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INTRODUCTION |
In any type of cell, a multitude of integral membrane proteins is
simultaneously synthesized and integrated into various membranes followed by association to homo- or heterooligomeric complexes. To
ensure specific assembly, their subunits must present complementary recognition domains to each other. These domains may be located on the
ectodomains and/or the transmembrane segments
(TMSs).1 Interactions between
TMSs are currently intensely studied, since they usually form
autonomous
-helices and have been found to direct subunit assembly
or support correct folding of many membrane proteins (1, 2).
Biochemical and functional analyses, molecular modeling, and structural
studies indicated that the self-assembly of transmembrane helices is
driven by a close packing of their characteristically shaped surfaces.
These packing interactions may result in pairs of
-helices with a
right-handed twist as exemplified by glycophorin A (3, 4) and probably
by synaptobrevin II (5). Other TMS interactions involve a leucine
zipper type of side-chain packing as known from certain soluble
proteins. Within soluble leucine zippers, the interacting residues form repeated heptad (abcdefg) motifs. Residues at a-
and d-positions constitute the hydrophobic core of the
interfaces; side-chains at the e- and g-positions
are frequently charged, form salt bridges to each other, and make
hydrophobic contacts to the core (6). Heptad motifs were also suggested
to form the TMS interfaces of phospholamban (7, 8) and the M2 proton
channel (9). Based on a quantitative evaluation of high resolution
structures, we recently confirmed previous observations (10, 11) in
demonstrating that TMSs primarily interact via a leucine zipper type of
packing within bacteriorhodopsin, the photosynthetic reaction center, and cytochrome c oxidase. There, the heptads are repeated on
average 2-3 times, and the motif gaxxdexgaxxdexga covers
the central parts of the membrane-spanning interfaces. Salt bridges are
absent due to the hydrophobic nature of most membrane-embedded residues
(12).
To establish a simplified model of membrane-spanning leucine zipper
domains, we designed artificial TMSs on the basis of leucine and
alanine residues. We show that an oligoleucine sequence or a
gaxxdexgaxxdexga motif of leucine residues elicits specific self-assembly in membranes and in detergent solution. Interestingly, variants of this motif are found within the TMSs of a diverse set of
natural membrane proteins, where they appear to be important for
oligomeric assembly.
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EXPERIMENTAL PROCEDURES |
Plasmid Constructs--
Construction of plasmids pToxR
TM and
pSNiR
TM was described previously (5, 13). All other pToxR constructs
were made by ligating synthetic oligonucleotide cassettes encoding the
desired sequences into the plasmid pHKToxR(TMIl4)MalE (14)
previously cut with NheI and BamHI. For the
nuclease A fusions, the oligonucleotide cassettes were ligated into
plasmids pSNiR (5) or pSNiR2 previously cut with
NheI and BamHI. Details on the pSNiR and pSNiR2
plasmids will be described elsewhere. All constructs were verified by
dideoxy sequencing.
ToxR Activity Assays--
Transcription activation was
determined upon expression of the pToxR constructs in the indicator
strain FHK12 as described (15). 0.4 mM isopropyl
1-thio-
-D-galactopyranoside was added to the cultures to
enhance the dynamic range of the produced
-galactosidase signals (in
Miller units (MU), means ± S.D.) elicited by the different constructs in several independent experiments. This effect is thought
to result from isopropyl
1-thio-
-D-galactopyranoside-induced expression of an
F'-plasmid-encoded truncated
-galactosidase, which competes with
full-length enzyme in the formation of functional tetramers. The
previously (15) described construct pToxR/GPA13 elicited 1240 ± 298 MU under these conditions.
