Protein-Protein Interaction Studied by Site-directed Mutagenesis

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 267, No. 20, Issue of July 15, pp. 14175-14182,1992 Printed in U.S.A.

Protein-Protein Interaction Studied by Site-directed Mutagenesis CHARACTERIZATION OF THE ANNEXIN 11-BINDING SITE ON p l l , A MEMBER OF THE SlOO PROTEIN FAMILY*

(Received for publication, February 5, 1992)

Eckhard Kube, Tilmann Becker, Klaus Weber, and Volker GerkeS From the DeDartment of Biochemistrv. Max Planck Institute for Biophysical Chemistry, P. 0. Box 2841,D-3400 Goettingen, “, Federal Republic of Germany

p l l , a member of the SlOO protein family, forms a stable heterotetrameric complex with annexin 11. The p l 1-binding site of annexin I1 resides in the N-terminal 14 residues, which form an amphiphatic a-helix with the hydrophobic face representing the contact site for p l l (Johnsson, N., Marriott, G., and Weber, K. (1988) EMBO J. 7,2435-2442). We show that a corresponding peptide can be used to purify recombinant p l l by affinity chromatography. To map the annexin 11-binding site on p l l , we have produced progressively truncated p l l derivatives by site-directed mutagenesis. Our analysis reveals that a highly hydrophobic region between residues 85 and 91 is indispensable for annexin 11-binding. It is located in the C-terminal extension, following the second distorted EF-hand. Using a series of single amino acid replacements, we have identified individual hydrophobic residues, which seem to represent contact points for annexin 11. Most notably, substitution of tyrosine 85 or phenylalanine 86 by alanine drastically reduces the affinity of p l l for annexin 11, whereas replacement of these residues by tryptophan has no or only a marginal effect. Thus, hydrophobic side chains on both annexin I1 and p l l are involved in complex formation.

p l l is a member of a recently identified multigene family of small dimeric proteins, which are characterized by two consecutive Ca2+-binding motifs of the helix-loop-helix struc- ture, the so-called EF-hand. The entire protein family was named SlOO after the small acidic SI00 proteins originally isolated from brain (for review, see Hilt and Kligman, 1991). In addition to p l l and SlOOa and p, the SI00 family includes proteins which are expressed in a cell-cycle or growth-dependent manner, e.g. calcyclin and 18A2, as well as proteins which are induced during certain inflammatory processes, e.g. the calgranulins A and B (also known as MRP 8 and 14). The SlOO proteins are a subgroup of the EF-hand superfamily of Ca2+-modulated proteins, which also contains proteins with a 4-fold repetition of the EF-hand motif, e.g. troponin C and calmodulin (for review, see Persechini et al., 1989). Although the precise biological functions of the SI00 proteins are not known, the structural relation to calmodulin and troponin C indicates that they might act as Ca2+-sensitive modulators of certain cellular target proteins.

p l l is so far the only member of the family for which a

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

4 To whom correspondence should be addressed.

cellular protein ligand has been identified unambiguously. It binds in a highly specific manner to annexin 11, a major cellular substrate for the tyrosine kinase encoded by the src oncogene (for review, see Gerke et al., 1990). However, in contrast to the interaction between other EF-hand proteins and their cellular targets, p l l binding to annexin I1 does not depend on the presence of Ca2+ (Gerke and Weber, 1985a). This is because p l l has suffered crucial amino acid deletions and substitutions in both EF-hand loops. The mutations render the Ca2+-binding sites inactive and probably fix the p l l molecule in an “interaction-competent’’ conformation (Gerke and Weber, 1985a; Glenney, 1986). Annexin 11, the cellular ligand of p l l , is a member of a second multigene family, the so-called annexin family of Ca2+-dependent phospholipid- and membrane-binding proteins (for review, see Klee, 1988; Gerke et al., 1990; Moss et al., 1991). Interaction of a p l l dimer with annexin I1 leads to the formation of a heterotetrameric annexin I12pl12 complex, which shows a high stability (dissociation constant below 30 nM, Johnsson et al., 1988). The complex has a significantly increased affinity toward Ca2+ and/or phospholipid as compared to monomeric annexin I1 (Powell and Glenney, 1987). In addition, the annexin I12pl12 complex is able, unlike the annexin I1 mon- omer, to induce the fusion of chromaffin granule vesicles at physiologically relevant Ca2+ concentrations, indicating again the regulatory importance of p l l binding (Drust and Creutz, 1988).

