showing the methyl-aromatic proton cross-peaks of subunit c
in chloroform/methanol/water (4 : 4 : 1) The spectrum was recorded
with 150 ms mixing times on a Bruker DRX800 spectrometer.
Assignments of the signals are indicated.
Trang 4We hypothesize that monomeric subunit c may be
considerably prone to structural polymorphism Regardless
of whether the protein is dissolved in organic solvents or
embedded into detergent micelles, these environments are
not a good mimic of the natural situation where the protein
forms strong protein/protein contacts to assemble into rings
of 10, 11 or 14 c subunits These rings can be extremely stable,
and resist boiling in SDS for 5 min in the case of the
undecameric c ring of P modestum [41] This stability of the
ring makes structural flexibility rather unlikely, indicating
that the structural polymorphism of monomeric subunit c is
due to the interaction of the monomer with the artificial
environment lacking the stabilizing protein/protein contacts
within the ring The undecameric c ring of the ATP synthase
from Ilyobacter tartaricus, a close relative to P modestum,
has recently been crystallized in two dimensions and
subjected to structure determination by cryo-transmission
electron microscopy [20] All c subunits of the ring show the
hairpin-like folding and the structure of all monomeric units
appears to be the same Hence, the structural flexibility
observed for the subunit c monomer is apparently lost upon
assembly into the ring, probably because rings of the stability
observed require defined structures of the monomeric units
in order to generate strong protein/protein interactions
A C K N O W L E D G E M E N T S
We thank Georg Kaim and Mark E Girvin for critical reading of the manuscript We are grateful to Torsten Herrmann for his support in CANDID, Reto Horst for introduction into the DRX800 spectrometer and Mark Girvin for providing the detailed protocol of the NMR sample preparation We express a special thank to Kurt Wu¨thrich for his support and the provision of NMR equipment.
R E F E R E N C E S
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2 Abrahams, J.P., Leslie, A.G.W., Lutter, R & Walker, J.E (1994) Structure at 2.8 A˚ resolution of F 1 -ATPase from bovine heart mitochondria Nature 370, 621–628.
3 Noij, H., Yasuda, R., Yoshida, M & Kinosita, K (1997) Direct observation of the rotation of F 1 -ATPase Nature 386, 299– 302.
4 Duncan, T.M., Bulygin, V.V., Zhou, Y., Hutcheon, M.L & Cross, R.L (1995) Rotation of subunits during catalysis by Escherichia coli F 1 -ATPase Proc Natl Acad Sci USA 92, 10964– 10968.
5 Sabbert, D., Engelbrecht, S & Junge, W (1996) Intersubunit rotation in active F-ATPase Nature 381, 623–625.
Fig 3 Alignment of c subunits from E coli and P modestum and location of the a helical secondary structures in different subunit c preparations (a) Amino-acid sequence of E coli subunit c and location of a helices in the structure in chloroform/methanol/water (4 : 4 : 1) [23], amino-acid sequence of P modestum subunit c with the helix locations in (b) SDS micelles [24], and (b¢) in chloroform/methanol/water (4 : 4 : 1) Identical amino acids are denoted by white letters on dark grey background and conservative substitutions are indicated by light grey background The conserved carboxylate residues involved in ion binding (E coli D61, P modestum E65) are indicated with an asterisk.
Fig 2 Amino-acid sequence of P modestum subunit c and survey of selected NMR data Sequential NOE connectivities are displayed by continuous bold lines extending over the residues that showed these connectivities Medium-range NOEs are represented by lines connecting the two interacting residues In the row Dd( 13 C a ) the difference between the observed shifts and the corresponding random coil values [34] is shown The sequence locations of the a helical secondary structures derived from these data are indicated by bars at the bottom.
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