Prediction of transmembrane topology of F 0 proteins from Escherichia coli FIF 0 ATP synthase using variational and hydrophobic moment analyses

The a subunit, a membrane protein from the E. coli F1FO ATP synthase has been examined by Fourier analysis of hydrophobicity and of amino-acid residue variation. The amino-acid sequences of homologous subunits from Vibrio alginolyticus, Saccharomyces cerevisiae, Neurospora crassa, AspergiUus nidulans, Schizosaccharomyces pombe and Candida parapsilosis were used in the variability analysis. By Fourier analysis of sequence variation, two transmembrane helices are predicted to have one face in contact with membrane lipids, while the other spans are predicted to be more shielded from the lipids by protein. By Fourier analysis of hydrophobicity, six amphipathic t~-helical segments are predicted in extra-membrane regions, including the region from Glu-196 to Asn-214. Fourier analysis of sequence variation in the b- and the c-subunits of the Escherichia coli F1F0 ATP synthase indicates that the single transmembrane span of the b-subunit and the C-terminal span of the c subunit each have a face in contact with membrane lipids. On the basis of this analysis topographical models for the a- and c-subunits and for the F 0 complex are proposed

© 1992 Elsevier Science Publishers B.V All rights reserved 0005-2728/92/$05.00

BBABIO 43728

Prediction of transmembrane topology of F 0 proteins

from Escherichia coli FIF 0 ATP synthase using variational

and hydrophobic moment analyses

Department of Biological Sciences, Southern Methodist University, Dallas, TX (USA)

(Received 10 April 1992)

Key words: Alpha helix; a-Helical periodicity; Fourier transform; Hydropathy analysis; ATP synthase, FIF0-; Proton channel

The a subunit, a membrane protein from the E coli F1F O ATP synthase has been examined by Fourier analysis of hydrophobicity and of amino-acid residue variation The amino-acid sequences of homologous subunits from Vibrio alginolyticus, Saccharomyces cerevisiae, Neurospora crassa, AspergiUus nidulans, Schizosaccharomyces pombe and Candida parapsilosis were

used in the variability analysis By Fourier analysis of sequence variation, two transmembrane helices are predicted to have one face in contact with membrane lipids, while the other spans are predicted to be more shielded from the lipids by protein By Fourier analysis of hydrophobicity, six amphipathic t~-helical segments are predicted in extra-membrane regions, including the

region from Glu-196 to Asn-214 Fourier analysis of sequence variation in the b- and the c-subunits of the Escherichia coli F1F 0

ATP synthase indicates that the single transmembrane span of the b-subunit and the C-terminal span of the c subunit each have

a face in contact with membrane lipids On the basis of this analysis topographical models for the a- and c-subunits and for the

F 0 complex are proposed

Introduction

The F t F 0 A T P synthase from Escherichia coli is a

prototype of the A T P synthases found in mitochondria

and chloroplasts [1,2] It comprises an F~ complex,

which contains the catalytic subunits and an F 0 com-

plex, which conducts protons across the m e m b r a n e

T h r e e different subunits, a, b and c form F 0 with a

stoichiometry of 1 : 2 : 9 - 1 0 [3]

T h e n u m b e r and relative location of the transmem-

brane spans of the F 0 subunits is still at issue Based on

a variety of evidence [4,5], the c subunit is thought to

contain two m e m b r a n e spanning a-helices connected

by a tight turn that faces the cytoplasm The b subunit

contains a single hydrophobic region that is long enough

to span a m e m b r a n e as an a-helix and that is located

at the extreme amino-terminus T h e bulk of the pro-

tein extends into the cytoplasm and is thought to

interact with F 1 subunits [6-9] T h e a r r a n g e m e n t of the

largest subunit, a, is less certain Models have been

Correspondence to: S.B Vik, Department of Biological Sciences,

Southern Methodist University, Dallas, TX 75275-0376, USA

Abbreviations: TID, 3-trifluoromethyl-m-(iodophenyl)diazirine; GES,

hydrophobicity scale of Engleman et al [16]

offered ranging from four to eight t r a n s m e m b r a n e spans [6,10-15] based on considerations of hydro- phobicity and charge and on alkaline phosphatase gene fusion experiments The low solubility of this protein has hindered efforts to learn more about its arrange-

m e n t in the m e m b r a n e and within the F 0 complex Two different models for the organization of a, b and c subunits in the F 0 complex have been offered Based primarily upon the labeling of all three subunits

by the h y d r o p h o b i c r e a g e n t 3-trifluormethyl-m- (iodophenyl)diazirine (TID), H o p p e and Sebald [4] have

proposed an oligomer of c subunits adjacent to an a-b 2

complex Alternatively, Cox et al [11] have proposed

that an a-b 2 complex is surrounded by a ring of c

subunits, shielding them from the lipids

We have applied Fourier analysis of hydrophobicity and of variation within a group of related proteins to detect a-helical periodicity within F 0 subunits The resulting hydrophobic m o m e n t s can identify transmem- brane helices that have segments with a polar face They also can identify e x t r a m e m b r a n e segments that are located at a polar-nonpolar surface The variational

m o m e n t s can identify t r a n s m e m b r a n e helices that in- teract with m e m b r a n e lipids on one face and with other t r a n s m e m b r a n e helices on the opposite face

