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INTRODUCTION |
S100 proteins comprise a family of low molecular weight
calcium-binding proteins, which are specifically expressed in a variety of tissues and cell types. It is thought that they are primarily involved in Ca2+-mediated signal transduction. Changes in
intracellular calcium levels alter the structure and function of these
proteins. However, the specific functions of individual members of the
S100 protein family in cell metabolism are not yet clear. They have
been frequently described in association with cell growth and
differentiation, with cell cycle regulation and with several human
diseases as cancer and skin disorders (see Refs. 1-4, for review).
Recent investigations have given much emphasis on the structure of S100
proteins (5, 6). The primary sequence, around 90-100 amino acids long,
consists of two calcium-binding EF-hand domains bridged by a so called
hinge region. Particularly the sequences of the N-terminal 14-amino
acid variant EF-hand and the 12-amino acid C-terminal invariant EF-hand
are highly conserved within the family and among family members of
different species (3, 4). The three-dimensional structure of calcyclin
(S100A6), a typical S100 protein, has been determined by NMR
spectroscopy (5, 6). These data revealed, that a single monomer is
composed of two calcium-binding helix-loop-helix motifs and forms a
globular monomer containing four helices (helix I, II, III, and IV).
Two monomers are oriented in an antiparallel fashion forming a
noncovalent antiparallel homodimer. Dimerization, which is a
characteristic feature for the S100 proteins, appears to be mediated by
several evolutionary conserved hydrophobic residues located in helix I and IV, which contribute to the dimer interface. This interface has
been called a homodimeric fold and appears to be unique among calcium-binding proteins (5, 6). The dimeric complex is widely regarded
as the biologically active form of most S100 proteins.
The S100 proteins MRP8 and MRP14 (calgranulin A and B; S100A8 and
S100A9) are expressed in myeloid cells and in epithelial cells
like keratinocytes upon inflammatory activation (see Refs. 4, 7, and 8,
for review). There is overwhelming evidence that they are involved in
inflammatory processes (4, 7, 9). On the amino acid level the sequences
show 60% homology among different species (10). Based upon the
conserved amino acid sequence, the structural similarities, their
tissue-specific expression pattern, and their ability to form dimeric
complexes (11) they are regarded as typical members of the S100 protein family.
MRP8 and MRP14 represent an ideal model system to study the
dimerization of S100 proteins. MRP8 and MRP14 homologues are known from
a number of species, which facilitate the identification of important
residues by sequence comparison and "domain swap" experiments.
Additionally, a naturally occurring "deletion derivative" is known
from human MRP14, the so-called MRP14* isoform, which is 4 amino acids
shorter using a second ATG encoding methionine at position 5 as
alternative translational start site (12). Similarly, different MRP14
isoforms were observed by two-dimensional gel electrophoresis in murine
cells.1 With respect to the
biological relevance of these isoforms it might be interesting to know
whether the dimerization properties of the two proteins are different.
Last, these S100 proteins are members of a subgroup of the S100 family,
which are all coexpressed in the myeloid cell lineage as well as in
activated and/or fetal keratinocytes. Beside MRP8 and MRP14 two other
proteins belong to this group, namely human S100A12 (homologous to
bovine CAAF1) (13, 14) and psoriasin (S100A7, homologous to bovine
CAAF2) (15, 16). Although these four S100 proteins are expressed by the
same cells and are structurally highly homologous, they do not randomly
interact with each other. Despite the potential biological relevance of
the interaction specificity nothing is known about how this specificity
is mediated in S100 proteins.
Using the yeast two-hybrid system (17) this report presents evidence
that the C-terminal domain of S100 proteins is primarily involved in
protein-protein dimerization. The results are discussed with respect to
recently published structural data (6) and may have implications for
S100 proteins in general.