Gel Filtration Chromatography--
pSNiR and pSNiR2 fusion
proteins were expressed in BL21(DE3)pLysS cells (Novagen), solubilized
in 25 mM HEPES, pH 7.9, 0.5 M NaCl, 2% CHAPS,
1 mM EDTA and quantitated as described (5). Volumes of 300 µl at concentrations of 4 or 20 µM fusion protein were
separated on a Superdex 200HR 10/30 column (Amersham Pharmacia Biotech
FPLC system) using a flow rate of 0.5 ml/min and 25 mM HEPES, pH 7.9, 0.5 M NaCl, 1% CHAPS, 1 mM EDTA
as running buffer. Fractions of 0.5 ml were collected and analyzed for
fusion protein with a dot blot procedure (16) using the 9E10 monoclonal
antibody directed against the c-myc marker epitope for
detection. The elution profiles were constructed from the antigen
content, and the apparent molecular weights were calculated with
reference to standards given in the legend to Fig. 3.
Data Base Searching--
The Swiss-Prot data base (release 35.0)
was searched with the
LLXXLLXLLXXLLXLL motif
using the Findpatterns option of the HUSAR sequence analysis package
made available by the German Cancer Research Center (Heidelberg). Up to
three mismatches were allowed. To selectively retrieve TMSs, any amino
acid except the charged residues lysine, arginine, glutamate,
aspartate, or the helix-breaker proline was allowed for those positions
not occupied by leucine.
Miscellaneous Methods--
Western blotting was done as
described with an antiserum recognizing the maltose-binding protein
(MalE) moiety of the constructs, and the bands were quantitated
densitometrically (13, 15). The ability of our constructs to complement
the MalE deficiency of PD28 cells was tested by measuring the cell
densities of transformed bacteria in minimal medium containing maltose
at 640 nm after different growth periods (13). NaOH extraction was done
as described (17) by vortexing whole bacteria with cold 0.1 M NaOH followed by centrifugation to separate soluble from
membrane-bound proteins.
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RESULTS |
A Model of Membrane-spanning Leucine Zipper Domains--
Leucine
is the most prevalent amino acid within the interface of leucine
zippers (18), which is probably related to its ability to adopt
multiple conformations (19). We therefore reasoned that the flexible
leucine side chain may be particularly well suited to form a well
packed membrane-spanning leucine zipper. The methyl side chain of
alanine, in contrast, is expected to be too small for efficient
interaction with other alanine residues. This prediction was tested by
comparing the self-association of oligoleucine and oligoalanine
sequences, which are known to form stable
-helices (20, 21).
One of the experimental approaches we used is based on an engineered
version of the ToxR transcription activator. This protein is anchored
by a single TMS of choice within the inner membrane of expressing
Escherichia coli cells, where it is thought to exist in a
monomer/dimer equilibrium. The dimeric form binds to the cholera toxin
promoter, thus activating expression of a downstream lacZ
gene in a reporter strain (Fig. 1; Ref.
14).
-Galactosidase expression is therefore diagnostic of ToxR
self-assembly in the membrane. We previously established this system as
a sensitive tool to study TMS interactions using the structurally well
characterized glycophorin A TMS dimer for reference (13, 15).

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Fig. 1.
Functional organization of ToxR chimeric
proteins. The cytoplasmic domain ToxR is linked via a TMS of
choice to the periplasmic MalE moiety. Upon dimerization, ToxR binds to
the ctx promoter, thus initiating lacZ
transcription in the indicator cells. TM, transmembrane
segment; MalE, maltose-binding protein; OM, outer
membrane; IM, inner membrane.
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Here, we found that a sequence of 16 leucine residues (designated L16)
elicited strong transcription activation (924 ± 209 MU; mean ± S.D.). In contrast, a sequence of 16 alanine residues (designated
A16), elicited only a weak signal (210 ± 53 MU) (Fig. 2, A and B). This
suggests that the oligoleucine sequence self-assembles in the membrane,
whereas the oligoalanine sequence stays largely monomeric. Thus, the
latter can be used as host for a leucine zipper motif. Based on the
gaxxdexgaxxdexga motif representing the central parts of
most transmembrane helix-helix interfaces within crystallized membrane
proteins (12), a simplified version of a membrane-spanning leucine
zipper interaction domain was designed. In this model, the
a, d, e, and g positions
are occupied by leucine and all others by alanine. The construct with
this hybrid sequence (AZ2) self-interacted to a similar degree
(929 ± 186 MU) as the parental L16 protein (Fig. 2, A
and B). To demonstrate that the leucine residues contained
within AZ2 constitute the helix-helix interface, we mutated some of
them to alanine and assessed the consequences for self-interaction.