Similarly, they can also identify surface helices in glob-

ular proteins or extra-membrane segments of mem-

brane proteins

Materials and Methods

Hydropathy profiles were generated using the

hydrophobicity scale (GES) of Engelman et al [16] and

a window of 21 residues Potential amphipathic a-

helices were detected using the GES scale and a win-

dow of 11 residues Hydrophobic moment power spec-

tra/z(o~) [17] were calculated for stretches of 11 amino

acids, according to

o)(tx ) = [kL_ l( hk hk ) cos( ko)) ]2+ [k~-~Sl( hk hk) sin( ko) ) ] 2

where n = 11, h k is the hydrophobicity of the k th

residue and h is the mean hydrophobicity of the se-

quence of n residues:

1 n

n ~,k=l /

The a-helical amphipathic index, ~h,(g(o~)), measures

how much of the power spectrum /z(to) resides in the

vicinity of 100 °, i.e., 3.6 residues per turn for an a-helix,

as compared to the entire spectrum

1 ,'120

3~J9 ° ~(o)) do)

I r180

8oJ0 /x(o)) do)

Fourier analysis of the variability of amino-acid se-

quences was performed as described by Komiya et al

[18], using a window of 11 or 21 residues The calcula-

tions are formally the same as above, except that

variability, which is defined as the number of different

amino-acid residues that appear at a single position

among a group of aligned sequences, replaces hydro-

phobicity This analysis is based on the observation

that surface residues are less highly conserved than are

buried residues [19] In the case of a family of related

transmembrane proteins, such as the reaction centers

of photosynthetic bacteria, it has been found that

residues in contact with the membrane lipids tend to

be more variable than those in contact with protein

[20] For this reason, surface transmembrane a-helices

can be detected by a periodicity in the variability

profile consistent with an a-helix For variability analy-

sis, the alignment of sequences of a-subunits from E

coli and Vibrio alginolyticus was that of Krumholz et al

[21] and fungal a-subunits were aligned essentially

according to Gu61in et al [22] Bacterial b-subunits

were aligned by inspection The alignment of c-sub-

3.5 2.5

"~ 1.5

~ I).5

"~ 0.5 -1.5 -2.5

A

"%/Iso ~'i ~ 2s0

3.5

Residue n u m b e r

2.5 :o 1.5

~ 0.5

2

~ 0.5 -1.5 -2,5

B

i,,r , v rflllllq~llllE[]]lll¢lllfl] J ~ " II]]llllmllllllglllllllflllLtlll~J~][~ [~I~[E~]'~[[~ '!]i]Tll]![:,ii~ii

Residue n u m b e r

Fig 1 Hydropathy profiles of (A), a-subunit from Escherichia coil

and (B), a-subunit (ATP 6) from Saccharomyces cerevisiae using a window of 21 residues and the GES [16] hydrophobicity parameters Underlined regions correspond to the aligned sequences shown in

Fig 2

units was according to Sebaid and H o p p e [4], with the following additional sequences included: I~ alginolyti- cus [21], Propionigenium modestum [23], Bacillus mega- terium [24], Synechococcus [25], Anabaena [26],

Marchantia chloroplast [27], sunflower mitochondria [28], rice mitochondria [29], sugar beet mitochondria [30], petunia mitochondria [31], pea mitochondria [32], tobacco mitochondria [33] and maize mitochondria [34] Secondary structure predictions were carried out using Chou-Fasman parameters [35] with a window of 5 residues

Results

Hydropathy analysis of the a-subunit

A hydropathy profile of the E coli a-subunit is shown in Fig 1A Five prominent hydrophobic regions with average hydrophobicity values of greater than 1.5 can be seen The first three are well-resolved, while the last two are nearly contiguous In addition, there are two minor peaks of value about 0.7, one following the first major peak and the second following the third major peak Previous authors have interpreted such results as indicating 5, 6 or 7 transmembrane spans For comparison, the hydropathy profile of the yeast, S

cerevisiae., mitochondrial a-subunit ( A T P 6) is shown

in Fig lB The peaks corresponding to the minor peaks seen in the E coli a-subunit profile are some- what different The first one is no longer hydrophobic, while the second one is a major peak with an average

