From the Department of Medical Protein Research, Flanders Interuniversity Institute for Biotechnology and the Department of Biochemistry, Faculty of Medicine and Health Sciences, Ghent University, B-9000 Ghent, Belgium
Received for publication, August 14, 2002, and in revised form, February 25, 2003
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ABSTRACT |
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We propose phage display combined with
enzyme-linked immunosorbent assay as a tool for the systematic analysis
of protein-protein interactions by investigating the binding behavior
of variants to a partner protein. Via enzyme-linked immunosorbent assay
we determine both the amount of fusion protein presented at the phage surface and the amount of complex formed, the ratio of which is proportional to the affinity. Hence this method enables us to calculate
the relative affinities of a large number of mutants. As model systems,
we investigated actin-binding motifs conserved in a number of proteins
binding monomeric or filamentous actin. The hexapeptide motifs LKKTET,
present in thymosin As a consequence of various genome-sequencing projects, novel
proteins are being identified, many of which may take part in new
interactions. Efforts are going on to tackle the discovery of
protein-protein interactions globally (1), but there is also need for
novel methods for the analysis of these interactions, preferably easy,
reliable, and high through-put techniques. Several means to probe amino
acids that contribute to the binding of two proteins have been
developed. X-ray crystallography and NMR may yield superior
information about the interface of proteins at atomic resolution (2,
3). However, both methods are intrinsically difficult (even for
noncomplexed proteins) and time-consuming and require expensive and
sophisticated equipment, as well as large amounts of biological
material. Cross-linking of interacting proteins followed by mass
spectroscopy or conventional sequence determination of covalently
coupled peptide fragments has limited application (4-6). Probing the
accessible surface in protein complexes by deuterium exchange has also
been employed (7). But by far the two most popular methods are the use
of peptide mimetics either in solution (8) or on membranes (9, 10) and
especially site-directed mutagenesis (11, 12). However, in the latter
powerful technique to study functions of proteins or interactions
between proteins, one often faces the difficulty of choosing the
position and type of amino acid exchange to be introduced. To
circumvent this, one may perform a saturation mutagenesis at several
positions thought to be important for the interaction. Obviously this
creates a new problem, i.e. screening a large number of
mutants in a systematic way.
We propose to combine saturation mutagenesis, phage display and
ELISA1 for the systematic
screening and quantification of protein-protein interactions. Phage
display is traditionally used for the selection of stronger binding
ligands against target molecules (13); here we employ it differently.
We generate defined libraries of mutants and subsequently analyze all
of the different recombinant phages using ELISA. In this ELISA we
determine both the amount of fusion protein on the phage and the amount
of complex formed. The ratio of both gives information about the
binding strength of the different mutants. We apply this method to the
presentation of thymosin We selected these actin-binding modules because for thymosin Here we show that a combination of mutagenesis, phage display, and
ELISA may be a powerful new tool to systematically investigate protein-protein interactions by evaluating the relative binding strengths of mutants. Next to mutational tolerance at a position, structural information on the bound conformation can be inferred from
mutants displaying increased affinity. In addition, our results show
that some of the charged crown residues in the villin headpiece, hypothesized to be important for actin interaction, are dispensable for
actin binding.
Construction of the Libraries and Rescue of the Recombinant
Phages--
Enzymes and reagents for molecular cloning were purchased
from New England Biolabs or Invitrogen and were used following the manufacturer's instructions. The oligonucleotides were synthesized by
Eurogentec. The phagemid vector pCANTAB5E, the detection module, and
the Escherichia coli strain TG1 were from Amersham
Biosciences. The helper phage M13KO7 was from Promega. The Sequenase
version 2.0 kit was from U.S. Biochemical Corp., and
[
We used the phagemid pCANTAB5E harboring the wild type human thymosin
Biotinylation of Actin on Cys374 or
Gln41--
Actin was prepared from rabbit skeletal muscle
(25) and isolated as calcium-G-actin by chromatography over
Sephadex G-200 in G buffer (5 mM Tris-HCl, pH 7.7, 0.1 mM CaCl2, 0.2 mM ATP, 0.2 mM dithiothreitol, and 0.01% NaN3). For
biotinylation on Cys374, we applied actin to a NAP10 column (Amersham
Biosciences) and eluted it with G buffer without dithiothreitol. We
immediately derivatized actin, on Cys374, with 5-fold molar
excess Immunopure Iodoacetyl-LC-Biotin (Pierce) in the dark at room
temperature for 2 h. The biotinylated actin was dialyzed four
times against G buffer with dithiothreitol to remove excess of labeling
reagent. We biotinylated actin (1 mg) on Gln41 using
transglutaminase (1 unit; Sigma) as in Ref. 26 except that we used
biotin cadaverin (Molecular Probes). In both cases we checked the
incorporation of biotin by a Western blot using streptavidin-conjugated
alkaline phosphatase (Sigma).
Theoretical Considerations Relating Absorbance to Relative
Affinity--
The set up of the method relies on monovalency of the
phage display system and on the possibility to measure two absorbance values in an ELISA. The first criterion is fulfilled (see main text and
Refs. 24 and 27). In wells, coated with E-tag antibody, one measures
absorbance A0, which is proportional to
the total amount of recombinant phages, ergo to the total amount of
presented protein [Ytot]. In wells coated with target
protein (here G- or F-actin), one measures absorbance A,
which is proportional to the amount of protein-protein complex [XY].