The pll-binding site on annexin I1 is contained within the N-terminal 10 to 14 amino acids, which form an amphiphatic a-helix upon p l l binding (Glenney et al., 1986; Johnsson et al., 1986, 1988). Thus, p l l binding occurs in the so-called N- terminal tail (residues 1-29) of annexin 11, which also harbors the src kinase phosphorylation site (Tyr-23) and forms a domain distinct from the protein core (residues 30-338). The core domain, which is resistant to limited proteolysis and characterized by a 4-fold repetition of the annexin-type seg- ment of 70-80 amino acids, harbors the binding sites for Ca2+ and phospholipid (for review, see Gerke, 1989). By analyzing a large number of synthetic peptides, which differed from the 14 N-terminal residues of annexin I1 by single amino acid substitutions, Becker et al. (1990) showed that the N-acetyl group of Ser-1 and the hydrophobic side chains on one side of the amphiphatic helix contribute most to the p l l binding. Thus, hydrophobicity seems the major stabilizing factor of the annexin 11-pll interaction. Despite the detailed knowl- edge about the pll-binding site on annexin 11, the contact site of the N-terminal annexin I1 helix on p l l has not been identified. However, chemical modification experiments sug- gest that Cys-82 or its immediate environment is involved in annexin I1 binding, while the other cysteine residue in p l l , Cys-61, does not influence the binding (Johnsson and Weber,

14175

14176 p l l -Annexin 11 Interaction

1990a). Cys-82 is located in the so-called C-terminal extension, which follows the last helix of the second EF-hand (for review, see Gerke, 1991). Since this C-terminal extension (residues 78-96) is highly conserved among p l l molecules from different vertebrates, it could play an important role in annexin I1 binding (Kube et aL, 1991).

Here, we have employed site-directed mutagenesis and the expression of recombinant p l l molecules in Escherichia coli to map the annexin 11-binding site. C-terminal truncations of increasing lengths revealed that the unique C terminus of p l l is important for binding. The contribution of individual residues was determined by analyzing a series of point mutants for their affinity to annexin 11. This approach led to the identification of Tyr-85 and Phe-86 as two hydrophobic amino acids most likely directly involved in annexin I1 binding. Introduction of an alanine in place of Tyr-85 or Phe-86 resulted in a significantly reduced annexin I1 affinity, whereas the replacement by tryptophan, another bulky hydrophobic amino acid, had only a marginal effect. The results strongly support the hydrophobic nature of the protein-protein interaction in the annexin 11-pll complex.

EXPERIMENTAL PROCEDURES

Materials-Restriction enzymes and T4-ligase were obtained from New England Biolabs, and calf intestinal phosphatase and ampicillin were from Boehringer Mannheim (Federal Republic of Germany (F. R. G.)). IPTG,' chloronaphthol, EGS, and tricine were purchased from Sigma (Deisenhofen, F. R. G.); H202, dimethyl sulfoxide were from Baker (Deventer, Netherlands), and the fluorophore Prodan from Molecular Probes (Eugene, OR).

Synthesis and Purification of Synthetic Oligonucleotides-01igo- nucleotides were synthesized on a Biosearch 8700 DNA synthesizer (New Brunswick, Heusenstamm, F. R. G.). They were purified by electrophoresis in 16% (w/v) polyacrylamide, 7.5 M urea gels in 1 X TBE (90 mM Tris-borate, 90 mM boric acid, 2 mM EDTA). Bands migrating at the expected size were visualized by UV shadowing on silica gel plates, cut out, and eluted overnight at room temperature in 300 mM sodium acetate, pH 7.0, 1 mM EDTA. The eluted oligonucleotides were desalted with SEP-PAK C18-columns (Millipore, Esch- born, F. G. R.) and stored at -20 "C in sterile water.