TABLE I

Comparison of potential span IV sequences

Average hydrophobicity is calculated from the GES parameters [16]

Sequences were obtained from the indicated references: maize mito-

chondria [66] S cerevisiae mitochondria [4], Drosophila yakuba [67]

bovine mitochondria [68], E coli [10], pea chloroplast [69] and B

megaterium [25]

hydro- phobicity Maize mitochondria FFSFLLPAGVPLPLAPFLVLL 2.08

S cerecisiae FFSLFVPAGTPLPLVPLLVII 2.03

Drosophila yakuba MFAHLVPQGTPAILMPFMVCI 1.63

Bovine mitochondria SLAHFLPQGTPTPLIPMLVII 1.33

E coli FTKELTLQPFNHWAFIPVNLI 0.72

Pea chloroplast GLAYFGKYIQPTPILLPINIL 0.59

B megaterium FSAYTKDYFKPMAFLFPLKII - 0.11

S.cerevisiae 29 I D L S C L N L T T F S L Y T I I V L L V I T S L Y T L

E.coli 3 8 F W T I N I D S M F F S V V L G L L F L V L F R S V A K

S.cerevisiae 9 O K N W G L Y F P M I F T L F M F I F I A N L I S M I P Y

E.coli % G K S K L I A P L A L T I F V W V F L M N L M D L L P I

S c e r e v i s i a e l l S I P Y S F A L S A H L V F I I S L S I V I W L G N T I L

E.coli 1 4 7 D V N V T L S M A L G V F I L I L F Y S I K M K G I G G

S c e r e v i s i a e I 4 8 G W V F F S L F V P A G T P L P L V P L L V I I E T L S

E.coli 1 7 2 G G F T K E L T L Q P F N H W A F 1 P V N L 1 L E G V S

S.cerevisia~ I f f ? L G S N I L A G H L L M V l L A G L T F N F M L I N L F

E.coli 2 I l L F G N M Y A G E L 1 F 1 L I A G L L P W W S Q W I L N

S c e r e v i s i a e 2 2 5 1 L A 1 M I L E F A I G l I Q S Y V W T 1 L T A S Y L K

E.coli Z 3 S N V P W A I F H I L I l T L Q A F 1 F M V L T 1 V Y L S

Fig 2 Predicted transmembrane-spanning regions of the S cere- visiae a-subunit and corresponding regions in the E coli a-subunit

Identical residues are shown in boldface

hydrophobicity p e r residue o f 2.0 I n addition, the

second and third m a j o r peaks are contiguous, due to a

s h o r t e r polar region T h e six p r e d i c t e d t r a n s m e m b r a n e

spans in the yeast p r o t e i n are shown in Fig 2 In each

case, spans o f 28 residues are shown, a l t h o u g h it is

expected that the actual lengths m a y vary f r o m 22 to 30

residues C o r r e s p o n d i n g s e q u e n c e s from the E coli

p r o t e i n are also shown, with four to eight identical

residues f o u n d b e t w e e n each pair O n e o f the least

similar pairs, that o f span IV, also contains the E coli

span with the lowest hydrophobicity In Table I, this

region o f the a - s u b u n i t f r o m several diverse organisms

is c o m p a r e d A p a t t e r n o f low s e q u e n c e similarity can

be seen t h r o u g h o u t the series, but the average hydro-

phobicity p e r residue varies f r o m 2.08 to - 0 1 1 T h e r e -

fore, b a s e d on h y d r o p a t h y data alone, the identifica-

tion o f this region as a t r a n s m e m b r a n e span is some-

what uncertain, assuming that all a-subunits have the

same n u m b e r o f t r a n s m e m b r a n e spans

A m p h i p a t h i c a-helices in the a - s u b u n i t

T h e F o u r i e r analyses o f the hydrophobicities o f the

E coli and yeast a-subunits are p r e s e n t e d in Fig 3

T h e a m p h i p a t h i c index ~ h , ( / z ( w ) ) is plotted as a func- tion o f the amino-acid residue number A value o f

> 2.0 is indicative that an a-helix is a m p h i p a t h i c [36] S e g m e n t s with potential h y d r o p h o b i c m o m e n t s within the predicted t r a n s m e m b r a n e spans are listed in Table II Most o f the t r a n s m e m b r a n e spans contain regions with h y d r o p h o b i c m o m e n t s and there is consid- erable similarity b e t w e e n the two proteins In each case, there are no h y d r o p h o b i c m o m e n t s f o u n d in span III In each pair o f spans II, IV, V and VI, there are

h y d r o p h o b i c m o m e n t s at similar locations In general, the polar faces of these predicted helices contain sev- eral polar but u n c h a r g e d residues

M a n y potential a m p h i p a t h i c a-helices are f o u n d in the the e x t r a - m e m b r a n e regions and these are listed in Table III A m o n g these segments, there is m u c h less