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EXPERIMENTAL PROCEDURES |
Construction of Plasmids--
Polymerase chain reaction
(PCR)2-amplified cDNA
fragments encoding the entire murine and human MRP's were fused
in-frame to either the DNA-binding domain or activation domain of yeast
plasmids pAS2-1 or pACT2 (CLONTECH Laboratories
Inc., Palo Alto, CA), respectively. Vector pAS2-1 encodes the
C-terminal GAL4 DNA-binding domain (amino acids 1 to 147) and the TRP1
selection marker. pACT2 encodes the N-terminal acidic GAL4 activation
domain (amino acids 768 to 881) and the LEU2 selection marker. To
facilitate directional cloning of the PCR products into the yeast
expression vectors the 5'-primers contained either a NcoI or
NdeI restriction site and the 3-primer contained a
BamHI restriction site. This resulted in the in-frame fusion
of each MRP to the 3' end of either the GAL4 (1-147) DNA-binding domain (pAS2-1), or the GAL4 (768-881) activation domain (pACT2).
To construct a vector encoding a chimeric S100A12/mMRP14 a PCR fragment
of the N-terminal domain of human S100A12 was generated, which
contained an EcoRI site at the 3' end. The murine MRP14 fragment was restricted with EcoRI (human S100A12 amino acid
lysine, position 32, murine MRP14 position 39, amino acid
phenylalanine) before ligation (Fig. 1). The chimeric murine/human
MRP14 encoding constructs hm14-pAS2-1 and mh14-pAS2-1 were also cloned
by enzymatic restriction of the PCR-amplified fragments of human MRP14
and murine MRP14 with EcoRI and subsequent ligation of the
fragments. The desired chimeric fragments were isolated via PCR with
the corresponding primers and cloned into pAS2-1 (Fig.
1).

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Fig. 1.
Schematic presentation of the chimeric MRP14
molecules. Murine/human chimeric MRP14 molecules were constructed
using a naturally occurring EcoRI site. Additionally, a
human S100A12/murine MRP14 molecule was cloned by PCR generating an
EcoRI site at the homologous position. Black box,
murine MRP14; hatched, human MRP14; gray box,
S100A12; m, murine; mh, N-terminal murine,
C-terminal human; hm, N-terminal human, C-terminal
murine.
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Deletion mutants (Fig. 2) were cloned
into the pAS2-1 yeast expression vectors as described above. The
deletions were generated by PCR using the murine MRP14 cDNA as
template and the corresponding primer pairs. Amino acid substitutions
located at the very ends of the MRP14 molecule were generated by using
mutated primers for the PCR reaction. Construction of the internal
MRP14 substitution mutants (Fig. 2) were achieved with the
QuickChangeTM Site-directed Mutagenesis Kit (Stratagene,
Heidelberg). Briefly, two oligonucleotide primers, which encode the
mutation and are complementary to each other, extend the entire plasmid
during temperature cycling by means of Pfu DNA polymerase. Parental DNA was digested with DpnI endonuclease (target sequence:
5'-Gm6ATC-3'), while the unmethylated PCR-amplified DNA
could not be restricted by DpnI. The PCR-generated plasmid
DNA was then transformed into Escherichia coli and the
inserts of several clones analyzed. The integrity of all constructs has
been verified by sequence analysis.

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Fig. 2.
Amino acid substitution and deletion mutants
of the murine MRP14. Residues supposed to be involved in
protein-protein interaction (bold letters) (6) were
exchanged to alanine (A). Additionally, the N- and
C-terminal deletions are shown. Numbers in
brackets indicate the position of the N- and C-terminal
amino acid. The sequences of both EF-hands are underlined,
the C-terminal tail of MRP14, which is unusual among S100 proteins, is
shown (black background). The table summarizes
the data of the -galactosidase filter lift assay resulting from the
interaction between various murine MRP14 mutants and murine wild type
MRP8 and MRP14, respectively. ++, strong; +, weak; , no blue
color.