Expression of Human pll-cDNA-10 pg of human pll-cDNA (Kube et al., 1991) was digested with 2 units of NcoI and HindIII following standard protocols (Sambrook et al., 1989). A 450-bp fragment covering nucleotides 134-584 (Kube et al., 1991), i.e. most of the protein coding plus 3'-nontranslated region, was isolated and ligated to two complementary oligonucleotides (pll-8+: 5"GGAAT-

TTCCATTTGAGATGGCATGAATTCC). pll-8+ and pll-8- rein- stall the 5' part of the p l l protein-coding region (nucleotides 112- 134 of the cDNA), which was removed by the NcoI-Hind111 digestion of the pll-cDNA. In addition, the two oligonucleotides introduce an EcoRI restriction site directly 5' to the ATG start codon. Following ligation the DNA was digested with EcoRI and HindIII to yield a 479-bp fragment containing the entire p l l protein coding region. This fragment was ligated into the EcoRI/HindIII linearized expression vector pkk223-3 (Pharmacia, Freiburg, F. G. R.). After transformation of competent E. coli JMlOl cells, clones containing the plasmid pkkpl l were identified by growth on LB-agar containing 0.1 mg/ml of ampicillin (Sambrook et al., 1989). Clones expressing recombinant p l l were identified by analyzing total protein extracts of bacteria, which were grown overnight in LB containing 0.1 mg/ml ampicillin and 1 mM IPTG, in 15% (w/v) polyacrylamide tricine gels (Schagger and von Jagow, 1989).

Purification of Recombinant pll by Conventional Chromatogra- phy-E. coli cells expressing recombinant p l l were grown at 37 "C overnight in 500 ml of LB medium containing 0.1 mg/ml ampicillin and 1 mM IPTG. Cells were collected by centrifugation (30 min at 5,000 X g, 4 "C). The cell pellet was washed once in Mg2+ buffer (0.2

The abbreviations used are: IPTG, isopropyl-P-D-thiogalactopyr- anoside; DTT, dithiothreitol; EGS, ethyleneglycolbis-(succinic acid N-hydroxysuccinimide ester); EGTA, [ethylenebis(oxyethyleneni- tri1o)ltetraacetic acid; SDS, sodium dodecyl sulfate; WT, wild-type.

TCATGCCATCTCAAATGGAACACGC;pll-8-: 5"CATGGCGTG-

M Tris-HC1, pH 7.5, 0.2 M NaCl. 10 mM MgCl,, 1 mM NaN3, 2 mM DTT) and then resuspended in 25 ml of Mg2+ buffer. The suspension was sonified six times for 1 min at 30 W (Branson sonifier). After ultracentrifugation (35,000 X g, 30 min, 4 "C) the supernatant was dialyzed overnight against DE buffer (20 mM imidazole-HC1, pH 7.5, 50 mM NaCl, 2 mM DTT, 0.5 mM EDTA, 0.5 mM EGTA, 1 mM NaN3), and then applied to a DE52 column (2.5 X 10 cm, Whatman) equilibrated in DE52 buffer. The flow-through fractions containing the recombinant p l l were dialyzed overnight against CM buffer (20 mM sodium acetate, pH 5.6, 2 mM DTT, 0.5 mM EDTA, 0.5 mM EGTA, 1 mM NaN3), clarified by centrifugation, and applied to a Mono S column connected to an FPLC system (Pharmacia, Freiburg, F. R. G.). Recombinant p l l was eluted with a linear NaCl gradient at approximately 250 mM salt. Subsequently, the sample was concentrated in Centricon tubes (Amicon, Witten, F. R. G.) , dialyzed against p l l buffer (25 mM Tris-HC1, pH 7.5, 2 mM DTT, 0.5 mM EDTA, 0.5 mM EGTA, 1 mM NaN3) containing 9 M urea and applied to a Sephacryl S-200 column (1 X 100 cm, Pharmacia) equilibrated in the same urea buffer. S-200 fractions containingpll were dialyzed against p l l buffer to renature the protein (Gerke and Weber, 1985b). p l l was stored at -20 "C.