TABLE II

Transmembrane segments of the a subunit with high hydrophobic moment

Segments that appear to be homologous are listed on the same line The numbers in parentheses indicate the positions of the residues within the transmembrane spans ~ is the a-helical amphipathic index as defined in Materials and Methods Polar face residues are listed in the order in which they appear in the sequence

I (8-19) 45- 56 2.31 SSG

I (18-31) 46- 59 2.41 TTSN

II (16-28) 111-123 2.52 AVND II (16-31) 105-120 3.57 IANS

VI (6-16) 230-240 1.97 EGQ

~u

A

Residue number

4

3

~ 2

0

Residue number Fig 3 Amphipathic a-helical index plots for (A), the E cell a-sub-

unit and (B), the S cerevisiae a-subunit using a window of 11

residues Peaks indicate segments with hydrophobic moments, if

a-helical

correspondence between the E coli and yeast proteins,

consistent with the more limited sequence similarity in

these regions One exception is the region between

spans IV and V, EGVSLLSKPVSLGLRLFGN, (E

coli resdiues 196-214) This is a highly conserved re-

gion among all a-subunits and contains an arginine

residue (Arg-210) shown by mutagenesis to be essential

for proton translocation [37,38]

Near the amino-terminus of the E coli a-subunit is

a segment of 13 residues, QDYIGHHLNNLQL (9-21),

predicted to be an amphipathic a-helix In the region

between spans I and II, the E coli a-subunit has three

segments, LFRSVAKKATSG (59-70), QTAIELVIGF

(75-84) and KDMYHG (91-96) with predicted amphi-

pathic a-helices, separated by two probable turns: VPGK (70-73) and VNGSV (86-90) Between spans II and III is a segment of 16 residues predicted to form

an amphipathic a-helix: L P I D L L P Y I A E H V L G L (121-136) In addition, a second segment, which would

be contiguous with the transmembrane span, could also form an amphipathic helix, PALRVVPSADV (137- 147), with arginine and aspartic acid on the polar face The segment connecting span III and IV, MKGIG- GFTKE, while amphipathic if projected as an a-helix, contains a region GIGG likely to be a turn

Variational moment analysis of a-subunits

Fourier analysis of sequence variation is plotted in Fig 4 In Fig 4A, five fungal sequences (Refs 22, 40-42 and Lang, B.F., personal communication) have been analyzed, in which the pairwise sequence identi- ties are about 40-70% The alignment of these se- quences is shown in Fig 5 Two of the predicted transmembrane spans, II and VI, are associated with peaks of value greater than 2.0, indicative of an a-helix with a single face in contact with membrane lipids In each span, the most polar residues are conserved: N-110, E-233 and Q-240 The other four transmem- brane spans are associated with regions having values

of 1.0 or less

Similar results, shown in Fig 4B were obtained from

a variability analysis of the a-subunit from E coli and

V alginolyticus, the only a-subunit known with greater

than 40% identity with the E coli protein [23] Again,

spans II and VI have values greater than 2.0, indicating

a face in contact with membrane lipids In the regions

of spans I, III, IV and V and at the amino-terminus there are a large number of peaks against a broad background To resolve these peaks, the calculations were redone with a window of 11 residues This allows detection of a-helical periodicity that is exhibited by segments shorter than a transmembrane helix These results are presented in Fig 4C

TABLE III

Extramembrane segments in the a subunit with high hydrophobic m o m e n t

Loop I-II refers to the extramembrane segment connecting the putative spans I and II ~ is the a-helical amphipathic index as defined in Materials and Methods hav e is the average hydrophobicity per residue using the GES parameters [16]

Region residues ~ h ave region residues ~ h ave

N-terminus 9 - 21 2.68 - 1.60

Loop I-II 5 9 - 73 2.36 - 0.72

Loop I-If 75- 88 2.51 0.38

Loop I-II 86- 96 1.96 - 1.39

Loop II-III 121-136 2.96 0.24

Loop II-III 135-147 2.27 - 0.28

Loop III-IV 168-179 2.51 - 0.62

Loop IV-V 191-215 2.82 0.13

N-terminus 6 - 17 2.89 - 1.22 Loop I-II 73- 86 3.08 - 1.27

Loop III-IV 136-148 2.08 0.02 Loop IV-V 166-190 3.48 0.13 Loop V-VI 208-225 2.82 2.22

