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INTRODUCTION |
Specific protein-protein interaction is an important process in
biology, not only in the formation of a stable quaternary structure but
also in transient interactions such as those in signal transduction and
control of gene expression. Although several organisms' complete
genomes are available, understanding the functions of many gene
products requires identifying interactions among the encoded proteins.
A variety of approaches have been developed in recent years to study
such interactions, including the development of the two-hybrid system
in yeast (1). Whereas the two-hybrid approach is well established for
soluble proteins, measuring the interaction of integral membrane
proteins is more difficult, and it is only recently that useful
approaches have emerged (2-5).
The finding by Kolmar et al. (6) that the transmembrane part
of the transcription activator ToxR from Vibrio cholerae
drives the dimerization of the DNA binding domains to activate the
ctx promoter and that this TM domain can be functionally
replaced by any other oligomerizing protein or peptide led to the
development of an Escherichia coli two-hybrid approach
measuring the homo-oligomerization of single
TM1 helices in the inner
membrane of E. coli (7, 8). Although these systems are very
useful for the measurement of homo-oligomerization (7-13), they cannot
report heterologous interactions.
Here we present a system to measure the heterologous association of
transmembrane helices in E. coli. Recently, an E. coli two-hybrid system based on the LexA DNA binding domain was
developed, and it was shown that coupling of this domain to any soluble
protein can be used to measure homo-oligomerization of a soluble
protein (14). LexA is a transcription factor with an N-terminal DNA binding domain containing about 70 amino acids and a C-terminal dimerization domain (for a review on LexA, see Ref. 15). LexA dimerization is required to repress transcription efficiently, and the
LexA DNA binding domain does not contribute to the dimerization of the
protein. The discovery of LexA DNA binding domain variants that bind to
different DNA sequence enabled the development of a system to analyze
heterodimerization of soluble proteins in E. coli (16).
To study the interaction of two different transmembrane domains, the
GALLEX system presented here was developed. Two LexA DNA binding
domains with different DNA sequence specificity were coupled to wild
type (WT) and mutated glycophorin A (GpA) TM helices and the
promoter/operator sequence contained one specific binding site for
each. Association of two separately expressed chimeras that interact in
the membrane represses the synthesis of a reporter gene,
galactosidase. The correct membrane insertion of the chimeric proteins
was tested as well as the ability of the system to measure either homo-
or heterodimerization of single TM helices. Using GALLEX, different
interactions between WT and mutated glycophorin A TM helices were
measured. By this method, it could be shown that the WT TM domain
interacts with other investigated TM helices to a certain degree, and a
strongly heterodimerizing pair of glycophorin A mutants was found. The
heterodimerization of two TM helices in a glycophrin A-like manner was
recently described as key to the formation of heterodimeric integrin
complexes (17). Integrins are a family of single spanning membrane
proteins that from
/
heterodimers. The analysis of the
interaction of the
4 and the
7 integrin
TM helices using GALLEX showed that both TM domains form weak homo- as
well as hetero-oligomers. Although heterodimer formation results also
from the interaction of a large extracellular domain (18), the role of
the transmembrane domains for the formation of these heterodimers was
recently discussed (17, 19).
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MATERIALS AND METHODS |
Plasmid Construction--
Molecular cloning was carried out
using standard techniques described in Ref. 20. All enzymes used for
PCR and cloning and plasmids were purchased from New England Biolabs.
The N-terminal part (corresponding to residues 1-87) of the WT
lexA gene from E. coli (21) was amplified by PCR,
introducing an NdeI site at the 5'-end and an
SacI site at the 3'-end. The restriction-digested PCR
fragment was ligated to the NdeI/SacI restriction-digested plasmid pMal-p2, resulting in the plasmid pLEX. A
fragment from the plasmid pccGpA (8), carrying the TM region of GpA
C-terminally fused to the maltose-binding protein (MBP) domain, was
amplified by PCR, introducing an XbaI site at the 3'-end of
the fragment. After digestion with SacI and XbaI, the fragment was ligated into the
SacI/XbaI-digested plasmid pLEX, resulting in the
plasmid pLGM. In construction of pLKM, for cloning reasons, a
K
cassette was amplified by PCR and
ligated to the SacI/BamHI restriction-digested
vector pLGM, replacing the GpA TM region and introducing a
SpeI site at the 5'-end of the malE gene.