Purification of Recombinant pll by Affinity Chromatography- Recombinant p l l or p l l mutant proteins (see below) were also purified by affinity chromatography on an Acl-14 peptide matrix (see below). 25 ml of E. coli lysate containing p l l or a particular p l l mutant were incubated with 10 ml of the peptide matrix in a small column for 30 min at room temperature. Subsequently, the column was washed extensively with TBS (20 mM Tris-HC1, pH 7.4, 0.15 M NaCl), Triton buffer (0.1 M Tris-HC1, pH 7.5, 2 mM DTT, 2 mM NaN3, 0.2% (w/v) Triton X-loo), and NaCl buffer (0.1 M Tris-HC1, pH 7.5, 2 mM DTT, 2 mM NaN3, 5 M NaCl). Bound wild-type or mutant p l l was released from the column with p l l buffer containing 8-10 M urea (depending on the mutant). It was concentrated, chro- matographed on S-200 (see above), and renatured by dialysis to remove the urea (Gerke and Weber, 198513). Tryptophan-containing p l l mutants for analysis in fluorescence emission spectroscopy were subjected to an additional purification step on a reverse-phase C18 column (Vydac, Hisperia, CA). The column was developed with a linear acetonitrile gradient (0-90%) in 0.1% trifluoroacetic acid. p l l eluted at approximately 45% (v/v) acetonitrile and was dialyzed against p l l buffer.

Oligonucleotide-directed Mutagenesis-The modified pll-cDNA (see above) was inserted into the EcoRI/HindIII treated double- stranded form of M13 mp18 (New England Biolabs) using standard protocols (Sambrook et al., 1989). The single-stranded M13-DNA containing the complete p l l protein coding region was isolated according to Sambrook et al. (1989). Mutagenesis was performed on the single-stranded M13 construct with an oligonucleotide-directed in vitro mutagenesis kit (Amersham) following the manufacturer's protocol. All mutations were verified by dideoxy sequencing (Sanger et al., 1977).

Acl-14 Peptide Synthesis and Preparation of the Acl-14 Affinity Matrix-The peptide Acl-14 comprises the first 14 residues of annexin I1 and carries the N-terminal acetyl group. Its synthesis and purification has been described (Becker et al., 1990). For fluorescence assays, the peptide was labeled at Cys-8 with 6-proprionyl-2-dime- thylaminonaphthalene (Prodan, Prendergast et al., 1983) as described (Johnsson et al., 1988). To immobilize peptide Acl-14, cysteine 8 was replaced by serine, as in chicken annexin I1 (Johnsson et al., 19881, and the peptide was linked via a C terminally added cysteine (Cys- 15) to AH-Sepharose. 1 g of AH-Sepharose (Pharmacia) was washed with an excess of 1 M NaCl and then with 40 mM potassium phosphate, pH 7.2. 20 mg of maleimidobenzoyl sulfosuccinimide ester

sium phosphate (40 mM, pH 7.2) and incubated with the washed (Sulfo-MBS, Pierce Chemical Co.) was suspended in 5 ml of potas-

Sepharose for 2 h at room temperature. Subsequently, 10 mg of peptide Acl-14 was suspended in potassium phosphate (40 mM, pH 7.2) and incubated with the Sulfo-MBS-Sepharose conjugate for 1 h at room temperature and then for 12 h at 4 "C. The final product (Acl-14-Sepharose) was washed as described, incubated with 0.4 M P-mercaptoethanol for 2 h at room temperature to inactivate the unreacted cross-linking reagent, and then washed extensively with TBS containing 2 mM NaN3.

Peptide Matrix Assay-3 ml of E. coli lysate (i.e. the 100,000 X g supernatant) containing recombinant wild-type p l l , or a specific p l l mutant, was applied to 1.5 ml of Acl-14-Sepharose resin. After extensive washing with TBS, the beads were suspended in 4.5 ml of TBS and then divided into 9 aliquots. The peptide matrix was pelleted

p l l -Annexin I I Interaction 14177

hy a hrief spin (2 s, 10,000 X g) and resuspended in a buffer containing 100 mM Tris-HCI, pH 7.5, 2 mM DTT, 2 mM NaN3 plus gradually increasing urea Concentrations (increasing hy 1 M/atiquot). After incubation for 30 min at room temperature, the heads were pelleted and the protein eluted in the supernatant was analyzed in tricine SDS-l5% (w/v) polyacrylamide gels (see ahove).

F/uorrscmce Emission Spectroscopy-Fluorescence emission spectra were recorded on an SLM model 8000 spectrofluorometer (Ur- hana, 11,) hetween 305 and 420 nm (with the excitation wavelength set at 295 for the tryptophan fluorescence) or between 400 and 650 nm (with the excitation at 380 nm for the Prodan fluorescence, .Johnsson ct al., 1988; Recker et al., 1990). All emission data were corrected with respect to background signals. Spectroscopy was performed in 25 mM potassium phosphate, pH 7.1, 2.5 mM DTT in a total volume of 0.4 ml. Prior to the spectroscopic analysis, the proteins were incuhated in H20 containing 2.5 mM DTT to estahlish a fully reduced form of pl1. Spectra collected from samples with an increased volume (maximum 0.44 ml) were corrected with respect to the dilu- t ion.