None of the single mutations made (L2A, L5A, L9A) significantly reduced
the signal (data not shown). However, when either four a and
d (L2A/L5A/L9A/L12A) or four g and e
(L6A/L8A/L13A/L15A) positions were simultaneously mutated, the signal
dropped by about 50% (516 ± 106 or 596 ± 102 MU). Thus,
the leucine residues are critical for the interaction and, hence, most
likely make up the interface. Further, ad- and eg-positions seem to be of similar importance for
helix-helix packing. Introducing a glycine-proline pair into the center
of the AZ2 sequence (L9G/A10P) similarly affected the interaction (584 ± 100 MU), consistent with the known destabilization of
-helices by glycine (22) and their kinking by proline (23) residues. We also replaced the leucines of AZ2 by three different random sequences consisting of the most abundant residues found within TMSs
(leucine, isoleucine, valine, phenylalanine, alanine) (24) while
maintaining total hydrophobicity and side-chain surface (25). Compared
with AZ2, these random sequences also self-assembled much less
efficiently, thus emphasizing the superior suitability of the leucine
side chain for helix-helix packing (e.g. "random," 446 ± 72 MU; Fig. 2, A and B, and data not
shown). The reductions in signal strength of the mutants compared with
AZ2 are statistically highly significant (two-tailed Student's
t test, p < 0.001).

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Fig. 2.
Transcription activation,
expression, and membrane incorporation of ToxR constructs with
artificial TMSs. A, TMS sequences aligned to the
underlying heptad pattern. Leucine residues of the zipper variants are
shaded for clarity. B, different levels of
transcription activation elicited by the different constructs in FHK12
cells indicate sequence-specific TMS assembly in the membrane. The
bars represent mean specific -galactosidase activities
calculated from numbers of data points given for each construct;
error bars denote S.D. C, expression
level and membrane association in FHK12 cells. tot, the
total cell content of most ToxR proteins was similar as revealed by the
staining intensities of the 65-kDa proteins upon Western blotting
(densitometric quantitation of seven independent blots established that
the average levels of the mutant TMSs ranged from 98 to 111% of the
parental AZ2 protein), whereas ToxRA16 was overexpressed; P,
the alkali-extracted membrane pellet quantitatively retained all
constructs except ToxRA16; SN, the alkali supernatant
contained part of ToxRA16 but none of the other proteins. The order of
samples corresponds to that in B. D, functional
complementation of MalE deficiency to assess correct membrane
incorporation. All constructs except the control construct ToxR TM
allowed for similar rates of PD28 cell growth, thus confirming their
correct Nin-Cout integration. The individual
data points represent means from five independent experiments.
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Comparing the concentrations of our ToxR constructs by Western blot
analysis indicated that most of them were expressed at similar levels,
whereas consistent overexpression was noted for the A16 construct (Fig.
2C). When we extracted the cells with NaOH to separate
membrane proteins (pellet) from soluble proteins (alkali supernatant)
(17), all constructs cosedimented quantitatively with the membranes as
expected except A16, which could be partially alkali-extracted (Fig.
2C). Thus, a fraction of the A16 protein seems to remain in
a soluble compartment, which is probably due to the comparably low
hydrophobicity of the oligoalanine sequence. This fraction is thought
not to interfere with the assay. To assess correct integration of the
proteins into the inner membrane, we tested their ability to
functionally complement the MalE deficiency of PD28 cells. Due to a
MalE deletion, this E. coli strain is unable to grow in
minimal medium with maltose as the only carbon source (26). In cells
expressing correctly inserted ToxR membrane proteins with the ToxR
moiety facing the cytoplasm and the MalE domain exposed to the
periplasmic space (see Fig. 1), however, the MalE domain allows maltose
uptake and thus cell growth (13, 14). Here, expression of all
constructs including A16 complemented the MalE deficiency of PD28 cells
to comparable degrees (Fig. 2D). In contrast, a control
construct where the TMS is deleted (ToxR
TM) proved unable to support
cell growth as expected from its presumed cytoplasmic localization. In
sum, equivalent amounts of all ToxR proteins analyzed here for
self-assembly appear to be correctly integrated into the inner
bacterial membrane, and the obtained
-galactosidase activities can
thus be directly compared.