In addition, if the total concentration of the presented protein
[Ytot] is much lower than the total concentration of the
target protein [Xtot] (see below), we can state that the
concentration of free protein [X] equals [Xtot]. Thus,
in the dissociation equation we can replace [Ytot] by
kA0 and [XY] by kA (with
k being the same proportionality factor). It is important to
note that the Kd value of the E-tag antibody
(10
In a previous paper (24), we used a linear correlation between the
Kd and the ratio of phage titer (T) over
the absorbance (A) measured in a similar way as here, with a
formula of the following form:
(Kd/[Xtot]) + 1 = T/ANk (where N is Avogadro's number
and k is a proportionality factor). This formula is related
to the one above because T/N is equal to
kA0. Note that both formulae are also modified
Scatchard equations.
The above derivation relies on an excess of functional target protein,
coated in the wells, over the amount of phage presented protein. The
amount of biotinylated actin in the neutravidin-coated wells (see
below) was 1.4 pmol/well. This is 106-fold higher than the
amount of presented thymosin ELISA for Thymosin Other Binding Assays--
The Kd values of
chemically synthesized thymosin Evaluating the contribution of amino acids participating in the
interaction of two proteins is often difficult and time-consuming, especially when three-dimensional structures of the individual proteins
or of the complex are unknown. In the post-genome era, in which
researchers employ bioinformatics to predict new potential interactions, there is a need for relatively fast and easy ways to
investigate the binding of two proteins to confirm these predictions. In a previous study, we already explored the possibility of using a
combination of three existing methods: PCR, phage display, and ELISA to
study protein-protein interactions (24). In the present work we
improved this method and applied it to the interaction of thymosin Creation and Completeness of the Libraries of Thymosin An ELISA-based Method for Measuring Protein-Ligand
Interactions--
The method is schematically depicted in Fig.
1. Basically, we insert the gene of
interest (or a limited library of mutants) in the gIIIp gene coding for
the M13 minor coat protein pIII. We opted for the phagemid pCANTAB5E,
which has been engineered for monovalent display of heterologous
proteins (27), to avoid complications arising from avidity effects
during measurements of relative affinity. In addition, this vector
carries the coding information for the E-tag sequence, the epitope for
the anti-E-tag antibody, between the gene of interest and the gIIIp
gene. The presence of a M13 origin of replication in pCANTAB5E permits
the packaging of the phagemid DNA when bacteria are infected with a
helper phage (e.g. M13KO7) during the so-called rescue.
Because we purify by cloning, we avoid laborious purification steps
(note that this also avoids possible background absorbance values in the ELISA by religated phages containing no insert but expressing the
E-tag). Because the system is statistically monovalent (maximally one
of 2,000 recombinant phages will be divalent) (24), the resulting
recombinant phages only carry one copy of the heterologous protein and
can be used in an ELISA for presentation of this protein to its coated
partner. In this ELISA, the measured absorbance (A) is then
proportional to the amount of protein complex formed. On the other
hand, using the same phage preparations, the total amount of presented
protein (A0) can be measured because only the
recombinant phages carry the E-tag sequence. As derived under "Experimental Procedures" (see also Fig. 1), the formula predicts that there is a linear correlation between the equilibrium dissociation constant of the interaction and the ratio
(A0/A) of the number of recombinant
phages over the amount of complex formed, provided that the amount of
coated target protein is in large excess over the amount of presented
protein. As an alternative for A0, one can
determine the phage titer (T) (24), assessing the sum of the
recombinant and nonrecombinant phages. It is, however, important to
note that only 1% of the total phage population carries a recombinant protein (31).
In a previous paper (24) we determined the T/A
values of 18 mutants of Lys18 in the actin binding motif of
thymosin Correlation between the Kd and the
A0/A Values of the Different Thymosin The Relative Affinities of the Different Thymosin
If we take into account the number of mutants displaying reduced
affinity and the extent of decreased binding, simple visual inspection
of the graphs in Fig. 3 allows deriving the mutational tolerance at
each position in the thymosin The Profile of Relative Affinities of the Different Villin
Headpiece Mutants Is Different for Interaction with F- and
G-actin--
We prepared phages displaying the villin headpiece
mutants and measured their relative affinities for F-actin using wild
type villin headpiece as reference (Table
III and Fig. 4).
Because we only analyzed two-thirds of
all possible mutants, we interpret our results cautiously. The first
three residues of the motif appear more important because most of the
analyzed mutants display significantly reduced binding, whereas at
positions 72, 73, and 74 one or more mutants have a significantly
higher affinity than wild type.
Although the villin headpiece is an F-actin-binding protein, we assayed
the same recombinant phages for their relative affinity for G-actin in
view of the observed competitive actin binding with thymosin An Alternative Use for Phage Display--
Phage display is a
widely used technique for the selection of antibodies against antigens
(34) or for searching protein variants with a higher affinity than wild
type (13, 35, 36). We here employ it differently, i.e. not
for the purpose of selecting stronger binding mutants but rather for
systematically investigating the interaction of two proteins. In
combination with PCR mutagenesis and ELISA, it yields a reliable and
easy way to determine relative affinities for mutants, even for those
cases where partners interact weakly (we still obtained a positive
signal for a low affinity interaction with a Kd
equal to ~0.2 mM). In solid phase assays, such as the
ELISA used here, there may be bias toward slowly dissociating mutants.
Indeed, within the incubation time, equilibrium between the
kon and koff values is
reached, but during subsequent washing steps the
koff plays a major role in determining the
amount of complex remaining on the plates. Nevertheless, the observed
ratios of A0/A (representing the
relative affinities in our solid phase assay) correlate very well with
the corresponding Kd values for the six chemically
synthesized thymosin
The relative affinities can be determined by two related methods.