Other T~chniyue,s-Immunoblots were performed according to Towhin ct 01. (1979) and Burnette (1981). N-terminal sequence analysis was carried out on an Applied Riosystems gas-phase sequenator (model 470A) with an on-line phenylthiohydantoin analyzer. Protein concentrations were determined according to Bradford (1976). The UV spectrum of p l l was measured on a UV240 photometer (Shi- madzu) in 25 mM Tris-HCI, pH 7.5, 2.5 mM DTT. Chemical cross- linking of p l l dimers WAS carried out using EGS as the cross-linking reagent. 50 pg of p l l in 10 M urea solution (volume 0.1 ml) was dialyzed overnight against 0.1 M potassium phosphate, pH 7.1. The renatured protein was incuhated with 10 pg of ECS in 6 p1 of dimethyl sulfoxide. Aft.er 30 min at room temperature, the reaction was stopped with 1.5 pg of glycine in 50 pl of 0.1 M potassium phosphate, pH 7.1. The sample was dialyzed overnight against 0.1 M (NH,)HCO,, pH 7.0, dried in a Speed Vac concentrator (Savant Instruments, Far- mingdale, CT), resuspended in SDS gel sample huffer (Laemmli, 1970), and analyzed in a Tricine SDS-15% (w/v) polyacrylamide gel (Schagger and von Jagow, 1987). The p l l overlay of porcine annexin I1 immobilized on nitrocellulose was performed as described (Gerke et a/., 1991). Briefly, 2 pg of annexin II/lane was suhjected to electrophoresis in an SDS-12.5% (w/v) polyacrylamide gel (Laemmli, 1970) and transferred t.o nitrocellulose (see above). Free binding sites on the nitrocellulose were hlocked with 3% (w/v) serum albumin in TRS (see above). Suhsequently, the nitrocellulose was incuhated with 1 ml of crude extracts of E . coli containing recornbinant wild-type p l l or p l l stop mutants (20 pg of total protein/O.l ml, 1 h at room temperature). After extensive washing, hound wild-type or mutant p l l was detected immunologically with the monoclonal antibody HZ1 (Osborn c't al., 1988).

RESULTS

Expression, Purification, and Characterization of Recombi- n a n t p l I-To install recognition sites for restriction enzymes, which would allow straightforward cloning into procaryotic expression vectors, the human p l l cDNA (Kube et al., 1991) was modified. A NcoI/HindIII fragment of the cDNA, which contained with the exception of the first 23 nucleotides the entire protein coding region plus 179 nucleotides of 3'-nontranslated sequence, was ligated to two complementary oligonucleotides, which reinstalled the 23 nucleotides at t h e 5' end. The oligonucleotides also created a unique EcoRI restric- t ion site directly 5' to the ATG start codon. Subsequent digestion with EcoRI and Hind111 yielded a fragment, which could be cloned directly into the bacterial expression vector pkk223-3 (Amann et al., 1983; Amann and Rrosius, 1985). Transformation of E. coli strain JMlOl wi th t he pkkp l l plasmid led to the synthesis of recombinant pll . When total bacterial proteins were analyzed in SDS gels a strong hand at approximately 11 kDa was visible in the pkkpll-transformed strain but not in the strain bearing the parental pkk223-3 plasmid (Fig. lA, lanes I and 2). High speed centrifugation of the disrupted bacteria revealed that most of the recombinant p l l exists in a soluble form, i.e. it is recovered in the supernatant (Fig. lA, lane 3 ) . Recombinant p l l was purified from