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Yeast Transformation--
To exclude nonspecific interactions,
each vector was also solely transformed into yeast. Additionally, yeast
containing human or murine MRP8 and MRP14 encoding vectors and the
corresponding two-hybrid vector encoding human S100A12 were transformed
as negative control. Transformation was performed by the
high-efficiency lithium acetate method modified by Gietz et
al. (20) using yeast strain Y190 (MATa, ura3-52, his3-20,
ade2-101, lys2-801, trp1-901, leu2-3, 112, gal4D, gal80D,
cyhr2, LYS2::Gal1UAS-His3TATA-His3,
URA3::GAL1UAS-GAL1TATA-lacZ). Generally, vectors were sequentially transformed into yeast. A human
leukocyte MATCHMAKER cDNA library (CLONTECH)
was transformed into yeast Y190 carrying human MRP14 as bait. Screening
procedure was performed according to the manufacturer's instructions.
Blue colonies were isolated, inserts amplified via PCR, and sequenced according to standard methods.
-Galactosidase Activity--
-Galactosidase activity was
assayed on plates and in liquid following the manufacturer's
instructions (CLONTECH). Colony filter lifts were
performed by submersion of nitrocellulose transfer membranes lifted
from each plate into liquid. Subsequently filters were incubated at
30 °C for up to 8 h upon Whatman filter paper, which was
presoaked in Z-buffer (60 mmol/liter Na2HPO4 × 7H2O, 40 mmol/liter
NaH2PO4H2O, 10 mmol/liter KCl, and
1 mmol/liter MgSO4, pH 7.0) supplemented with 0.27%
-mercaptoethanol and 1.67% 5-bromo-4-chloro-3-indoyl
-D-galactoside in 100-mm Petri dishes.
To evaluate the
-galactosidase activity quantitatively, a single
yeast colony was inoculated into 5 ml of SD medium without tryptophan
and leucine and agitated overnight at 30 °C. Then, 2 ml were added
to 8 ml of YPD liquid and grown until the OD600 was 0.5 to
0.7. The yeast cells were processed for quantification of
-galactosidase activity following the CLONTECH
protocol. Briefly, 1.5 ml of the culture was centrifuged, resuspended
in Z-buffer, and the cells were lysed. Subsequently
O-nitrophenyl-
-D-galactopyranoside was added
to the protein extract, incubated at 30 °C, and the OD420 was measured. The
-galactosidase activity is
expressed in units according to Miller (18). Calculations were
performed following the manufacturer's instructions
(CLONTECH), also described by Ausubel et
al. (19). At least five independent colonies per mutant were
measured; mean values and standard errors were calculated. Differences
between values, which were above background (S100A12 as negative
control), but below 10 units were not quantitatively interpreted.
Western Blot Analysis--
In order to confirm that each desired
fusion protein was properly expressed in yeast, Western blot analysis
with the appropriate GAL4 mAb were performed. The GAL4 activation
domain and GAL4 DNA-binding domain monoclonal antibodies
(CLONTECH), which bind specifically to the major
activation domain (amino acid 768-881) or the DNA-binding domain
(amino acid 1-147) of the GAL4 protein, were used. The preparation of
yeast protein extracts and performing of the Western blot followed the
manufacturer's instructions (CLONTECH). For immunodetection an alkaline phosphatase-conjugated secondary antibody was used.
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RESULTS |
The yeast two-hybrid system (17) was used to investigate the
interaction between MRP8 and MRP14 qualitatively and quantitatively. Both human MRP8 and MRP14 and murine MRP8 and MRP14 coding regions were
cloned in-frame with the corresponding GAL domains of the two-hybrid
vectors. Additionally, the coding region of the human S100A12 cDNA
was cloned in-frame with the GAL-activating domain serving as a
negative control. We could not detect any Gal activity (values <1
unit) with any mutant or wild type MRP alone or in combination with
S100A12. Furthermore, Western blot experiments with protein extracts
from yeast clones containing the constructs described below could
confirm the integrity of the construct and proved the proper expression
of the fusion proteins (data not shown).
Human and Mouse MRP8 and MRP14 Differ in Their Dimerization
Properties--
Analysis of all possible combinations of MRP8 and
MRP14 encoding plasmids revealed that the heterodimerization of
MRP14/MRP8 is strongly preferred over any homodimerization. This was
true for both the human and murine MRP proteins. Surprisingly, human and murine MRPs appear to behave differently with respect to
homodimerization. The mouse MRP8 and MRP14 readily form homodimeric
molecules, although this interaction was significantly weaker than the
heterodimeric complex formation. We could not detect homodimerization
of the human MRPs (Fig. 3). To test the
possibility that human MRP14 may interact with so far unknown MRP14
isoforms, deletion derivatives, or other members of the S100 family,
which occur in leukocytes, a leukocyte library was screened to search
for such human MRP14 interacting proteins. Sixteen blue colonies were
analyzed in detail and all of them encoded the human MRP8 protein.