Either one calculates the ratio phage titer (T) over the absorbance (A) (24), or one determines the ratio
A0 over A (this study). The second
approach is faster, and the correlation between the
Kd and the measured values no longer contains a
proportionality factor that is dependent on a variety of
instrumentation and environmental parameters. The most important
improvement, however, is that the results become independent of the
fraction of phages presenting the partner protein. An ELISA-based
method for protein-ligand interaction was previously developed for the
analysis of the interaction of zinc fingers with DNA (37). Similar to
our strategy it relies on presenting protein in concentrations much
lower than those of the target, and Kd values can be
determined by coating several concentrations of target ligand
(preferably in 100-fold excess of the Kd value). For
the low affinity interactions studied here (1 µM to 200 µM), this would have required milligram amounts of actin,
which in practice is difficult to achieve (in our assays we coat
nanogram amounts). Because, in our method, we measure the amount of
displayed partner protein, we avoid this problem.
Several other tools and methods have been developed to study
protein-protein interactions, such as multi-use peptide libraries (8)
and spot synthesis (9, 10), alanine (11, 12) or cysteine (38) scanning,
and two-hybrid analysis (39, 40). Our technique has important
advantages over these methods. Multi-use peptide libraries and spot
methods may yield very similar, but less quantitative, scan information
and are limited by the length of peptides that can be chemically
synthesized (generally 15-20 amino acids). There may also be size
limitations for phage display, especially when using the pVIII coat
protein (41); however, presenting small proteins or single domains is
usually no problem unless they precipitate as inclusion bodies or are
not compatible for translocation through the membrane into the
periplasm. In this respect, we recovered three thymosin
Alanine scanning is a powerful tool to study protein-protein
interactions (12), but one may overlook residues participating in the
interaction because this type of mutation may be rather neutral. In
addition, it is not indicative for the tolerance of a functional
residue (42). This is exemplified here in the thymosin
Another advantage is that the heterologous mutant proteins are purified
by cloning and do not require extensive purification prior to testing
their binding characteristics. Recovering and analyzing all possible
mutants from a library remains time-consuming. However, this may not
always be necessary because our results with thymosin Probing Structural Information Using Libraries of Mutants--
One
of the reasons we embarked on this project was to use the binding
characteristics obtained for the extensive set of mutants to distill
structural information about the mutated regions. For the discussion
below, we base our interpretation solely on mutants having similar or
increased affinities compared with wild type, thereby assuming they are
properly folded and stable.
First, comparing the profiles per position in thymosin
Second, NMR experiments showed that the motif in noncomplexed thymosin
Thus, using this technique one can obtain useful hints on structural
information such as the type of interaction that occurs at the
interface or the secondary structure required for recognition. Both are
based on the tolerance of amino acids at particular positions and/or on
those mutants displaying higher affinity.
Thymosin Some Charged Crown Residues in Villin Headpiece Are Dispensable for
F-actin Binding--
We correlated our results with the prediction
that charged residues (Lys65, Lys71,
Glu72, Arg37, and the terminal carboxyl group
of Phe76) form a charged crown necessary for actin binding
(20, 22). Because the villin headpiece (WT and mutants) can be
presented as a C-terminal fusion protein, the charge of the carboxyl
function is not absolutely essential for actin interaction. Likewise,
the charges of Glu72 and Lys73 appear not
necessary for binding. Hardly any of the mutants at these positions
displays a dramatically decreased affinity, and more curiously,
substitutions to aromatic amino acids at the latter position result in
an even higher affinity. Of the charged crown side chains studied here,
only Lys71 is important because the tolerance for mutation
at this position is very low. We note that substituting RVDN in the
protovillin headpiece domain by a charged crown-like sequence, KKEK,
does not increase its affinity for F-actin (20). This is entirely consistent with our data, demonstrating that at least three charges in
the crown are dispensable for F-actin binding.
Conclusion--
We describe a technique based on phage display
technology in combination with PCR mutagenesis and ELISA for the
systematic investigation of protein-protein interactions. As a model we
chose the mutagenesis of the hexapeptide motif in thymosin 4, and LKKEKG, present in the villin headpiece,
were mutated, and the variants were analyzed. Study of the positional
tolerance allows postulating that the motifs, although similar in
primary structures adopt different conformations when bound to actin.
In addition, our data show that the second and the fourth amino acid of
the thymosin
4 motif and the first three residues of the villin
headpiece motif are most important for actin binding. The latter result challenges the charged crown hypothesis for the villin headpiece filamentous actin interaction.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
4, analyzing monomeric actin
(G-actin) binding, and of the villin headpiece, probing both
G-actin and filamentous actin (F-actin) binding.
4 the
actin binding information is relatively well characterized, but its
structure is poorly defined. By contrast the villin headpiece has a
well resolved NMR structure, but information on those residues that
interact with actin is still elusive. Thymosin
4 interacts with
actin via residues in an
-helix and a conserved hexapeptide motif
(LKKTET) (14-16). NMR studies of thymosin
4 reveal no unique structure for this motif; however, it is evident that the motif must
become structured upon binding actin (17-19). A seemingly related
sequence (LKKEKG) is present in a set of C-terminal headpiece domains
(15, 16) implicated in F-actin binding (20). NMR of the villin
headpiece showed that the first five amino acids of the motif are in a
-helical conformation (21, 22), and some of these are part of a
charged crown suggested to be important for actin binding (20).
Although both modules appear to have analogous motifs in their primary
structure, they display different specificities for G- and F-actin.
This suggests that local structural changes in these actin-binding
units govern the recognition of the different conformational states of actin.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-35S]dATP was from ICN.