To examine self-assembly of our artificial TMSs by an independent
approach, their oligomeric states were directly compared in detergent
solution (Fig. 3). The L16, A16, AZ2,
TM, L9G/A10P, and "random" sequence segments were genetically
fused to the C terminus of a fusion moiety based on
Staphylococcus aureus nuclease A, a monomeric soluble
protein. The fusion proteins were overexpressed in E. coli,
solubilized with CHAPS, and subjected to gel filtration chromatography
at concentrations of 4 or 20 µM. When injected at 20 µM, both L16 and AZ2 fusion proteins eluted as broad
peaks with mean apparent molecular masses of ~300 kDa plus minor
peaks at 47 kDa. At 4 µM, the 300-kDa peaks were
decreased in favor of the 47-kDa peaks, indicating equilibrium between
both forms of the proteins (data not shown). Whereas the 300-kDa peaks
clearly indicate assembly to multimers whose stoichiometry is currently not clear, the 47-kDa peaks most likely reflect monomers that may
migrate at increased apparent molecular weights due to bound detergent
(calculated molecular masses: L16, 21.2 kDa; AZ2, 20.9 kDa). In
contrast to that, the
TM, A16, L9G/A10P, and "random" constructs
gave rise to major peaks at 17, 22, 31, and 41 kDa at both
concentrations. These peaks are consistent with monomers (calculated
masses: 19.5, 20.5, 20.9, and 20.9 kDa, respectively) whose migration
may be influenced by different amounts of bound detergent depending on
the presence and the hydrophobicity of the hydrophobic segments.

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Fig. 3.
Oligomeric assembly in detergent
solution. Nuclease A fusion proteins were expressed, solubilized
in CHAPS, and subjected to gel filtration chromatography at
concentrations of 20 µM. A, the L16 and AZ2
constructs assembled to ~300-kDa oligomeric complexes accompanied by
47-kDa minor peaks probably representing monomers. B, the
major fractions of all other analyzed proteins migrated at apparent
molecular weights consistent with monomers containing different amounts
of bound detergent. Elution profiles are compared with the positions of
marker proteins (vitamin B12, 1.35 kDa; carbonic anhydrase; 29 kDa;
bovine serum albumin, 67 kDa; alcohol dehydrogenase, 150 kDa;
thyroglobulin, 669 kDa). The chromatograms are normalized relative to
their highest peaks.
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Taken together, two independent experimental approaches indicate that
both the oligoleucine sequence and the model leucine zipper motif AZ2
self-assemble in a sequence-specific way in membranes as well as in
detergent solution.
Self-assembly of Leucine-rich Natural Transmembrane
Segments--
Given the self-assembly of the AZ2 model, we assessed
whether TMSs with similar leucine patterns exist in naturally occurring proteins. The Swiss-Prot data base was searched for hydrophobic sequence segments with the motif
LLXXLLXLLXXLLXLL allowing
for up to three mismatches. This search yielded 38 predicted N-terminal signal sequences, 30 TMSs predicted within polytopic membrane proteins,
and 15 predicted TMSs from bitopic membrane proteins when homologous
proteins from different species were counted only once. Whereas the
signal sequences and TMSs of polytopic proteins were not further
investigated here, the TMS sequences corresponding to the bitopic
proteins are shown in Table I.