Using a window of 11 resdiues, only spans II and VI

have broad peaks with values greater than 2.0 In other

regions, several narrow peaks can be identified, which

correspond to segments of approx 11 or fewer residues

with a-helical periodicity in sequence variation The

previously identified amphipathic helix at the amino-

terminus, Q D Y I G H H L N N L Q (9-20) exhibits a sharp

peak in the variability plot, indicating a conserved

helical face, shown in projection in Fig 6A Examina-

tion of the sequence reveals that the conserved face

contains most of the nonpolar residues, including the

two histidines, while most of the polar residues vary

The carboxy-terminal half of span I, L G L L F V L F R S V

(52-63), shows a very high variational moment (3.92),

in which the two polar residues arginine and serine

vary The region connecting spans III and IV,

I K M K G I G G F T K (166-177) has a high variational mo-

ment that corresponds closely to the region (168-179)

previously shown to posess a hydrophobic moment

The carboxy-terminal half of span IV, A F I P V N L I L E G

(187-197), also has a very high variational moment

(3.61), but this region does not contain a significant

hydrophobic moment Finally, a region (200-211) with

a high hydrophobic moment containing the essential

2

1

Residue n u m b e r

3

2

1

0

Residue n u m b e r

4

2

1

R e s i d u e n u m b e r

Fig 4 a-helical index plots of variability for (A) the S cerevisiae

a-subunit and (B) the E coil a subunit using a window of 21

residues In (C) the E coli a subunit is analyzed using a window of

11 residues Peaks indicate segments that would exhibit a conserved

face if a-helical, i.e., a variational moment

Arg-210, thought to connect spans IV and V, also has a significant variational moment (3.17), shown in projec- tion in Fig 6B

Analysis o f b- a n d c-subunits

Among bacterial F1F 0 A T P synthases, some are thought to contain two identical b-subunits [10,23- 25,43,44], while others have one each of two similar subunits b and b' [26,27,45] Each b- or b'-subunit is predicted to contain a single transmembrane spanning region near its amino-terminus For variability analysis, the sequences of six different b-subunits, from bacteria with a single type of b-subunit, were used as shown in Fig 7 The ~ value for this segment of twenty one residues is 2.80, indicating an a-helix with a face in contact with membrane lipids Examination of the se- quences via helical wheel projection revealed that the conserved face contained two polar residues, Gin-10 and Lys-23

The sequences of 24 c-subunits were examined for variational moments and the results are presented in Fig 8 The two anticipated transmembrane helices show quite different results At the carboxy-terminus, including the conserved acidic residue (Asp-61 in E

coli), is a broad region with values of 2-3, indicating an a-helical face in contact with membrane lipids The maxima centered near residues 55 and 63 might indi- cate two a-helical regions, separated by a proline or glycine in most species [4] However, at the amino- terminus, no a-helical segments can be found with a conserved face The most highly conserved residues correspond to positions 14, 20, 23, 25, 27 and 29, which are always alanine or glycine in the 24 cases examined here, with a single exception These positions would appear on opposite faces in an a-helical projection, as shown in Fig 9A

Discussion

The use of variational analysis has previously been

applied to reaction center proteins from Rhodopseu-

d o m o n a s viridis and Rhodobacter pseudomonas [18,20]

and results were confirmed by the high resolution

structure determined for the R viridis proteins [46]

We have applied a similar analysis to a pair of Gram- negative bacterial a-subunits and to a group of 5 homologous proteins from fungal mitochondria for which high-resolution structural data are unavailable

In each case, two segments of 20-30 amino acids of low overall polarity were found to display sequence variation with a period of about 3.6 residues, consistent with the face of an a-helix This suggests that these segments form transmembrane a-helices that interact