4 or human villin headpiece cDNA, inserted between the
SfiI and NotI restriction sites, as starting
material to create the libraries. We constructed this library by
independently mutating each of the six codons of both hexapeptide
motifs into the 63 other possible ones, using overlap extension
PCR mutagenesis (23) with synthetic oligonucleotides completely
degenerated at the desired positions. The obtained PCR products were
SfiI- and NotI-ligated in pCANTAB5E. The
resulting phagemid contains under control of the lac promotor, the gene
III (gIII) signal coding sequence, the thymosin
4 or the villin
headpiece gene, the E-tag coding sequence, an amber codon (TAG), and
the rest of the gIII coding region. The ligation was electroporated in
TG1 cells. Phagemid DNA of individual transformants, was purified with
WizardTM minipreps (Promega) and sequenced. These isolated
transformants were also used to produce phages, displaying thymosin
4 or villin headpiece variants, according to the protocol available
from the company (Amersham Biosciences). We determined phage titers as described previously (24). Rescued phages were also used in the ELISAs
described below.
7 M see below) for the presented protein
is significantly lower than the Kd value of the
coated protein for the presented protein. We can then write:
Kd = [Xtot](kA0
kA)/kA or
(Kd/[Xtot])+ 1 = A0/A, i.e. the
Kd value is in a linear correlation with the ratio
A0/A. Although our results, presented
below, indicate that this correlation exists, the formula cannot be
used to calculate Kd values because
[Xtot] represents a solution concentration, and we here
use a solid phase assay. Coating of functional biotinylated monomeric
actin or filamentous actin was reproducible.
4 (in general attomol/well). We
performed a similar assay coating monomeric actin (5 µg/100 µl)
directly to the wells. Although 29 pmol of actin was coated, the
read-out in the ELISA, using the same phage preparations, consistently
resulted in lower signals (not shown), indicating most of the directly
coated actin is not functional. Therefore, and because
biotinylation on Cys374 of actin does not interfere with
thymosin
4 interaction,2
we used the biotinylated form in all of the thymosin
4-G-actin interaction assays. For the villin headpiece we used a previously documented procedure of F-actin coating (28).
4 or Villin Headpiece--
We coated
microtiter plates (Nunc Maxisorp) either with 400 ng/100 µl
Immunopure neutravidin (Pierce) or 1 µg/100 µl of E-tag antibody
(Amersham Biosciences) overnight at 4 °C. Excess binding sites were
blocked with 200 µl of blocking buffer (2% solution of skimmed milk
powder in phosphate-buffered saline). Neutravidin-coated wells were
incubated with 1 µg/100 µl biotinylated actin for 1 h at room
temperature. We washed the microtiter plate three times with
phosphate-buffered saline containing 0.05% Tween 20 (washing buffer)
and added 1010 rescued phages in 200 µl of blocking
buffer to the biotin-actin-neutravidin wells or a 1:100 dilution of the
phages to the E-tag antibody-coated wells. After 2 h of incubation
at room temperature, we washed the plates four times with washing
buffer and supplied the wells with 200 µl of 1:2500 diluted anti-M13
antibody, conjugated with horseradish peroxidase (Amersham Biosciences)
for 1 h at room temperature. We washed the wells three times with
washing buffer, incubated the enzyme with the substrate
2',2'-azinobis(3-ethylbenzothiazoline-6) sulfonic acid (Amersham
Biosciences) and measured absorbance at 405 nm using a microtiter plate
reader (Dynatech). For the villin headpiece mutant analysis, we coated
12 µM polymerized actin overnight at 4 °C (28). The
ELISA was carried out as above with similar phage dilutions as used for
thymosin
4. Based on the higher A0 values,
the amount of presented villin headpiece was a factor 2-3-fold higher
than that of thymosin
4.
4 variants (24) were determined as
in Ref. 15 except that we used phosphate-buffered saline, the buffer
also used in the ELISA. The genes coding for wild type or mutant villin
headpiece were recloned from the pCANTAB5E phagemid in pET11d
(Novagen), with a new stop codon inserted at the 3'-end of the villin
head-piece coding sequence and expressed in E. coli MC1061
containing pSCM26 (29). The proteins, thus lacking the E-tag sequence,
were purified following the protocol in Ref. 22. The
Kd values for the villin headpiece mutants were
determined in a sedimentation assay. We incubated 12 µM
F-actin with various molar ratios of the villin headpiece, or mutant,
for 30 min at room temperature and sedimented the F-actin with an
Airfuge (Beckman) for 15 min at 100,000 × g. The
supernatant was removed, and the F-actin pellet was washed with G
buffer supplemented with 100 mM KCl and 1 mM Mg2Cl. Aliquots of the supernatant and pellet were analyzed
using a 10% Tricine gel. After staining the gels, we used
densitometric scanning to determine the amount of bound and free villin
headpiece. We used these values in a Scatchard plot to calculate the
Kd. The Kd for the WT-villin
headpiece F-actin interaction is ~10-fold higher than previously
reported using slightly different buffer conditions at 4 °C (20).
The Kd of the E-tag antibody for the E-tag sequence
was determined using a Biacore X. The antibody was coated to a CM5 chip
(Biacore, Sweden) using amine coupling with
1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride and
N-hydroxysulfosuccinimide chemistry according to the
manufacturer's instructions. Various concentrations of purified
thymosin
4, expressed from pCANTAB5E in HB2151 cells and thus
containing the E-tag sequence, were passed over the sensorchip in
HEPES-buffered saline (Biacore, Sweden). We derived the
Kd value (100 nM) from fitting the
association and dissociation curves. Note that this is 100-fold lower
than the Kd for the actin thymosin
4 interaction
in phosphate-buffered saline.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
4
with G-actin and of villin headpiece with G- and F-actin using
libraries of mutants.