Self-interaction of a subset was examined with the ToxR system. The TMS
eliciting the strongest signal was derived from the erythropoietin
receptor followed by the TMSs of the Friend spleen focus-forming virus envelope protein, E-cadherin, and hemagglutinin of canine distemper virus. Other TMSs corresponding to papillomavirus E5 protein, mouse
poliovirus receptor homolog, and chick asialoglycoprotein receptor gave
rise to intermediate values suggesting lower levels of self-assembly
(Fig. 4A and Table I). A
Western blot run for control revealed roughly similar expression levels
(Fig. 4B).

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Fig. 4.
Transcription activation and expression of
ToxR constructs with natural TMSs. A, transcription
activation in FHK12 cells reflects various levels of self-assembly of
the TMSs whose amino acid sequences are given in Table I. The
Swiss-Prot identifiers are explained in the legend of Table I. The
bars represent mean specific -galactosidase activities
averaged from 24-32 data points; error bars
denote S.D. Arrowheads indicate the signals elicited by the
L16 and A16 sequences for comparison (see Fig. 2). B,
Western blotting revealed roughly similar expression levels for the
different proteins. The order of samples corresponds to that in
A.
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The data predict that these TMSs are important for oligomerization of
the corresponding proteins. A survey of previously reported experimental evidence and our own experiments indicated this indeed to
be the case for several of these proteins or related homologs as
discussed below.
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DISCUSSION |
We demonstrate that an artificial TMS of leucine residues
efficiently self-assembles in membranes and in detergent solution. A
heptad motif of leucine residues suffices to elicit self-assembly, which therefore is thought to be driven by the type of side-chain packing known from leucine zipper interaction domains. The main implications of our results are 2-fold. (i) They establish a simplified model system of short membrane-spanning leucine zippers. (ii) They
suggest that similar interaction domains may play a role in
subunit-subunit recognition of certain natural membrane proteins.
Structural Aspects of Membrane-spanning Leucine Zippers
We assume that the L16 and AZ2 TMSs form
-helical bundles upon
self-assembly. Self-assembly is thought to involve self-complementary helix surfaces that associate which each other via a
"knobs-into-holes" type of side-chain packing characteristic of
leucine zippers (6). The highly flexible leucine side chain (19) may be
particularly well suited for this type of packing interaction.
Consistent with this concept, leucine-rich heptad motifs have
previously been applied in the design of helix bundles forming
transmembrane ion channels (27) or of a folded polytopic membrane
protein (28). On the other hand, leucine helices have frequently been
used as experimental models to study TMS interactions with lipid
bilayers. For some of these studies (20, 29-31), both termini of the
leucine helices were capped with lysine residues whose repulsive
interaction may keep them in a monomeric state (31). In other cases
(17, 32, 33), their self-assembly as implied by our data should be
considered in interpreting the results.
A leucine zipper type of side-chain packing also accounts for TMS
interactions within phospholamban (7, 8), the M2 proton channel (9),
and different polytopic membrane proteins (12). In contrast to our
leucine-based model, these heptad motifs are made up of different
hydrophobic amino acids, which may generate the characteristically
shaped helix surfaces ensuring specific, stoichiometric, and/or
heterophilic assembly of these natural proteins.
Leucine Zipper Motifs in Natural Membrane Proteins
Data base searching identified leucine-rich heptad motifs within
different naturally occurring TMSs, and an analyzed subset of these
indeed exhibited various levels of self-interaction. This predicts a
role of TMS interactions in the assembly of the corresponding membrane
proteins. This is also implied by studies on the corresponding
full-length proteins as will be briefly discussed below.
Cadherins--
Cadherins are calcium-dependent
homophilic cell-cell adhesion molecules. Their function depends on
lateral clustering within the plasma membrane (34), which is believed
to involve interactions between extracellular (35) and juxtamembrane
domains (36). On the other hand, leucine-rich heptad motifs are
evolutionarily conserved in the TMSs of different cadherin families,
and our data demonstrating self-interaction of the E-cadherin TMS
suggest a role of TMS interactions in clustering. Strong support for
this hypothesis is provided by our recent experimental evidence
indicating that mutations reducing the TMS interaction likewise affect
the adhesive properties of full-length E-cadherin expressed in
eukaryotic cells.2
Erythropoietin Receptor--
The erythropoietin receptor
(EpoR)is required for erythrocyte maturation. In analogy to other
growth factor receptors, erythropoietin binding is thought to trigger
homo-dimerization followed by receptor activation (37). Apart from the
case of the Neu oncogene product, where a point mutation within the TMS
triggers ligand-independent receptor activation (38), the role of the
TMS in growth factor receptor activation is currently not clear. Since
ligand binding is translated into activation of cytoplasmic domains, it
has been postulated that the subunit-subunit interface of growth factor receptors extends across the membrane and that TMS interactions contribute to ligand-induced subunit assembly in a nonspecific way (1).