B

\ !i"- . ~ . 1 1 . , 1 v l a - I",

FIG. 1. Purification of recombinant WTp11 from bacterial extracts and characterization of the purified protein. Itecom- binant p l l was purified from hacterial extracts as outlined under "Experimental Procedures." Samples from the different purification steps were analyzed in Tricine SDS-15ri (w/v)-polvacrylamiclc gels and stained with Coomassie Hrilliant Blue ( /anes I-6. 8, 9). A. E,'. coli JMlOl total extract (lane I ) , total extract from 5Ml01 cells expressing recornbinant WTpll (lane 2; note the strong additional hand at approximately 11 kDa), supernatant after sonification and high speed centrifugation (lane 3 ) , flow-through of the DE.52 column ( /one 4 ) . p l l pool after chromatography on Mono S (lane 5). p l l after final purification steps on S-200 and a ClH reverse-phase column (lanp 6 ) . lmmunohlot analysis reveals that purified p l l is decorated hv monoclonal antihody H21 (Oshorn et al., 198R. lane 7) . Lanes H and 9, cross-linking of the native p l l dimer hy EGS. Hecomhinant pl 1 was incubated for 30 min in the ahsence (lanv H ) or presence (/an#. 9 ) of cross-linking reagent and suhsequently analyzed in a Tricine SDS- 1.5% (w/v)-polyacrylamide gel. Note the very efficient Cross-linking of the p l l dimer hy EGS (lane 9). R, IJV spectrum of rccomhinant WTpll. The spectrum was recorded on a n L'V240 Spectrometer (Shimadzu) in 25 mM Tris-HCI, pH 7.5, 2.5 mM [>TI'. Sote the characteristic phenylalanine maxima.

wo...-o*".-,

the bacterial extract by a series of conventional chromato- graphic steps. Due to the basic nature of p l l , ion-exchange chromatography on DE52 separates pll from most of the bacterial host proteins (Fig. lA, lane 4 ) . Additional purification was achieved by ion-exchange chromatography on Mono S (Fig. lA, lune Fj), gel filtration on Sephacryl S-200 and H P L C o n a reverse-phase Clfl column (Fig. lA, l a w 6 ) .

Recombinant p l l is identical to the authentic molecule purified from porcine intestine as judged by various biochemical criteria. I t exhibits the same behavior throughout the purification scheme and has the same mobility in SDS-polyacrylamide gels. p l l hinds the monoclonal antibody H21 (Osborn et al., 1988), which recognizes human, porcine, bo- vine, and chicken hut not murine and rat p l l (Fig. l A , lane 7). Recombinant p l l can he cross-linked to dimers by dimethyl-suberimidate (not shown) or EGS (Fig. lA, lanes R and 9) as can the authentic protein (Gerke and Weher, 1985h). Thus, the recombinant protein most likely forms the same dimer as authentic pll. N-terminal sequencing of the protein purified from E. coli revealed the sequence PSQME (not shown), indicating that the bacterially synthesized protein shows the same posttranslational modification as p l l expressed in mammalian cells, i.e. the initiator methionine is removed (Gerke and Weber, 1985a). Porcine p l l shows a distinct UV ahsorption spectrum. Due to the ahsence of tryptophan the spectrum is characterized by the high content of phenylalanine (9 residues) compared to only 2 tyrosine residues (Gerke and Weber, 1985b). The characteristic phenylalanine absorption maxima are also visible in the UV spec- t rum of recombinant p l l (Fig. 1R). The spectra of authentic porcine and recombinant human pll are indistinguishahle.

Identical properties of recombinant and authentic pll are also observed when the interaction with the intracellular protein ligand annexin I1 is studied. Under native conditions p l l a n d a n n e x i n I1 form a tight heterotetrameric complex

14178 p l I-Annexin I I Interaction

(annexin I12pl12). Complex formation can be monitored by the fluorescence emission of Prodan (6-proprionyl-2-dimethyl- aminonaphtha1ene)-labeled annexin 11, which is significantly altered upon p l l binding (Johnsson et al., 1988). The annexin I1 peptide Acl-14, which covers the N-terminal14 amino acid residues of annexin 11, can substitute in this assay for annexin I1 since it contains the entire pll-binding site (Johnsson et al., 1988; Becker et al., 1990). Upon excitation a t 380 nm Prodan-bound Acl-14 shows an emission maximum a t 530 nm, which is shifted to 483 nm when p l l is added. T h e p l l - induced shift in the emission maximum is accompanied by a 5-fold increase in signal intensity (Johnsson et al., 1988). The same changes in the fluorescence emission of Prodan-labeled Acl-14 are observed when recombinant p l l is used instead of p l l from porcine intestine, indicating that the binding site for annexin I1 on recombinant p l l is folded properly (see below and Fig. 5). Binding of recombinant p l l t o intact annexin I1 is also indistinguishable from that of the authentic protein. In a ligand overlay assay (Gerke et d., 1991), both authentic and recombinant p l l bind to immobilized annexin I1 under identical conditions (not shown). Based on this structural and biochemical equivalence of authentic and recombinant p l l we chose to study the sequence parameters of the annexin 11-binding site on p l l by a mutational analysis of p l l molecules synthesized in bacteria.