4 and
Villin Headpiece--
Using double cycle PCR saturation mutagenesis
reactions (23) on the thymosin
4 and the villin headpiece gene,
inserted in the pCANTAB5E phagemid, we created libraries for each of
the six positions of the hexapeptide motifs of both actin-binding modules. In the case of thymosin
4, the motif is known to be important for the interaction with actin (14, 15); for the villin
headpiece this has been proposed (22, 30). For each of the 12 libraries
we obtained at least 2-5 × 106 transformants/µg
DNA in E. coli, far in excess over the expected 64 variants
for each position in the motif. From each of the six thymosin
4
libraries, we isolated on average 70 clones and sequenced them to
identify the created mutation. We recovered 103 of the 114 possible
mutants at the amino acid level. From the results presented below for
thymosin
4, it became evident that classes of mutants exist. For
this reason we isolated from the six villin headpiece libraries enough
mutants to get a member of each class allowing rapid screening of the
villin headpiece-F-actin interaction.
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Fig. 1.
A phage display strategy for studying
protein-protein interactions. Wild type and/or mutant genes are
ligated in the phagemid, here SfiI- and
NotI-restricted pCANTAB5E, between the coding sequence of
the gIIIp signal(s) and the coding sequence for the E-tag followed by
the remainder of the gIIIp gene. The phagemid is electroporated in the
appropriate E. coli strain. Individual clones are grown both
for rescue and preparing the phagemid DNA for sequencing. In ELISA one
measures, for the obtained recombinant phages,
A0 and A proportional to the total
amount of fusion protein (via the E-tag) and the amount of formed
complex with the target protein, respectively. The ratios
A0/A are in linear correlation with
the Kd value, and [Xtot] is the total
target protein concentration, here actin (for details see
"Experimental Procedures").
4. Here, we measured the A and
A0 values of the same mutants in an ELISA using
biotinylated actin and coated anti-E-tag antibody, respectively. We
plotted T/A (24) versus
A0/A values (this study) and observed
a correlation of 0.86, indicating that both measurements yield
essentially the same result independent of the manner in which the
amount of recombinant protein is determined (data not shown). This is
with the exception of those mutants where codon usage in E. coli influences the amount of expressed fusion protein. Indeed,
for thymosin
4 mutants in which Lys18 had been mutated
to Arg, the T/A values were higher for mutants with a less frequently used codon, whereas their
A0/A values were all very similar
(Table I). Similarly,
A0/A values of K18Q mutants, where
glutamine is encoded by TAG in the supE strain TG1 (this stop codon is
only partially suppressed resulting in lower expression levels) or by
the normal CAG codon, are comparable (83.7 and 82.6, respectively).
Consequently, the method using titer over absorbance (T/A) can only be employed if one takes into
account codon usage in E. coli. Thus, the purely ELISA-based
method (measuring A and A0) is
superior and exclusively used in the analysis presented below.
Codon usage influences T/A values but not
A0/A values
4 and
Villin Headpiece Variants, Proof of Principle--
We predict a
correlation between the Kd and the
A0/A ratio (see "Experimental
Procedures"). To validate this, we determined the
A0/A values of six phage displayed
thymosin
4 variants (WT, K18A, K18R, K18Y, K18E, and K19E) for which
the Kd values of chemically synthesized counterparts
were measured by a sequestration assay (15, 24). We also measured the
Kd values of bacterially produced and purified
villin headpiece wild type and mutants (K70R, K71D, E72S, and K73F) by a co-sedimentation assay and Scatchard analysis (data not shown). In
both cases we plotted the Kd values
versus the corresponding A0/A values of the same phage
presented mutants. There is a linear correlation of 0.99 for the
thymosin
4-G-actin interaction and of 0.98 for the villin
headpiece-F-actin interaction (Fig.
2). These values suggest that despite the
bias favoring dissociation inherent to washing steps in the ELISA,
apparent affinities can be measured, illustrating the robustness of the
method.
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Fig. 2.
The Kd-values
correlate with the A0/A
values for selected thymosin 4 and
villin headpiece mutants. A, we determined the
Kd values (µM) of chemically
synthesized wild type and five thymosin
4 variants (K18R, K18Y,
K18A, K18E, and K19E) for G-actin and plotted them versus
their A0/A values (Table II).
B, we did the same for the affinities of villin headpiece
wild type and four purified recombinant mutants (K70R, K71E, E72S, and
K73F) for F-actin. Linear correlations are 0.989 in A and
0.978 in B. The mutants were selected to represent a wide
range of A0/A values.
4 Variants Are
Indicative of the Mutational Tolerance--
We made recombinant phages
of the 103 mutants of thymosin
4, isolated from the six different
libraries. All of the variants could be displayed on the phage surface,
except for the Met, Arg, and Asp mutants at position 17, for reasons we
currently cannot explain. We performed the modified ELISA in duplicate,
starting from two independent phage preparations and calculated the
average A0/A values (Table
II). Mutants with higher
A0/A values have reduced affinities.
In Fig. 3 these affinities are shown
relative to wild type. In this representation a positive value
indicates stronger binding than wild type, a negative value indicates
weaker binding, and wild type is 0 (we consider values between +0.03
and
0.03 as wild type-like interaction based on the maximal variation
observed during our measurements). Two residues of the motif,
Lys18 and Thr20, appear to be essential because
no variant has a significantly higher affinity than wild type (K18R,
T20A, and T20Q have wild type-like affinity). At the other four
positions of the motif, we do observe one or more mutants that have a
higher affinity than wild type (see "Discussion").
A0/A values for the different thymosin 4 mutants
View larger version (44K):
[in a new window]
Fig. 3.
Relative affinities of the different
thymosin 4 motif mutants per position.