Our finding that the EpoR TMS is capable of self-interaction indeed
suggests its contribution to ligand-induced receptor assembly. Alternatively, the EpoR may exist as a preformed dimer activated by
ligand binding. Precedence for the latter model is given by the insulin
receptor or the aspartate chemoreceptor; in both cases, ligand-binding
activates preformed receptor oligomers (39). Ligand-independent
dimerization has also been proposed for the epidermal growth factor
receptor (40).
Viral Envelope Proteins--
Enveloped viruses enter the cytoplasm
of host cells upon fusion of viral and cellular membranes mediated by
fusogenic viral envelope proteins. These proteins exist as oligomeric
complexes (41), and both their fusogenicity and oligomerization appear to depend on their TMSs. For example, the influenza hemagglutinin TMS
is required for full membrane fusion (42) and stabilizes the trimeric
complex (43). Also, mutations of conserved leucine residues within the
TMS of the hemagglutinin-neuraminidase of Newcastle disease virus
affected tetramerization and fusion promotion (44). Extending these
findings, our data suggest a role of the TMS in oligomerization of
hemagglutinin-neuraminidase from canine distemper virus and of the
Friend leukemia virus envelope protein. Apart from homooligomerization,
a heterophilic and functionally important interaction has been reported
between the EpoR and the gp55 protein of Friend spleen focus-forming
virus, which is derived from its envelope protein (45). At the surface
of infected erythroid cells, the EpoR and gp55 form a noncovalent
complex, which results in erythropoietin-independent cell
differentiation (46). Complex formation is therefore thought to cause
persistent EpoR activation (47). Notably, both the gp55 TMS and the
EpoR TMS have been shown to be crucial for this heterophilic
interaction (48, 49). Since both TMS sequences have been identified by
our data base search and shown to self-interact, we propose that
formation of the heteromeric complex proceeds from preformed gp55 and
EpoR homomers.
E5 Protein--
The papillomavirus E5-protein is a transforming
membrane protein that exists as a disulfide-bonded dimer (50). Its
transforming activity presumably rests on interaction with, and
ligand-independent activation of, the receptors for epidermal growth
factor, colony-stimulating factor (51), or platelet-derived growth
factor (52). In the case of the platelet-derived growth factor
receptor, binding to the E5 protein has been directly demonstrated to
involve the TMSs plus extracellular flanking regions of both receptor
and E5 protein (53). Although the E5 protein extracellular region and
the glutamine residue within the TMS are important for activity (54),
we speculate that the leucine-rich surface of its TMS aids in homodimer
formation and/or binding to the various growth factor receptor TMSs.
Asialoglycoprotein Receptor--
The hepatic asialoglycoprotein
receptors remove abnormally glycosylated proteins from blood
circulation (55). The chick homolog exists as a homotrimer whose
formation and stability depends on the TMS and flanking sequences (56,
57). This is consistent with self-interaction of its TMS shown here.
These examples demonstrate that assembly of several different natural
membrane proteins depends on their TMSs as predicted by the presence of
leucine-rich heptad repeats. Future studies will show whether these TMS
interactions are based on the leucine zipper type of packing as
inferred for our self-assembling model TMSs L16 and AZ2. TMS
interactions may be modulated by the lipid composition of the
respective host membrane. Further, they may not be the exclusive cause
of subunit-subunit recognition but may be complemented by interactions
between extramembraneous domains in particular cases.