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Fig. 3.
A filter lift assay is shown demonstrating
the different dimerization behavior between human and murine MRP8 and
MRP14. Representative colonies of Saccharomyces
cerevisiae Y190 containing the plasmids as indicated have been
grown on nitrocellulose filter. Yeast colonies containing
-galactosidase activity were identified by the enzymatic conversion
of 5-bromo-4-chloro-3-indoyl -D-galactoside resulting in
blue colored yeast cells. Yeast cells without -galactosidase
activity remained colorless. Each letter represents a yeast
transformed with MRP encoding derivatives of pAS2-1 and pACT2,
respectively. A, mMRP8/mMRP14; B, mMRP14/mMRP8;
C, mMRP8/mMRP8; D, mMRP14/mMRP14; E,
hMRP8/hMRP8; F, hMRP14/hMRP14; G, hMRP8/hMRP14;
H, hMRP14/hMRP8.
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The C-terminal EF-hand Domain Determines the Specificity of
Interaction--
The amino acid sequence of the MRP8 and MRP14
proteins are highly homologous to each other among mouse and man. We
therefore examined whether mouse and human MRPs are able to form hybrid heterodimers. We could neither detect human MRP8-murine MRP14 nor
murine MRP8-human MRP14 nor any hybrid homodimeric complex formation.
Thus, the structural difference is obviously big enough to prevent any
protein-protein contact between a human and murine MRP. To investigate
which region of the MRP14 protein determines the specificity of
protein-protein interaction, we constructed chimeric mouse/human MRP14
molecules (Fig. 1). The hmMRP14 (N-terminal domain human/C-terminal
domain murine) interacts with murine MRP8 and murine MRP14, but not
with any human MRP. On the other side the mhMRP14 (N-terminal
murine/C-terminal human) complexed with human MRP8, but we could
neither detect any significant
-galactosidase activity with human
MRP14 nor with the murine MRPs (Fig. 4).
Thus mhMRP14 behaved like a human MRP14 and hmMRP14 like a murine MRP14 with respect to their dimerization specificity. Surprisingly, the
complex stability of the chimeric molecules with wild type MRP14 or
MRP8, respectively, was only slightly reduced compared with stability
of the wild type MRP14 hetero- or homodimer (Table I). A human S100A12/murine MRP14 chimeric
molecule did not dimerize with any human or murine MRP.

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Fig. 4.
Schematic presentation of the
-galactosidase activities in units (18) resulting from the
interaction between combinations of homo- and heterodimerization of
murine and human wild type MRPs and chimeric MRP14s indicating that the
specificity of interaction is encoded in the C-terminal half of the
MRP14. h, human; m, murine; mh,
N-terminal murine/C-terminal human; hm, N-terminal
human/C-terminal murine.
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Table I
Quantitative analysis of MRP8-MRP14 interaction
Overview about -galactosidase activities of all combinations, which
were tested for homo- and heterodimerization in the two-hybrid system.
In addition to the absolute -galactosidase activities in units (18)
calculated according to Ausubel et al. (19), the relative
Gal activities in percent are presented. Interaction of the murine and
human wild type molecules were set 100%. The strength of interaction
depends on the choice of vector used for MRP8 and MRP14, respectively.
This results in values above 100% for the interaction of
mMRP14-GAL4-DNA-BD/mMRP8-GAL4-AD and hMRP14-GAL4-DNA-BD/hMRP8-GAL4-AD.
All values indicate the range observed in at least five experiments.