For the measurements of heteroassociation, a two-plasmid system was
created, allowing the simultaneous low level expression of two
different chimeric proteins. The plasmid pLKM was restriction-digested with ScaI and PflMI, and the resulting termini
were filled by T4 DNA polymerase to generate blunt ends. The plasmid
pBR322 was restriction-digested with SspI/AvaI,
and the termini of the resulting 2743-bp fragment were filled with T4
DNA polymerase. The plasmid pACYC184 was restriction-digested with
AvaI/PvuII, and the termini of the resulting
2423-bp fragment were filled with T4 DNA polymerase. The fragment
originating from pLKM was cloned into the fragments of pBR322 and
pACYC184, finally producing the plasmid pBLM100 and pALM100,
respectively. These plasmids can be used for the measurement of hetero-
as well as homoassociation of single TM helices. For generating
plasmids expressing a chimera with a mutated LexA protein
(LexA408), the mutated gene was amplified by PCR from the plasmid
pSR659 (21). All cloning steps were identical as described above for
the WT gene. The fragment originating from pLKM(mut) was cloned into
the fragments of pBR322 and pACYC184, finally producing the plasmids
pBLM148 and pALM148, respectively. These plasmids can be used to
express a chimeric protein with the mutated LexA DNA binding domain,
and, in combination with pALM100 or pBLM100, they can be used to
measure heteroassociation of TM helices. The plasmids and E. coli strains used for testing the homo- and heteroassociation
capacity of given TM helices are shown in Fig.
1.

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Fig. 1.
Overview over the generated plasmids and the
used reporter strains. A homodimerizing fusion protein from one of
the WT LexA plasmids (pALM100 or pBLM100) will bind to the WT LexA
promoter/operator and represses expression of lacZ in
the genome of the reporter strain SU101. For monitoring
heterodimerization, one subunit is expressed from one WT LexA plasmid,
and the other one is expressed from the corresponding mutated
(mut.) LexA plasmid (combination 1 or
2). A heterodimerizing fusion will bind to the hybrid LexA
promoter/operator and represses the expression of
lacZ in the genome of the reporter strain SU 202. The
plasmids pALM are derived from pACYC184, and the pBLM plasmids are
pBR322-based.
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All further pBLM- and pALM-based constructs were made by ligating
synthetic oligonucleotide cassettes, which encode for the TM sequences
of interest, into the SpeI/SacI
restriction-digested vectors.
-Galactosidase Assay--
The association capacity of
different chimeric proteins was measured as the repression of reporter
gene (
-galactosidase) activity in the E. coli SU101
(homoassociation) and SU202 (heteroassociation) indicator strains. The
genotypes of these strains are described previously (16). To measure
dimerization, overnight cultures of freshly transformed SU101 cells
were grown in the presence of various IPTG concentrations, diluted to
an A600 = 0.1 and finally grown to an
A600 = 0.6 in LB medium containing the
appropriate antibiotics and IPTG. 100 µl of cells were used for the
measurement of the
-galactosidase activity as described in Ref.
21.
Test for Insertion and Orientation--
For maltose
complementation assays, a single colony of NT326 cells expressing the
LexA-TM-MalE chimeric proteins was cultured on M9 agar plates
containing 0.4% maltose, 1% ion agar, 0.02% IPTG, and 200 µg/ml
ampicillin. Plates were incubated at 37 °C for 3 days. Only if the
MBP portion of the chimeric protein is present in the periplasm will
the cells be able to use maltose as the carbohydrate source, since this
strain is deficient in endogenous MBP.