The relative affinities (left y axis) were
calculated using the A0/A values in
Table II and the following formula
log(AMut/A0 Mut × A0 WT/AWT). As a result
WT is 0, and WT-like activity (+) is between
0.03 and +0.03. Positive
and negative values indicate increased and decreased affinity compared
with wild type, respectively. The height of the bar is
proportional to the increase or decrease in binding. These values were
plotted versus the mutant amino acid (x
axis) at each position in the hexapeptide motif (right
y axis). Some mutants were not recovered (K18C, K19H,
K19C, T20Y, T20D, T20M, E21Y, E21N, E21M, T22E, and T22N), and some
were not displayed (ND). The amino acids are given in the
top and bottom panels only.
4 hexapeptide motif. Lys18
appears to be most important for the interaction with G-actin, followed
by Thr20, Leu17, Glu21,
Lys19, and Thr22. We note that this observed
tolerance agrees well with the conservation of hexapeptide motifs in
various
-thymosins (16, 32, 33), i.e. more mutations are
allowed in the C-terminal half. In addition, most of the naturally
occurring alterations result in a good binding thymosin
4 mutant
in our assay.
A0/A values for the interaction of different villin
headpiece mutants with F-actin
View larger version (39K):
[in a new window]
Fig. 4.
Relative affinities of the villin headpiece
mutants for F-actin. We used the formula in the legend to Fig. 3
to calculate the relative affinities from the
A0/A values listed in Table III and
employed the same representation as for Fig. 3. WT, WT-like activity
(+), mutants that are not displayed (ND), and displayed mutants with no
observed binding ( ) are indicated.
4 (15).
For none of the recombinant phages could we observe binding when we
used the same ELISA set up as for thymosin
4, i.e. with
G-actin biotinylated on Cys374. Possibly, the villin
headpiece-binding site on actin was occluded by the neutravidin moiety
because an ELISA with G-actin biotinylated on Gln41, which
is located at the opposite end of the actin monomer, yielded absorbance values (Table IV and Fig.
5). In general fewer mutants bind to
G-actin, and many of the ones that do bind have strongly reduced
affinities for monomeric actin consistent with the preference of this
module for F-actin. This is especially relevant for mutations in the
first three positions of the motif. However, similar to some of the
thymosin
4 mutants, these results show that very weak interactions
can be measured. Intriguingly, stronger G-actin-binding variants are
mainly found at position 72, whereas this is at position 73 for F-actin
(compare Figs. 4 and 5).
A0/A values for the interaction of different villin
headpiece mutants with G-actin
View larger version (38K):
[in a new window]
Fig. 5.
Relative affinities of the villin headpiece
mutants for G-actin. We used the formula in the legend to Fig. 3
to calculate the relative affinities from the
A0/A values listed in Table IV and
employed the same representation as for Fig. 3. WT, WT-like activity
(+), mutants that are not displayed (ND), and displayed
mutants with no observed binding ( ) are indicated.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
4 variants and for the five recombinant villin
headpiece variants tested (Fig. 2).
4 mutants,
of which the fusion protein is not presented.
4-G-actin
analysis where, with the exception of K18A, the mutation to alanine has
no dramatic effect on binding, even not for Thr20, a
residue important for the interaction. Also in two-hybrid systems large
numbers of mutants can be analyzed simultaneously, but certainly the
method described here allows easier quantification.
4 indicate
that mutants appear in affinity classes. For the villin headpiece we
tested two-thirds of all possible mutants, and these same groups can be
distinguished, albeit that at each position of the profile is
different. Consequently one can for each position analyze clones
randomly, measure their relative affinity, and only sequence a few of
each class. In this scenario, once optimized, the technique described
here is relatively fast and can in principle be improved because
several steps are adaptable to automation. As exemplified by the
thymosin
4 results, our technique allows probing positional
tolerance. As such, it can be applied as a screening tool prior to
choosing positions that can be mutagenized when constructing libraries
from which stronger binding variants will be selected.
4 and villin
headpiece allows to discriminate between residues participating in an
electrostatic interaction (T
4, Lys18; VHP,
Lys70 and Lys71) or not (T
4,
Lys19 and Glu20; VHP, Glu72 and
Lys73). This is because we observe that some charged
residues cannot be changed into any other amino acid without negatively
influencing the affinity (with the exception of similarly charged side
chains), and the most pronounced effect is observed for variants with
an opposite charge. By contrast other positions are more tolerant for
charge reversals. For thymosin
4 Lys18 an electrostatic
interaction is in agreement with previous studies (14, 15), and for
villin headpiece Lys70 this is evident from the NMR
structure because this residue is involved in making a buried salt
bridge with Asp39 (22). Intriguingly, in the latter case,
substitution with Arg results in increased affinity. Perhaps the
mutation to Arg yields a more stable domain enabling a better contact
with F-actin. The other charged residues in villin headpiece are
solvent exposed, and thus Lys71 is probably involved in an
ionic contact with F-actin. Surprisingly, charge reversal at position
73 leads to increased affinity. We suggest that the third lysine
residue in the motif is close to the interface with actin and that
substituting it with Glu creates a new (electrostatic) interaction.
Given the observation that microinjected K73E mutant still binds
F-actin in vivo (30), we predict that this happens with a
higher affinity.