Standard errors are shown. Values below 10 units were not interpreted
quantitatively. As negative control served the human S100A12 construct.
h, human; m, murine; mh, N-terminal murine/C-terminal human; hm,
N-terminal human/C-terminal murine; GAL4-AD, activating domain;
GAL4-DNA-BD, DNA-binding domain.
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C- and N-terminal Residues Are Not Essential for S100 Protein
Dimerization--
To investigate the role of the C- and N-terminal
residues, a number of deletion constructs were tested in the yeast
system. Deletions of the N-terminal 8 amino acids of murine MRP14
reduced the stability of the interaction (Fig. 2), but complex
formation was still detectable. This deletion mutant corresponds to a
potential murine MRP14* isoform. The corresponding deletion mutant was
also tested for the naturally occurring human MRP14* isoform, which is
4 amino acids shorter using a second methionine in position 5 as
translational start site. Deletion of these four N-terminal amino acids
of the human MRP14 also did not abolish the interaction with MRP8, but
again significantly decreased
-galactosidase activity. A single
amino acid exchange at position 3 from cysteine to serine (C3S) led to
a nearly identical reduction of interaction intensity (Table I).
A set of C-terminal deletions was constructed (Fig. 2). It is known
that the MRP14 molecule contains a C-terminal tail of about 12 amino
acids, which is unusual among S100 proteins. A deletion of these 12 C-terminal amino acids in murine MRP14 weakened, but did not inhibit
homo- or heterodimerization. Further deletions, however, namely mMRP14
(1-85) and mMRP14 (1-69), which affect helix IV located in the
C-terminal EF-hand domain, completely destroyed the ability to interact
with mMRP8 or mMRP14 (Fig. 2).
Amino Acid Exchanges of F89A, L95A, and F20A, but Not I17A Abolish
Dimerization--
Recent publications investigated the dimeric
structure of S100 protein calcyclin via NMR spectroscopy (6). Based
upon these data a limited number of evolutionary conserved amino acid
residues in helix I and IV seem to be responsible for the S100 protein dimerization. To investigate the functional relevance of these residues
several murine MRP14 mutants were constructed by exchanging single
amino acids (Fig. 2): phenylalanine at position 89 to alanine (F89A),
leucine at position 95 to alanine (I95A), isoleucine at position 17 to
alanine (I17A), and phenylalanine at position 20 to alanine (F20A).
Each amino acid exchange resulted in a dramatic decrease or complete
loss of
-galactosidase activity (Fig. 2). Only mutant (I17A) was
still able to dimerize significantly with MRP8 (Table I).
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DISCUSSION |
Complex formation is regarded to be an essential prerequisite for
the biological functions of S100 proteins. We therefore used the
two-hybrid system to analyze the dimerization of the S100 proteins MRP8
and MRP14. In contrast to the recently published data, which describe
the biophysical structure of S100 proteins in detail (6, 21) the
two-hybrid system allows a functional "in vivo" approach
to the molecular analysis of S100 dimers. Numerous studies have shown
that the two-hybrid system can be used to characterize and/or detect
protein-protein interactions (22). This report demonstrates that it may
also complement and enlarge our knowledge about the S100 protein dimerization.
We investigated the interaction between the murine and human MRP8 and
MRP14. Evidence is presented that heterodimerization is strongly
preferred to homodimerization in both species. This is in good
agreement with previous data, which suggest that the human MRPs form
preferentially heterodi- and -oligomers (11). Recent biophysical data
confirm, that there is a "unique complementarity in the interface of
human MRP8-MRP14 complex that cannot be fully reproduced in the MRP8
and MRP14 homodimers" (21). However, the formation of homodimers
could be demonstrated in vitro, if only one MRP species is
used. In mixtures of both proteins the heterodimer is at least
preferred 10:1 (21). According to our data, it seems questionable,
whether in vivo human MRP homodimers occur at all.
Murine MRPs are able to associate to homodimers, although the stability
of the homodimeric complexes is markedly reduced compared with the
heterodimer (Table I). This difference in homodimerization behavior
between human and murine MRPs is unexpected, because the primary
structure of the MRPs of both species is highly homologous. It would be
interesting to find out which structural property of the murine MRPs
provides the necessary flexibility that enables both homodimerization
and heterodimerization. In the case of murine MRP8, homodimerization
could recently be verified in vitro by electrospray
ionization mass spectrometry and cross-linking experiments. Unfortunately, heterodimerization was not investigated in this report
(23). We conclude, that also in mouse the heteromer is the naturally
occurring form at least in cells, which express both MRPs.