To prove that the chimeric proteins are associated with the membrane,
cells were extracted with NaOH as described in detail in Ref. 22. After
extraction, the pellet, containing stably associated membrane proteins,
was resuspended in SDS sample buffer. The proteins in the supernatant
fraction, which contains cytoplasmic, periplasmic, and peripheral
membrane proteins, were precipitated by 10% trichloroacetic acid and
finally resuspended in SDS sample buffer. Proteins were separated on
10% SDS-gels and blotted on nitrocellulose membranes for Western
analysis. Western analysis was performed using anti-MBP antibodies (New
England Biolabs), and blots were finally developed using goat
anti-rabbit alkaline phosphatase-conjugated antibody (Bio-Rad) with
nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate.
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RESULTS |
Outline of the Strategy--
A schematic of the system to measure
heterologous association of transmembrane helices in the E. coli inner membrane is shown in Fig.
2. The basic idea is to detect
association by suppression of
-galactosidase when two versions of
LexA domains bind to two adjacent, different DNA sequences. For initial
testing and optimization of the system, GpA TMs were used, because the
self-association of this transmembrane helix is very well characterized
(8, 23-25). Each of the individual single transmembrane fragments has an N-terminal LexA DNA binding domain and a C-terminal MBP domain from E. coli. The hydrophobicity of the transmembrane domain
functions as a membrane insertion signal, placing the LexA domain in
the cytoplasm and the MBP domain in the periplasm of E. coli. If the transmembrane domains interact, the LexA cytoplasmic
domains are in close proximity and can bind to the operator region,
repressing the expression of
-galactosidase. The expression of
chimera with noninteracting TMs results in a high
-galactosidase
activity due to the lack of repression. To measure heteroassociation,
two chimeras were simultaneously expressed from the plasmid pair
pALM148 and pBLM100. Whereas the pALM plasmids originate from the
plasmid pACYC184 and contain a tetracycline resistance gene, the pBLM plasmids are derived from pBR322 and contain the gene for
-lactamase, resulting in ampicillin resistance. In these plasmids,
the expression of the chimeric proteins is driven by the inducible
Ptac promoter, and until IPTG is added the transcription is
strongly repressed by the lacI gene encoding the Lac
repressor.

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Fig. 2.
The GALLEX assay for measuring TM helix-helix
interaction in a biological membrane. The TM domain anchors the
chimera in the cytoplasmic membrane of E. coli with the
C-terminal MBP domain located in the periplasm and the LexA DNA binding
domain in the cytoplasm. Interaction of the TM domains leads to the
formation of LexA heterodimers, which can bind to the operator region.
The binding of the LexA dimer results in repression of the reporter
gene (lacZ) activity.
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E. coli strain SU101 was used to measure the
homo-oligomerization capacity of a given TM helix. In this strain, the
lacZ gene is under the control of the wild-type LexA
recognition sequence (op+) stably integrated into the E. coli genome.
In E. coli SU202, the lacZ reporter gene is
placed under the control of a op408/op+ hybrid operator. This
asymmetric promoter is composed of half of the wild-type promoter plus
an altered half (16) (Fig. 2) and allows the binding of a LexA
heterodimer composed of one wild-type LexA DNA binding domain and one
altered domain (LexA408). It is reported that homodimers do not
recognize this hybrid operator so that heterodimeric proteins
alone efficiently repress the reporter gene activity (16). Therefore,
the capacity for heterodimerization should be measurable even if the
individual proteins homodimerize.
