Analysis of the MRP8-MRP14 Protein-Protein Interaction by the Two-hybrid System Suggests a Prominent Role of the C-terminal Domain of S100 Proteins in Dimer Formation*

Christian Pröpper, Xiaohua Huang, Johannes Roth, Clemens Sorg, and Wolfgang NackenDagger

From the Institute of Experimental Dermatology, Münster Medical School, Von-Esmarch Strasse 56, D-48149 Münster, Germany

    ABSTRACT
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ABSTRACT
INTRODUCTION
REFERENCES

Calcium-binding S100 proteins are thought to play a central role in calcium-mediated signal transduction pathways. They consist of two helix-loop-helix, calcium-binding EF-hand domains. A characteristic feature is their tendency to form homo- and/or heterodimeric complexes. This report presents for the first time a functional "in vivo" approach to the analysis of S100 protein dimerization. Using the two-hybrid system we analyzed the dimerization of MRP8 (S100A8) and MRP14 (S100A9), two S100 proteins expressed in myeloid cells. It is reported that the MRP8-MRP14 heteromer is the clearly preferred complex in both man and mouse. The ability to homodimerize, however, appears to be restricted to the murine MRPs. Interaction analysis of chimeric murine/human MRP14 proteins indicates, that the C-terminal EF-hand domain plays a prominent role in MRP8-MRP14 interaction and determines the specificity of dimerization. Site-directed mutagenesis of four evolutionary conserved hydrophobic amino acids, which have been recently supposed to be essential for S100 protein dimerization, suggests that at least one of these, namely the most N-terminal located residue, is not critical for dimerization.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
REFERENCES

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.

    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.

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 beta -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.

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.

beta -Galactosidase Activity-- beta -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% beta -mercaptoethanol and 1.67% 5-bromo-4-chloro-3-indoyl beta -D-galactoside in 100-mm Petri dishes.

To evaluate the beta -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 beta -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-beta -D-galactopyranoside was added to the protein extract, incubated at 30 °C, and the OD420 was measured. The beta -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.

    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 beta -galactosidase activity were identified by the enzymatic conversion of 5-bromo-4-chloro-3-indoyl beta -D-galactoside resulting in blue colored yeast cells. Yeast cells without beta -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.

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 beta -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 beta -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 beta -galactosidase activities of all combinations, which were tested for homo- and heterodimerization in the two-hybrid system. In addition to the absolute beta -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.

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 beta -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 beta -galactosidase activity (Fig. 2). Only mutant (I17A) was still able to dimerize significantly with MRP8 (Table I).

    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 beta -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.

    ACKNOWLEDGEMENTS

We thank B. W. Schafer for providing the S100A12 cDNA clone. T. Vogl, C. Kerkhoff, F. Schönlau, and M. Klempt for critical reading the manuscript and helpful discussions.

    FOOTNOTES

* This work was supported in part by Deutsche Forschungsgemeinschaft Grant Na 282/2-1 (to W. N.) and by a Sonderforschungsbereich 293 grant (to J. R.).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.

Dagger To whom correspondence should be addressed: Institute of Experimental Dermatology, Münster Medical School, Von Esmarch Str. 56, D-48149 Münster, Germany. Tel.: 0251-8356552; Fax: 0251-8356549; E-mail: hyperlink mailto:nacken{at}uni-muenster.de nacken{at}uni-muenster.de.

1 J. Roth, unpublished data.

    ABBREVIATIONS

The abbreviations used are: PCR, polymerase chain reaction; Gal, galactosidase..

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