on one face with membrane lipids

Similar analysis of the other two proteins that make

up the bacterial F0 complex, the b- and c-subunits,

S.cerevisiae M F N L L N T Y I T S

A.nidulans M Y Q F N F I L S

N.crassa M F N I L S

S.pombe M F Z T S

S.~re 5 2 S L Y T L T N N N

N c r ~ s a L L S E N Y

S ~ m ~ I I N L T P Y G S G

VarJ~ion 3 2 5 5 5 5 5 5 4 4

P L D Q F E I R T L

P L D Q F E I R D L

P L N Q F E I R D L

P L E Q F E L N N Y

P L D Q F E L K P L

1 1 3 1 1 1 2 3 4 2

N K I I G S R W L I

N K I I P N N W S I

N R L V S N S W S I

A K I V P Q K F G I

N Y L G S S R W G V

2 3 2 3 3 3 4 2 3 2

III

S ~ r e 1 0 1 T L F M F I F I A N L I S M I P Y S F A

An~ A L F I F I L V N N L I G M V P Y S F A

~ c r ~ $ 8 T L F I F I L I N N L I G M V P Y S F A

S ~ m ~ S L F V L I L F S N L L R L I P Y G Y A

C ~ a T I F N F I L I A N L I S M I P Y S F A

Vari~ion 3 2 1 4 2 1 2 3 3 1 1 2 3 2 2 1 1 2 2 1

S.ce~ 1 5 4 L F V P A G T P L P

A.n~ L F V P S G C P L G

N c r ~ s a L L V P A G C P L A

S ~ m ~ L F L P S G T P T P

C.p~a L F V P S G T P L A

V a r i ~ i o n 1 2 2 1 2 1 2 1 2 3

S ~ r e 2 0 7 N F M L I N L F T L

A.nid N I M T S G I L F F

N c r ~ s a N I M T S G I I F F

S ~ m ~ T F M G L N L I T F

~ a S L M S S S F L G F

V a r i ~ i o N 3 3 1 4 3 3 3 3 3 2

L V P L L V I I E T L

L L P L L V L I E F I

L L P L L V L I E F I

L I P L L V L I E F V

L V P V L V L I E A L

1 3 1 2 1 1 2 1 1 3 3

VI

V F G F V P L A M I L

F L G L I P L A F I I

F L G L I P L A F I I

I I G F L P I T V L V

V S G I I P I L A V V

3 4 1 3 3 1 2 3 4 3 3

I

F G L Q S S F I D L S C L N L T T F S L Y T I I V L L V I T

F S L N A N V L G N I H L S I T N I G L Y L S I G L L L T L G Y H

L S I D T L G N L H I S I T N I G F Y L T I G A F F F L V I N

F G F Y L F N Y H F D F S N F G F Y L G L S A L I A I S L A

L L I T D N L T F S I T N Y T L Y L I I V S L I I I F Y S

2 3 3 5 5 , 5 5 3 4 2 3 3 3 2 3 3 1 4 2 3 1 2 5 3 4 4 3 4 5 2 4 3 5

[!

S Q E A I Y D T I M N M T K G Q I G G K N W G L Y F P M I F

S Q E A I Y A T V H S I V I N Q L N P T K G Q L Y F P F I Y

S Q E S L Y A T I Y S I V T S Q I N P R N G Q I Y F P F I ¥

A M E A I Y F T M L N L V E N Q I H S S K T V S G Q S Y F P F I W

S V I A I Y D T I L N L V N G P I G R K G G Y Y F P L I F

2 3 2 2 2 1 3 1 3 4 2 3 2 5 3 2 2 3 4 5 5 5 4 4 2 3 4 1 1 1 3 2 3

IV

L S A H L V F I I S L S I V I W L G N T I L G L Y K H G W V F F S

S T 6 H F I L T F S M S F T I V L G A T F L G L Q R H G L K F F S

S T S H F V V T F A L S F T I V L G A T I L G F Q K H G L E F F S

T T A Q L I F T L G L S I S I L I G A T I L G L Q Q H K A K V F G

I S A Q L V A V V S F S L T L W I G N V V L G L Y L H G W G F F A

4 2 2 2 2 2 4 3 4 3 3 1 3 3 2 3 2 1 2 2 3 1 1 2 2 4 1 2 3 4 2 1 3

V

S Y I A R A I S L G L R L G S N I L A G H L L M V I L A G L T F

S Y L S R N V S L G L R L A A N I L S G H M L L S I L S G F T Y

S Y L A R N I S L G L R L A A N I L S G H M L L H I L A G F T Y

S Y I A R G L S L G I R L G A N I I A G H L T M S I L G G L I F

S Y A S R A I S L G L R L G A N I L S G H L L M L I L G S L I I

1 1 3 2 1 3 3 1 1 1 2 1 1 2 2 1 1 2 2 1 1 2 2 2 4 1 1 3 2 2 2 3

A I M I L E F A I G I I Q S Y V W T I L T A S Y L K D T L Y L H

A F S G L E L A I A F I Q A Q V F V V L T C S Y I K D G L D L H

A F S G L E L G I A F I Q A Q V F V V L T S G Y I K D A L D L H

A I S L L E F G I A F I Q A Y V F A I L T C G F I N D S L N L H

A I T I L E F G I A I I Q A Y V F S I L L S G Y I K D S V E L H

1 2 3 3 1 1 2 2 1 2 2 1 1 2 2 1 2 3 2 1 2 2 2 2 2 2 1 4 2 4 1 1

Fig 5 Amino-acid sequence alignment of five fungal a subunits The first aligned serine residue has been shown to be the first residue in the

processed protein of S cerevisiae and C parapsilosis [22] The predicted transmembrane spans (Fig 1) are designated by a line above the S cerevisiae sequence An alternate alignment of residues 25-52 (S cerevisiae) did not appreciably affect the variational analysis