Homodimerization of Glycophorin A--
To test the system's
ability to measure the homodimerization of a given TM, the strongly
dimerizing GpA WT sequence and the nondimerizing G83I sequence (8, 24)
were fused to the LexA DNA binding domain and the MBP domain as
outlined under "Materials and Methods." In GALLEX, the
IPTG-inducible lacUV5 promoter drives the expression of the
chimeric proteins. The ability of the fusion proteins to repress the
reporter gene's activity was determined at different levels of protein
expression obtained upon varying the IPTG concentration (data not
shown). In the range between 0.005 and 0.01 mM IPTG, a
50-80% difference was measured between the
-galactosidase activity
of the strains expressing either the GpA WT or the GpA G83I chimera.
Based on these results, an IPTG concentration of 0.005 mM
was used for all further experiments testing for homodimerization in
E. coli SU101 (if not indicated differently). Although
higher IPTG concentration can result in lower
-galactosidase
activities, the difference in the
-galactosidase activity between
interacting and weakly interacting TMs (GpA WT versus G83I)
becomes reduced. It should be noted that the absolute difference in the
-galactosidase activity between the GpA WT and G83I chimeric
constructs also depends on the plasmid used for the expression: using
pALM100 as the parental plasmid, a difference in the
-galactosidase
activity of about 50% was measured, whereas the use of pBLM100 led to
differences of up to 80%. This observed discrepancy is most probably
explained by the different copy numbers of the plasmids.
The optimal length of the incorporated TM for effective gene repression
was determined. For this purpose, GpA TM segments of 16, 17, 18, and 19 residues comprising dimerization motifs (23) were cloned into the
plasmid pBLM100, and the reporter gene activity was measured in the
E. coli strain SU101 after induction of protein expression
by the addition of 0.5 mM IPTG. As shown in Fig.
3, insertion of a TM of 17 residues led
to the strongest repression. Remarkably, the repression of the
-galactosidase activity was about 10-fold stronger than for the TM
helix of 19 amino acids.

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Fig. 3.
Optimal length of the GpA TM domain.
A, amino acid sequence of the GpA TM domain. The length of
the helix was altered by successive removal of a residue from the
C-terminal, shown in italics. The underlined
residues were mutated as described. B, repression
of the -galactosidase activity mediated by the dimerization of the
expressed constructs in E. coli SU101. The bars
represent data combined from three independent measurements. Expression
of the chimeric proteins was induced by the addition of 0.5 mM IPTG.
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To measure the relative homodimerization of the WT GpA TM and its
mutants at interfacial residues (L75V, I76A, and G83I), the TM domains
were expressed from pALM100, and the
-galactosidase activity was
measured in E. coli SU101. As a control, the parental plasmid pACYC184 was transformed in E. coli SU101, and the
maximal
-galactosidase activity measured in this strain was set to
100%. The results shown in Fig. 4
demonstrate that the GpA TM domain with the G83I point mutation
interacts very weakly. The different GpA TM helices interact in the
order WT > I76A > L75V
G83I.

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Fig. 4.
Homodimerization of GpA WT and mutated TM
sequences. As a control, the parental plasmid pBR322 was
transformed into E. coli SU101, and the -galactosidase
activity was set to 100%. Introduction of the G83I mutation leads to a
complete loss of the interaction capacity while the other sequences
interact. Bars represent the -galactosidase activities of
three independent measurements, and activities are shown relative to
the strain harboring the parental plasmid. The bars
represent data combined from three independent measurements.
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The Chimeric Proteins Are Located in the Membrane and Have the
Designed Topology--
Membrane localization of the chimeric proteins
was shown by NaOH extraction. This method was used to separate membrane
proteins from cytoplasmic and periplasmic proteins (26). After rigorous extraction of the cells with NaOH, the expressed chimeras are only
found in the membrane protein fraction (Fig.
5A), so almost all of the
chimeric protein is bound to the cytoplasmic membrane and not localized
in the cytoplasm or periplasm.

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Fig. 5.
Test for insertion and orientation of the
chimeric proteins in E. coli.