4 is structurally poorly defined (17, 18), whereas algorithms
predict a
-helical conformation We show that at certain positions in
the motif, some substitutions yield better or wild type binders. If we
correlate this with the secondary structure propensities of these
mutant amino acids (43), we can speculate on the local conformation of
the hexapeptide motif in the actin bound configuration. Interestingly
at several of the positions glycine and proline substitutions (L17G,
K19G, K19P, E21G, E21P, T22G, and T22P) are tolerated or result in
better binding. Because these two amino acids are usually not present
in
-helices or
-sheets, this may be an indication that the
hexapeptide motif adopts a loop or
-turn structure in the thymosin
4-actin complex. These results correlate well with the NMR studies
of thymosin
4 mutants in solution (i.e. not bound to
actin), which show that binding requires proper termination of the
N-terminal
-helix (residues 5-16) before the motif (19). Along the
same lines, NMR data on villin headpiece shows a helical structure for
the motif. Consistent with this is our observation that at every
position in the motif (with the exception of the last residue), glycine or proline substitutions result in weaker binding than wild type. Thus,
although the motifs in both proteins are rather similar in sequence,
their secondary structures when bound to actin appear to be completely
different, and this technique is capable of probing this. We also
observed differences in binding to G- and F-actin for the villin
headpiece mutants. This is most evident at the last four positions of
the mutated region. Better G-actin-binding mutants at positions 71, 72, and 74 have wild type affinities for F-actin, and vice versa, stronger
F-actin binders that are mainly found at position 73 behave similarly
to wild type for G-actin binding. Possibly, these mutants probe
conformational differences between G-actin and F-actin protomers.
4 and Villin Headpiece Interact Differently with
Actin--
We also show that thymosin
4 and the villin headpiece
interact differently with G-actin. This is based on our observation that thymosin
4 does interact with G-actin linked via biotinylated Cys374 on neutravidin, whereas the villin headpiece does
not. This suggests that the villin headpiece faces actin close to this
residue at the barbed end of the actin molecule and that the
biotin-neutravidin moiety sterically hinders binding. Thymosin
4 is
also proposed to contact the barbed end (5), but a model presented
recently shows a location of thymosin
4 shifted away from the barbed
end (44). Given the observed competition between thymosin
4 and the
villin headpiece (15), our results suggest that the binding sites only
partly overlap. In addition, the profiles of allowed substitutions in
the C-terminal halves of the hexapeptide for both actin-binding modules
are different, indicating that the G-actin binding capacity of the
villin headpiece is not the result of a conformational switch to a
thymosin
4-like structure.
4 and
probed its interaction with G-actin. We also applied the method to a proposed actin-binding motif in the villin headpiece. The method allows
for fast scanning of potential binding sites and determining relative
affinities of many mutants. With this technique, one may distinguish
between residues that are essential for a certain interaction and
positions that are more tolerant of mutation.
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FOOTNOTES |
---|
* This work was supported by Grants 9.0044.97 and 3G022598 from the Fund for Scientific Research-Flanders, Grant 9713-R7115B0 from the Koning Boudewijnstichting, and Grant 011B3496 of the Special Research Fund of Ghent University (to C. A.) and Grant 12051401 of the Concerted Research Actions of the Flemish Community (to J. V. and C. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by a Predoctoral fellowships from the Flemish Institute
for Promotion of Scientific-Technological Research and Industry.
§ Postdoctoral fellow of the Fund for Scientific Research-Flanders.
¶ To whom correspondence should be addressed: Dept. of Biochemistry, Faculty of Medicine and Health Sciences, Ghent University, Baertsoenkaai 3, B-9000 Ghent, Belgium. Tel: 32-9-3313336; Fax: 32-9-3313595; E-mail: christophe.ampe@rug.ac.be.
Published, JBC Papers in Press, February 26, 2003, DOI 10.1074/jbc.M208311200
2 S. Rossenu, S. Leyman, and C. Ampe, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are: ELISA, enzyme-linked immunosorbent assay; WT, wild type; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
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REFERENCES |
---|
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---|
1. | Von Mering, C., Krause, R., Snel, B., Cornell, M., Oliver, S. G., Fields, S., and Bork, P. (2002) Nature 417, 399-403[CrossRef][Medline] [Order article via Infotrieve] |
2. | Lo Conte, L., Chothia, C., and Janin, J. (1999) J. Mol. Biol. 285, 2177-2198[CrossRef][Medline] [Order article via Infotrieve] |
3. | Zuiderweg, E. R. (2002) Biochemistry 41, 1-7[CrossRef][Medline] [Order article via Infotrieve] |
4. | Vandekerckhove, J., Kaiser, D. A., and Pollard, T. D. (1989) J. Cell Biol. 109, 619-626[Abstract] |
5. | Safer, D., Sosnick, T. R., and Elzinga, M. (1997) Biochemistry 36, 5806-5816[CrossRef][Medline] [Order article via Infotrieve] |
6. | Bennett, K. L., Kussmann, M., Bjork, P., Godzwon, M., Mikkelsen, M., Sorensen, P., and Roepstorff, P. (2000) Protein Sci. 9, 1503-1518[Abstract] |
7. | Englander, S. W., Sosnick, T. R., Englander, J. J., and Mayne, L. (1996) Curr. Opin. Struct. Biol. 6, 618-623 |
8. | Meloen, R. H., Puijk, W. C., and Slootstra, J. W. (2000) J. Mol. Recognit. 13, 352-359[CrossRef][Medline] [Order article via Infotrieve] |
9. | Frank, R. (1992) Tetrahedron 48, 9217-9232[CrossRef] |
10. | Reineke, U., Volkmer-Engert, R., and Schneider-Mergener, J. (2001) Curr. Opin. Biotechnol. 12, 59-64[CrossRef][Medline] [Order article via Infotrieve] |
11. | Wells, J. A. (1991) Methods Enzymol. 202, 390-411[Medline] [Order article via Infotrieve] |
12. | DeLano, W. L. (2002) Curr. Opin. Struct. Biol. 12, 14-20[CrossRef][Medline] [Order article via Infotrieve] |
13. | Ehrlich, G. K., Berthold, W., and Bailon, P. (2000) Methods Mol. Biol. 147, 195-208[Medline] [Order article via Infotrieve] |
14. | Vancompernolle, K., Goethals, M., Huet, C., Louvard, D., and Vandekerckhove, J. (1992) EMBO J. 11, 4739-4746[Abstract] |
15. | Van Troys, M., Dewitte, D., Goethals, M., Carlier, M. F., Vandekerckhove, J., and Ampe, C. (1996) EMBO J. 15, 201-210[Abstract] |
16. | Van Troys, M., Vandekerckhove, J., and Ampe, C. (1999) Biochim. Biophys. Acta 1447, 323-348 |
17. | Czisch, M., Schleicher, M., Hörger, S., Voelter, W., and Holak, T. A. (1993) Eur. J. Biochem. 218, 335-344[Abstract] |
18. | Zarbock, J., Oschkinat, H., Hannapel, E., Kalbacher, H., Voelter, W., and Holak, T. A. (1990) Biochemistry 29, 7814-7821[Medline] [Order article via Infotrieve] |
19. |
Simenel, C.,
Van Troys, M.,
Vandekerckhove, J.,
Ampe, C.,
and Delepierre, M.