L

0

Fig 6 a-Helical projections of (A), residues 9-21 and (B), residues

198-215 of the E coil a-subunit The conserved residues indicated

are those conserved in E coli and V alginolyticus and in the case of

(B) are also generally conserved among the five fungal subunits

E coli

V alginolyticus

PS3

B megaterium

B firmus OF4

P m o d e s t u m

6 T I L G Q A I A F V L F V L F C M K Y V W

6 T L L G Q A I S F A L F V W F C M K Y V W

21 T I I Y Q L L M F I I L L A L L R K F A W

18 D I L F Q L V M F L I L L A L L Q K F A F

9 S A L Y Q L L A F S V L L F F L S K F A L

14 N M F W Q I I N F L I L M F F F K K Y F Q

Fig 7 Alignment of five bacterial b-subunit sequences for variability

analysis The numbers indicate the positions of the first residues

shown in each case Sequences used are from Escherichia coli [10],

Pibrio alginolyticus [23], thermophilic bacterium PS3144], Bacillus

rnegaterium [25], Bacillus firrnusOF4 [45] and Propionigenium modes-

turn [24]

Residue number

Fig 8 a-Helical index plots of variability for 24 c-subunits using a

window of 21 residues Numbering is that of the E coli protein

A

Fig 9 a-Helical projection of the amino-terminal residues of the E

coli c-subunit (10-27) The inner lines mark the two highly conserved regions Residues marked (*) are those labeled by the hydrepbobic

reagent TID [48]

showed that these proteins also exhibit a-helical se-

quence variation in regions that have been identified as

transmembranous These results suggest that the two

b-subunits, each with a single transmembrane span, are

on the periphery of the F 0 complex and have extensive

contact with lipids Hydrophobic labeling studies with

TID support this location of the b-subunits H o p p e et

al [47] found that all of the amino-terminal residues of

the b-subunit (1-35) could be labeled to some extent,

but that residues Gln-10 and Lys-23 were especially

resistant As shown here, these are conserved residues

that could be on a conserved face of an a-helix in-

volved in protein-protein interactions

The 9-10 c-subunits are thought to have two trans-

membrane regions separated by a highly conserved

turn Variational analysis indicates that the carboxy-

terminal region would form an a-helix with a con-

served face, or possibly two a-helical segments flanking

the conserved acidic residue, corresponding to the two

peaks in Fig 8 The amino-terminal region does not

exhibit a-helical variation, but would have two con-

served faces due primarily to six highly conserved

glycine or alanine residues if it were a-helical Other

evidence points to an a-helical amino-terminus The

length of the hydrophobic segment in E coli, 23 con-

secutive amino-acid residues without a hydrogen-bond-

ing side-chain, is consistent with a transmembrane a-

helix, but is too long for an extended structure like a

/3-strand Labeling by the lipophilic reagent T I D indi-

cates that both hydrophobic regions of the c-subunit

are exposed to the lipid phase [47] and the pattern of

labeled residues at the amino-terminus is consistent

with the face of an a-helix, with one exception Tyr-10

(see Fig 9) Therefore, we propose a model of the

c-subunit oligomer in which the amino-terminal a-helix

contacts two carboxy-terminal helices on opposite faces,

while the carboxy-terminal helix contacts two amino-

terminal helices on a single surface In this model,

residues Tyr-10, Ala-24 and Asp-61 all face the outside

of the oligomer, consistent with T I D labeling [47] and

mutagenesis [48,49] experiments

The Fourier analysis of the sequence variation of a-,

b- and c-subunits used different groups of homologous

proteins The sequences of c-subunits from a highly

diverse group of organisms was used, while the less

conserved a- and b-subunits required more closely

related sources It was determined empirically that

sequences with a pairwise identity of 40-70% were

optimal for the detection of a-helical variation This is

reasonable, since a single conserved residue per turn of

an a-helix (1/3.6), on average, will yield 28% sequence

identity Likewise, a single variable residue per turn

will yield 72% overall sequence identity Since only one

other a-subunit was suitably similar to the E coli

protein, a second group of five fungal proteins was also

analyzed

The methods of locating transmembrane a-helices

by computation of average hydrophobicity per residue have been proposed and analyzed by numerous authors [16,50-52] The recently published structure of bac- teriorhodopsin [53] confirms that seven of the eight predicted helices of Engelman et al [16] lie within the determined transmembrane regions, but that one is slightly shifted The 27-residue F-helix has an average hydrophobicity of only 0.695 (GES scale), considerably less than a typical transmembrane span This seems to confirm that transmembrane helices, like secondary structure in general, are subject to some nonlocal influ- ences and simple predictive schemes are not perfect In the a-subunit, five transmembrane helices can be pre- dicted with confidence from all organisms examined, but in some cases a sixth span (span IV) must be considered somewhat uncertain (see Table I) In gen- eral, it seems likely that the number of transmembrane spans must be conserved among a group of homolo- gous proteins In this case the region in question sepa- rates two conserved regions, span II and spans V and