A, Western analysis of E. coli cell extracts
after NaOH extraction. C, whole cells; S,
supernatant after NaOH extraction (soluble proteins); M,
pellet after NaOH extraction (membrane proteins). The expressed
chimeric proteins with a molecular mass of 54 kDa are found solely in
the membrane protein fraction (pellet). *, proteolyzed chimera.
B, malE complementation assay to test for
LexA(TM)MBP orientation. E. coli NT326 were transformed with
various constructs and cultivated on M9 agar containing 0.4% maltose
and 0.02% IPTG. The GpA TM sequences inserted were WT, L75V, I76A, and
G83I. The MBP expression from pMAL-p2 results in periplasmic location
of the MBP domain, whereas after expression from pMAL-c2, the MBP is
located in the cytoplasm. No TM, pBLM100-based
plasmid encoding for a non-TM-spanning domain.
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The topology of the chimeric proteins was examined by complementation
assays on M9 minimal medium. The E. coli strain NT326 lacks
endogenous MBP, resulting in the inability of the cells to transport
maltose into the cytoplasm. If the chimeric proteins are inserted into
the cytoplasmic membrane with the MBP portion localized in the
periplasm, the MBP domain will compensate for this deficiency, and the
cells will be able to grow on minimal medium with maltose as the only
carbon source. The results shown in Fig. 5B demonstrate that
all chimeric proteins with a TM domain can complement the endogenous
MBP deficiency, consistent with the correct insertion of the protein
into the E. coli cytoplasmic membrane with the MBP domain
located in the periplasm. And since the assay shows repression, the
LexA domains are located in the cytoplasm, and the topology is established.
Heterodimerization of WT Glycophorin A and Mutants--
For
initial testing and optimization of the system to measure
heterodimerization, the TM domains of GpA WT and GpA G83I were cloned
into the vector pair pALM148/pBLM100 (see Fig. 1). After transformation, strains were obtained that measure the
heterodimerization of the chimeric proteins (with respect to the LexA
DNA binding domains) driven by the homodimerization of the GpA WT TM
region. The other strain expresses the GpA G83I TM domain from both
plasmids, which should result in little association. As expected, when
the GpA WT chimeric proteins were co-expressed from the two different plasmids, the reporter gene was highly repressed at 0.01 mM
IPTG, in contrast to the strain expressing the GpA G83I chimeric
proteins (data not shown).
Although the GpA TM domain strongly self-dimerizes, which adds some
complexity to the measurement, this TM was used for a more thorough
characterization and optimization of the system, since the interaction
of these helices is already well documented (8, 23, 25). Further, it is
desirable that the system should be able to measure heteroassociation
of other TMs that strongly homodimerize.
The relative heterodimerization of the WT GpA TM and its mutants at
interfacial residues (L75V, I76A, and G83I) was measured in E. coli SU202. The results are shown in Fig.
6. The data obtained using the
heterosystem show that the WT GpA TM helix is able to form heterodimers
with each mutation in the order WT > I76A > L75V > G83I.

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Fig. 6.
Heterodimerization of GpA WT and mutated TM
helices measured in E. coli SU202.
Bars in A represent the -galactosidase
activities of three independent measurements and activities are shown
relative to the strain expressing the G83I chimera from both plasmids.
GpA TM sequences shown under the black
bar are expressed from pBLM100; the ones above
the bar are expressed from pALM148. In B, the
values of A are summarized in a table.
C, expression level of the chimera in each strain tested by
Western analysis. Antibodies were directed against the MBP
domain.
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The GpA WT sequence forms strongly interacting homodimers, resulting in
a high repression of the reporter gene (about 80% repression).
Interestingly, the WT sequence also forms heterodimers with each of the
other tested sequences, resulting in low
-galactosidase activity.