C-terminal Truncations Identify a Region Important for Annerin I1 Binding-Previous experiments employing chemical modification of t h e p l l molecule identified Cys-82 as a residue whose structural integrity is critical for annexin I1 binding. Alkylation of Cys-82 with vinylpyridine leads to a p l l derivative which has lost the ability to form the annexin 11-pll complex (Johnsson and Weber, 1990a). This observation, as well as the fact that the C-terminal extension of the pl l molecule (residues 78-96) is highly conserved among species (Kube et al., 1991), made this region a prime candidate for annexin I1 binding. Therefore, we removed increasing portions of the C-terminal region of p l l by site-directed mutagenesis and studied the annexin I1 binding properties of the truncated p l l derivatives. The amino acid sequences of the three C-terminal truncation mutants are given in Fig. 2 A . All three mutants, GWStop, KSlStop, and Y85Stop, are expressed efficiently as soluble proteins in E. coli (Fig. 2B).

A I a 7u 30 4 0

They can be purified (Fig. 2C) with minor modifications of the procedure developed for recombinant wild-type pll . These take into account the less basic nature of the truncated molecules (4 lysine residues but no acidic amino acid are present in the extreme C terminus of wild-type p l l ; see Fig. 2A 1.

To determine the ability of the mutants to interact with intact annexin 11, we performed a ligand overlay with the C- terminal deletion mutants K91Stop and Y8fjStop. Porcine annexin I1 (kindly provided by Dr. N. Johnsson, Max-Planck- Institute) was transferred from a SDS-12.5% polyacrylamide gel to nitrocellulose and processed for t h e p l l overlay as described under "Experimental Procedures." The immobilized annexin I1 was incubated with porcine p l l (positive control), with E. coli crude extract without recombinant protein (neg- ative control), or with E. coli crude extracts containing recombinant wild-type p l l or the two truncation mutants, respectively. Bound p l l was specifically visualized by decoration with the monoclonal antibody H21, which recognizes the p l l mutants. While porcine p l l , recombinant wild-t-ype pl l , and K91Stop are able to bind to the immobilized annexin I1 Y85Stop yields no signal (not shown). To confirm this result, we incubated the same E. coli crude extracts with an affinity matrix containing immobilized Acl-14, Le. the acetylated N- terminal annexin I1 peptide which harbors the pll-binding site. Fig. 3 shows that recombinant wild-type p l l , G94Stop, and K91Stop bind specifically to the peptide matrix. Y85Stop, on the other hand, does not interact with immobilized Acl- 14 and remains together with E. coli proteins in the unbound fraction (Fig. 3). Thus, the ability to form a tight complex with annexin I1 is lost in a p l l molecule in which amino acids 85-96 have been deleted. In contrast, a shorter truncation, which deletes only residues 91-96, has either no or only a marginal effect on annexin I1 binding. Two possibilities can account for these findings: (1) because of the relatively large C-terminal deletion in the Y85Stop molecule (12 residues of the approximately 18 residues of the C-terminal extension) the annexin 11-binding site is perturbed in its general three- dimensional folding, and (2) the annexin 11-binding site or crucial parts of it are removed in the Y85Stop mutant. To distinguish between these possibilities and to localize the annexin 11-binding site more precisely, we created a series of

50 no ro nn m ( C I

WTpll P S O M E H A M E T M M F T F H K F A G D K G Y L T K E U L R V L M E K E F P G F L E N O K D P L A V ~ K I M K D L U ~ C R D G K V G T ~ S ~ F S L I A G L T I A C ~ J D Y F V V H M K ~ K ~ K K ~

G94Slop PSOMEHAMETMMFTFHKFAGDKGYLTKEDLRVLMEKEFPGFLENOKDPLAVDKIMKDLDOCRDGKVC.TOSFFSLIAGLT1ACt IDYFVV~IMKC)K~

K91Slop PSOMEHAMETMMFTFHKFAGDKGYLTKEDLRVLMEKEFPGFLENOKDPLAVDKIMKDLDOCRDGKVGFOSFFSLIA~;LT IACNDYFVVI I IA*

Y85Slop P S ~ M E H A M E T M M F T F H K F A G D K G Y L T K E D L R V L M E K E F P G F L E N O K D P L A V D K I M K D L D O C R U G K V G F ~ S F F S I . I A G L . T I A C ~ J D ~