(2000)
Eur. J. Biochem.
267,
3530-3538 |
20. | Vardar, D., Chishti, A. H., Frank, B. S., Luna, E. J., Noegel, A. A., Oh, S. W., Schleicher, M., and McKnight, C. J. (2002) Cell. Motil. Cytoskelet. 52, 9-21[CrossRef][Medline] [Order article via Infotrieve] |
21. | McKnight, C. J., Matsudaira, P. T., and Kim, P. S. (1997) Nat. Struct. Biol. 4, 180-184[Medline] [Order article via Infotrieve] |
22. | Vardar, D., Buckley, D. A., Frank, B. S., and McKnight, C. J. (1999) J. Mol. Biol. 294, 1299-1310[CrossRef][Medline] [Order article via Infotrieve] |
23. | Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., and Pease, L. R. (1989) Gene (Amst.) 77, 51-59[CrossRef][Medline] [Order article via Infotrieve] |
24. | Rossenu, S., Dewitte, D., Vandekerckhove, J., and Ampe, C. (1997) J. Prot. Chem. 16, 499-503[CrossRef][Medline] [Order article via Infotrieve] |
25. |
Spudich, J. A.,
and Watt, S.
(1971)
J. Biol. Chem.
246,
4866-4871 |
26. | Takashi, R. (1988) Biochemistry 27, 938-943[Medline] [Order article via Infotrieve] |
27. | Bass, S., Greene, R., and Wells, J. A. (1990) Proteins Struct. Funct. Genet. 8, 309-314[Medline] [Order article via Infotrieve] |
28. |
Bourdet-Sicard, R.,
Rüdiger, M.,
Jockusch, B. M.,
Gounon, P.,
Sansonetti, P. J.,
and Tran Van Nhieu, G.
(1999)
EMBO J.
18,
5853-5862 |
29. | Mertens, N., Remaut, E., and Fiers, W. (1995) Biotechnology 13, 175-179[CrossRef][Medline] [Order article via Infotrieve] |
30. | Friederich, E., Vancompernolle, K., Huet, C., Goethals, M., Finidori, J., Vandekerckhove, J., and Louvard, D. (1992) Cell 70, 81-92[Medline] [Order article via Infotrieve] |
31. | Saggio, I., Gloaguen, I., and Laufer, R. (1995) Gene (Amst.) 152, 35-39[CrossRef][Medline] [Order article via Infotrieve] |
32. | Safer, D., and Chowrashi, P. K. (1997) Cell Motil. Cytoskelet. 38, 163-171[CrossRef][Medline] [Order article via Infotrieve] |
33. | Huff, T., Muller, C. S., Otto, A. M., Netzker, R., and Hannappel, E. (2001) Int. J. Biochem. Cell Biol. 33, 205-220[CrossRef][Medline] [Order article via Infotrieve] |
34. | Hoogenboom, H. R., de Bruine, A. P., Hufton, S. E., Hoet, R. M., Arends, J. W., and Roovers, R. C. (1998) Immunotechnology 4, 1-20[CrossRef][Medline] [Order article via Infotrieve] |
35. | Lowman, H. B., Bass, S. H., Simpson, N., and Wells, J. A. (1991) Biochemistry 30, 10832-10838[Medline] [Order article via Infotrieve] |
36. | Rhyner, C., Kodzius, R., and Crameri, R. (2002) Curr. Pharm. Biotechnol. 3, 13-21[Medline] [Order article via Infotrieve] |
37. |
Choo, Y.,
and Klug, A.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
11168-11172 |
38. | Doering, D. S., and Matsudaira, P. (1996) Biochemistry 35, 12677-12685[CrossRef][Medline] [Order article via Infotrieve] |
39. | Fields, S., and Song, O. (1989) Nature 340, 245-246[CrossRef][Medline] [Order article via Infotrieve] |
40. | Serebriiskii, I. G., Khazak, V., and Golemis, E. A. (2001) BioTechniques 30, 634-636[Medline] [Order article via Infotrieve] |
41. | Kishchenko, G., Batlwala, H., and Makowski, L. (1994) J. Mol. Biol. 241, 208-213[CrossRef][Medline] [Order article via Infotrieve] |
42. | Jin, L., and Wells, J. A. (1994) J. Protein Sci. 12, 2351-2357 |
43. | Swindells, M. B., MacArthur, M. W., and Thornton, J. M. (1995) Nat. Struct. Biol. 2, 596-603[Medline] [Order article via Infotrieve] |
44. | Ballweber, E., Hannappel, E., Huff, T., Stephan, H., Haener, M., Taschner, N., Stoffler, D., Aebi, U., and Mannherz, H. G. (2002) J. Mol. Biol. 315, 613-625[CrossRef][Medline] [Order article via Infotrieve] |
45. | Gribskov, M., Devereux, J., and Burgess, R. (1984) Nucleic Acids Res. 12, 539-549[Abstract] |