The relative activities are shown in Fig. 6, A and
B. Also, the L75V and I76A sequences form homodimers as well
as heterodimers with the other TM helices. Besides the formation of
heterodimers between GpA WT and any mutant, the results also show that
the two mutants L75V and I76A homodimerize to a significant extent. If
GpA I76A is expressed from pBLM100 and GpA L75V is expressed from
pALM148, the repression reaches about the level of the GpA WT homodimer
(22%), whereas if the TMs are expressed in the other orientation, the
activity is only repressed to 44%. Nevertheless, in this case, the
lacZ repression is almost as high as the GpA WT/I76A
interaction, and the general lower
-galactosidase activity seems to
be caused by the intrinsic asymmetry of the GALLEX system as discussed
below. The amino acid substitution G83I is highly disruptive, resulting
in high
-galactosidase activities, and none of the other mutations
formed a strong dimer with the G83I TM, resulting in high
-galactosidase activities for each heterodimer studied (Fig. 6,
A and B).
Heteroassociation of
and
Integrin TM Helices--
To
analyze whether an GpA-like interaction of the
and
integrin TM
helices can contribute to the formation of the heterodimeric
/
integrin as suggested in Ref. 17, the sequences encoding the two
integrin
4 and
7 TM helices were cloned,
and the tendency to form homo- and heterodimers was measured with the
GALLEX system. The membrane insertion and orientation of
the expressed chimera was tested as described above for the GpA TM
helices. The results of the GALLEX measurements shown in Fig.
7 indicate that each integrin TM helix
has a tendency to form dimers. Whereas the
4 TM helix
shows a slight tendency to form homodimers, the TM helix of the
7 integrin homodimerizes more strongly. The measurement of the formation of
/
heterodimers shows that the two TM helices tend to form heterodimers, although these are relatively unstable compared with the GpA WT homodimer as shown above.

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Fig. 7.
Homo- and heterodimerization of the
integrin 4 and
7 TM domains measured in E. coli SU202. A, the sequences of the TM
domains used for the GALLEX measurements. B, association
capacity of integrin 4 and 7 TM domains.
Bars, the -galactosidase activities of three independent
measurements. To test for homodimerization, one subunit was expressed
from both plasmids, and the capacity to form heterodimers was tested by
expressing the two TM domains simultaneously from both plasmids in
either orientation. C, expression level of the chimera in
the reporter strain tested by Western analysis.
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 |
DISCUSSION |
Advantages of the System--
The homodimerization of TM helices
has been intensively studied in recent years using reporters of
homo-oligomerization in the E. coli inner membrane (7, 8,
27). The ToxR-based systems have been used to measure the
homo-oligomerization of given TM domains (8-10) and to select for
sequence motifs that drive homo-oligomerization from random libraries
(11, 28). Although useful, these systems are limited to the
investigation of homo-oligomerization. The published systems are based
on the formation of a homodimeric ToxR DNA binding domain from V. cholerae, which is driven by the interaction of two identical TM
helices. The basic principles used are similar to the GALLEX system
presented here. Although with GALLEX, the repression of a reporter gene is measured in contrast to its activation, the activity of the reporter
gene (lacZ) is directly related to the formation of LexA (hetero-) dimers driven by the association of the TM segments. In
contrast to the ToxR-based systems, this system can measure homo-oligomerization of TM helices (using E. coli SU101) as
well as hetero-oligomerization (using E. coli SU202) even if
one or both of these helices homo-oligomerize. It was recently shown that the expression of chimeric soluble proteins from different plasmids can result in conflicting results (21) and that the rate of
repression seems to depend on the plasmid from which each TM helix is
expressed. Since controlling the level of protein expression from the
two different plasmids (pALM and pBLM) simultaneously is difficult and
the slightly different expression levels can obviously cause some
variations, comparison of experiments with the reciprocal fusions of
the TM helices is always recommended.