Nevertheless the difference between human and murine MRP8/MRP14
dimerization characteristics suggests, that there are not only
structural, but also functional differences. In this context it is
worth noting that the murine MRP8 displays a strong chemotactic activity on myeloid cells (24), a property which has not been observed
with human MRP8. It needs further investigation to clarify the
structural basis and a potential functional relevance of this difference between human and murine MRPs.
A comparison of the primary structure of S100 proteins reveals that the
MRP14 sequence contains a C-terminal tail, which is unique among S100
proteins (Fig. 2). It was speculated that this glycine and
histidine-rich peptide sequence is involved in target protein
recognition and subsequent to phosphorylation might exhibit neutrophil
immobilizing activity (25). To study the influence of this moiety on
dimerization a set of C-terminal deletion experiments were carried out.
The deletion of the terminal 12 residues containing the His/Gly-rich
tail does not prevent murine MRP14 homo- and heterodimerization.
Further deletions which affect helix IV completely abolish MRP
interaction confirming the importance of this region for MRP complex
formation. This result implies that the unique tail is not essential
for MRP14/MRP8 association, and may argue for the hypothesis that this
region might be involved in interaction with target protein(s) other
than MRP8.
There is a second MRP14 form present in human myeloid cells, the
so-called MRP14* isoform lacking four N-terminal amino acids. It was
speculated that MRP14* may fulfil different biological functions (12).
The deletion experiments reported here demonstrate that the naturally
occurring human MRP14* (MRP14 (5-114)) is able to dimerize with human
MRP8. However, complex stability is reduced (Table I). The analysis of
the mutant MRP14 C3S implicates that the cysteine at position 3 of the
full size MRP14 is involved in complex formation (Table I). This
finding is surprising since other studies demonstrate that disulfide
bridges do not contribute to the S100 complex formation (11, 21).
Similarly, the N-terminal deleted murine MRP14, the potential murine
MRP14*, is also able to form homo- and heterodimers. The murine MRP14 N
terminus does not encode a cysteine. Whether the reduced stability may
be of biological relevance remains to be clarified. We conclude that neither the N-terminal nor the C-terminal residues of MRP14 are critical for the quality of MRP dimerization.
Despite their structural similarity the murine and human MRPs are not
able to interact across species borders. Analysis of chimeric
mouse/human MRPs indicate that the C-terminal domain determines the
specificity of interaction with respect to species specificity as well
as homodimerization characteristics (Fig. 4). Even the intensity of
protein-protein association is only slightly weaker using the chimeric
molecules (Table I). The chimeric human S100A12/murine MRP14 molecule
did not dimerize with MRPs in yeast, suggesting that not any N-terminal
S100 domain can substitute the homologous one.
Structural models indicate that several conserved hydrophobic residues
located in both the N-terminal EF-hand domain (helix I) and the
C-terminal EF-hand domain (helix IV) mediate the S100 dimer formation
(6). Consequently, we mutated the corresponding amino acids by
site-directed mutagenesis (Fig. 3). The analysis of three of these
mutants, F20A, F89A, and L95A, seems to confirm the importance of these
residues. Only with mutant I17A could a considerable
-galactosidase
activity (20 units) be detected in the assay (Table I). Thus, one of
these evolutionary conserved residues located in the C-terminal domain
which were recently identified by NMR spectroscopy to be directly
involved in S100 protein-protein association (6) is not essential for
MRP8-MRP14 complex formation. The data obtained with the chimeric MRP14
and the single amino acid substitutions suggest that the structural prerequisites for dimerization seem to be far more relaxed for the
N-terminal domain of S100 proteins. We conclude, that the C-terminal
domain, most likely residues of helix IV, determines the specificity of
interaction and is the critical domain for S100 protein dimerization.