VI, suggesting that the number of spans in the inter- vening region must be the same or differ by an even

number The span IV regions of a-subunits from Bacil-

lus species are significantly more polar than the bac- teriorhodopsin F-helix, bringing into question its loca- tion in the membrane Alternatively, since no complete amino-acid sequence of an a-subunit has ever been reported, the possibility of post-transcriptional or translational modification cannot be ruled out

Other proposals for the membrane topology of the

E coli a-subunit have been made on the basis of alkaline phosphatase gene fusion experiments In two different studies, largely similar results have been ob- tained, but have been interpreted in different ways Lewis et al [13] proposed four transmembrane spans similar to the first four here, but also proposed four shorter spans at the carboxy-terminus Bj~rb~ek et al [14] proposed eight spans, six similar to the six de-

e oligomer

Fig 10 Topological model of the a, b and c-subunits of the E coli

F o complex The bold lines connecting spans I and II, III and IV and

V and VI represent extramembrane loops on one side of the mem- brane, while the thin lines represent those on the opposite side In

an alternative model, where span IV is omitted, span II1 and one b-subunit would slide closer to span V and the last two spans would

have reversed orientation

scribed here, but two additional spans, one between I

and II and one between II and III The alkaline

phosphatase gene fusion technique [54] has been shown

to be reliable when tested on proteins with known

topology [55], but ambiguities in interpretation some-

times occur [56] Our results show that the two extra

spans proposed by Bj~rbaek et al [14] would have

segments with a-helical hydrophobic moments, but

have low overall hydrophobicity The four spans at the

carboxy-terminus predicted by Lewis et al [13] consist

of ten to thirteen residues each, which is conceivable if

they span the membrane in an extended conformation,

e.g., as /3-strands, or if they are a-helical but do not

extend to the membrane surface, as discussed by Lodish

[57] The variational analysis done here indicates that

most of those segments should be a-helical and located

at the surface of the protein Therefore, it is difficult to

reconcile these two sets of results

Amphipathic helices have been proposed to be com-

ponents of ion channels, such as in the acetylcholine

receptor [58], but the a-subunit may not conform to

that type of ion channel Recent work [53] has shown

that the proton channel in bacteriorhodopsin is formed

primarily from three of its seven transmembrane he-

lices, but only half of the residues in the channel have

polar character (13 of 26) Furthermore, another 25

residues with polar character are found in the trans-

membrane region outside of the proton channel

Therefore, the detection of amphipathic a-helices

within membrane spanning regions could identify ei-

ther ion channel pathways or helix-helix contacts Polar

residues involved in helix-helix interactions might be

important in stabilizing the tertiary structure of mem-

brane proteins The a-subunit is likely to be more

similar to bacteriorhodopsin, since it constitutes at

least part of a proton channel The role of retinal as

transducer might be played by the c-subunits In the E

coli a-subunit, amphipathic helical segments were de-

tected in all of the putative transmembrane spans

except III In both the E coli and yeast proteins, span

II is predicted to be the most amphipathic helix and

spans V and VI contain glutamyl and histidyl residues

thought to be essential for proton translocation [59-61]

Recent mutagenesis [62] of the E coli a-subunit sug-

gested that residues Asp-124 and Arg-140 are part of

the proton channel Therefore, in analogy with the

proton channel of bacteriorhodopsin, we propose that

these three spans provide most of the residues in the

proton channel Other amphipathic segments, such as

those found in spans I and IV might be involved in

protein-protein interactions

On the basis of these results, a model showing the

relative location of transmembrane segments is pre-

sented in Fig 10 Because of short connecting loops,

span V must be close to span VI and span III must be

close to span IV (assuming six transmembrane spans)

Span V is close to the c-subunits because of the importance of Glu-219 and Arg-210 (in the loop) in proton translocation Spans II and VI are adjacent to span V to form the channel and are exposed to lipid Both b-subunits are also exposed to lipid One b-sub- unit is located next to span VI because Pro-240 muta- tions in the a-subunit can partially suppress the effects

of Gly-9 -, Asp mutation in the b-subunit [63,64] Of the remaining two transmembrane spans, I is placed more peripheral to the complex than is III on the basis

of its hydrophobic moment In the model, span III has two opposite faces in contact with protein and two in contact with lipid, which could account for the lack of both hydrophobic and sequence variational moments The highly conserved region between spans IV and V

is proposed to lie parallel to the membrane, near the c-subunit surface

This model is intended to be a guide for site-di- rected mutagenesis and for site-specific chemical label- ing Such experiments are currently underway

Acknowledgements

We thank B Franz Lang for his unpublished DNA

sequence of S pombe ATP 6 This work was supported

by the Southern Methodist University Research Coun- cil and by USPHS Grant GM-40508

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