Other possible limitations of the system include the potential
inhibition of DNA binding of the LexA DNA binding domain caused by its
fusion to nonnative proteins as discussed in Ref. 29. Also, the length
of the TM helices and the localization of the oligomerizing interface
seem to be a critical factor. The results shown in Fig. 3 suggest that
the relative orientation of the soluble domains is important. Whereas a
high
-galactosidase activity is seen for the GpA TM helices of 18 or
19 residues, the TM helices of 16 or 17 residues seem to place the two
DNA binding domains in the right orientation for optimal DNA binding.
The 10-fold difference in the
-galactosidase activity of the
different TM lengths seen in Fig. 3 is not to be directly compared with
the 2-fold changes observed in the later experiments (Figs. 4, 6, and
7), since a higher concentration of IPTG was used for induction (0.5 versus 0.005 mM).
An optimized orientation of the interaction helical surface relative to
the soluble domains was also found in earlier studies with the ToxR DNA
binding domain coupled to the GpA TM helix (7). The observation of an
optimized spatial orientation of the interaction helical surface
relative to the soluble domains may make it necessary for an individual
experiment to optimize the length of a TM helix and the orientation of
the interacting interface relative to the soluble domains.
The analysis of the GpA TM helices that exhibits both homo- and
heteroassociation introduces an additional level of complexity to the
system, since multiple equilibria must be taken into account. If two
single TM helices do not homodimerize, the observed degree of
-galactosidase activity directly reflects the level of
heterodimerization of the two TM helices. If one or both helices
homodimerize, the observed level of interaction depends on the
individual strengths of dimerization, since the tendency of each TM to
form homodimers influences the concentration of free monomers for
heterodimer formation. In such a case, the measured values of
heterodimerization cannot be taken absolutely but demonstrates the
ability of two given TM helices to heterodimerize. In any case, the
simultaneous expression of the identical TM from both plasmids should
always be done to check for homodimerization and to evaluate the
results for the heterodimers properly. In the case of the analysis of the heteroassociation of one or two homodimerizing TM helices, the
observed strength of the interaction can be taken not as absolute but
as a trend.
Heterodimerization of Glycophorin A--
The dimerization of
glycophorin A is one of the best characterized TM helix-helix
interactions to date. The plethora of data available on the
homodimerization of GpA WT and mutants made it possible to optimize the
GALLEX system and to compare the results with data obtained by other
methods. The results shown in Figs. 4 and 6 clearly show that GpA WT
and the two GpA TM domain mutants I76A and L75V strongly homodimerize
in the E. coli inner membrane in contrast to the GpA G83I TM
helix when tested in E. coli SU101 or SU202.
The general trend of homodimerization described under "Results"
(WT > I76A > L75V
G83I) (Figs. 4 and 6) is identical
to the data obtained using the TOXCAT system (8). Nevertheless, it
should be mentioned that the relative strength of the interaction of
the two mutants I76A and L75V is higher than measured with TOXCAT. In
GALLEX, even weaker interactions seem to cause a strong response
(repression) of the reporter gene. Although the relative stabilities
measured by different methods vary, the hierarchy of the stability for
GpA sequence variants is conserved, and it was recently shown that this
hierarchy is conserved, even if the hydrophobic environment of the TM
helices is altered (25).
The different conditions for measuring the homoassociation in E. coli SU101 and measuring the heteroassociation of the DNA binding
domains driven by the homoassociation of the TM domains (expression of
the same TM domain from two plasmids) allow only a qualitative
comparison of the strength of the interaction but do demonstrate
whether heteroassociation exists. The absolute degrees of repression
should not be compared directly between different experimental setups.
Using GALLEX, the heterodimerization of GpA WT with each of the mutant
TMs was also observed, although to a lesser extent than the WT-WT
interaction. The GpA L75V and the GpA I76A chimeric proteins seem to
interact more strongly with some of the other GpA TM helices than with
themselves. Specifically, the pair GpA L75V/GpA I76A shows an
interaction that is remarkably strong. In earlier work, the heterodimer
formation between L75V and I76A was already observed on SDS gels (23).
Using SDS-PAGE for the identification of heterodimerizing GpA pairs did
not lead to the observation of any pair besides L75V/I76A, whereas an
interaction between virtually all of the GpA variants with the
exception of the G83I TM is seen in the present assay. The fact that
GALLEX measures the interaction in a biological membrane, in contrast to the relatively harsh conditions of SDS-PAGE, may explain why the
other interactions could not be observed on SDS gels.
As shown already in the case of the L75I/I76V pair, a point mutation on
one TM can be partly compensated by another point mutation on the
second GpA TM (23). In previous studies with the GpA TM domain, the
effect of single point mutations on the homodimerization was monitored
(7, 8, 23). The results presented here indicate that the disruptive
effects on homodimer formation of the investigated mutations
L75V, I76A, and G83I can be reduced by formation of heterodimers with
the WT sequence or with other, more stably associating helices.
Therefore, some of the stabilizing interactions disrupted in the
homodimer of the three mutant GpA TM helices can be compensated to a
certain degree by heterodimer formation. This observed compensatory
effect is of particular importance, since the interaction of two
different TM helices in a GpA-like manner is proposed to play an
important role in the formation of heterodimeric complexes (17, 30). The destabilizing effect of amino acids on the homodimerization of one
TM could be partly compensated by the formation of a heterodimer with
an other TM helix. By this mechanism, the formation of homodimeric complexes can be repressed, favoring the formation of the more stable heterodimers.
Heterodimerization of the
4 and
7
Integrin TM Helices--
Integrins mediate cell-cell and cell-matrix
interactions by forming
/
heterodimers. Each subunit of the
heterodimer consists of a large extracellular domain, a single
transmembrane helix, and a short cytoplasmic domain (reviewed in Ref.
31). Although it has been shown that the formation of heterodimers can
be driven by interactions of the large cytoplasmic extracellular
domains (18), the role of the transmembrane domains in the formation of
hetero-oligomers has recently been discussed as an additional factor
(17, 19). Computational studies showed that the TM domains of the
IIb/
3 heterodimer are likely to have a
structure similar to the GpA homodimer (17). Almost all integrin TM
domains show a GG4-like motif similar to GpA, and a global search of
helix-helix interactions indicated that this motif could mediate the
heterodimer formation of
/
integrins. Protein fragments
encompassing the transmembrane and the cytoplasmic domains of the
IIb and
3 integrins showed little or no
tendency to form heterodimers; instead, the
2 fragment
was found to form a homodimer, and the
3 fragment was
found to form a homotrimer (19). The analysis presented here shows
that, although the
4 and
7 TM domains
have a tendency to form homodimers, the integrin TM domains also form
heterodimers. Each of these dimers is relatively unstable compared with
the GpA homodimer. It has been suggested that the relative orientation of the TM helices to each other may change during signal transduction (17). If such a mechanism occurs, very strong association of two TM
helices would probably prevent such a reorientation. The observed
homodimerization of the single TM helices is most likely due to the
fact that in this assay no attention was paid to the extracellular
domain of the integrins, a region that is known to mediate interaction
and that might control the specificity of the heterodimer formation.
The results presented here suggest that the interaction of the
transmembrane parts of the integrins could contribute to the formation
and/or stabilization of heterodimeric
/
integrins.
Conclusions--
The GALLEX system presented in this study
provides a new approach to investigating helix-helix interactions in a
biological membrane. By using two versions of the LexA DNA binding
domain and a promoter/operator with two different sequences, the
association of GpA WT and mutated TM helices was detected using a
reporter gene,
-galactosidase. Also, the formation of heterodimers
between the integrin
4 and
7 TM domains
as well as homodimer formation of the two integrin TM helix alone was
shown. In principle, the interaction of any two parallel TM helices
should be assessable. As applications emerge, GALLEX should expand our
understanding of the chemistry of membrane protein folding and oligomerization.