B C

1 2 3 4 5 1 2 3 4

FIG. 2. Amino acid sequence, expression, and purification of the C-terminal truncation mutants. A , the amino acid sequences of the pll truncation mutants are shown helow the sequence of the wild-type molecule (WTp11). The C termini are indicated by asfrr i sk . and the mutated EF-hand-like loops are marked by lines ahove the WTpl l sequence. R, expression of C-terminal truncation mutants in h'.

coli. I m w 1, purified recoml)inant WTpll; lanes 2-5, total bacterial extracts of J M l O l cells expressing WTpll (lane 2). KSIStop (lane 3 ) . YR5Stop (Innr 4 ) , and JM101 cells carrying no expression plasmid (lane 5). C, purification of the C-terminal truncation mutants. Proteins were purified as under "13xperimental Procedures" and analyzed in a Tricine SDS-15% (w/v)-polyacrylamide gel: 1 pg lVTpl1 (lonp 1 ) is shown in comparison to 1 pg of the truncated molecules GS4Stop (lane 2). K91Stop (lone 3 ) . and YR5Stop (lone 4 ) .

p1 I -Annexin I I Interaction 14179

point mutations in the p l l molecule and studied their ability t o bind annexin I1 in different assays.

Identification of Individual Amino Acid Residues Necessary for Annexin II Binding-An analysis of 37 different Acl-14 peptides, which contained individual amino acid substitutions compared to the wild-type annexin I1 sequence revealed that certain hydrophobic residues in the amphiphatic a-helix are indispensable for high affinity p l l binding (Becker et al., 1990). Thus, it seems that hydrophobic interactions are the driving force for the annexin 11-pll complex formation. Con- sequently, we chose to substitute hydrophobic amino acids in p l l to identify residues directly involved in annexin I1 binding. Several hydrophobic residues in the C-terminal half of t h e p l l molecule, which are highly conserved in different vertebrates (Kube et al., 1991), were replaced by alanine using site-directed mutagenesis. If the contacts between p l l a n d annexin I1 were dependent on hydrophobic interactions, the substitution of a crucial hydrophobic amino acid by alanine should lead to a substantial reduction in affinity. Based on the previous observation that the structural integrity of Cys- 82 is important for annexin I1 binding (Johnsson and Weber, 1990a) we also introduced amino acid substitutions at this position. To assess the influence of a bulkier side chain, we replaced Cys-82 by glutamine. In addition, we introduced the closely related serine residue in place of Cys-82 to examine a n influence of the sulfhydryl group. The positions of the individual point mutations are summarized in Fig. 4.

All mutant proteins were synthesized efficiently as soluble

kDn WTpll O f )

G94Stop K91StoD YHSSloD

14 :

I D l 1

1 2 3 1 2 3 1 2 ' 1 1 2 . 5

FIG. 3. Interaction of recombinant WTpl1 and the C terminally truncated p l 1 derivatives with the immobilized Acl- 14 peptide. 1.5 ml of Acl-14 affinity matrix was incuhated with 3 ml of E. coli extract containing WTp11, G94Stop. KSlStop, and Y85Stop, respectively. After extensive washing, proteins hound to the matrix were eluted by boiling the heads in sample buffer (Laemmli, 1970). Total bacterial extracts are shown in lanes I , the unhound fractions are given in lanes 2, and the material eluted from the heads is shown in lanes 3. Note that WTpll, G94-Stop, and K91Stop are specifically bound hy the affinity matrix, while Y85Stop is not hound.

1 10 70 30 40

molecules in E. coli (not shown). Immunohlot analysis revealed that all point mutants were recognized by the monoclonal H21 antibody indicating that the H21 epitope is not perturhed. The ability of the different pl1 mutants to interact with annexin I1 was determined in a rapid and convenient screening assay which employed the Acl-14 affinity matrix (see "Experimental Procedures"). The immohilized annexin 11-peptide Acl-14 was incuhated with total bacterial extracts containing the recombinant p l l mutants (or wild-t-ype p l l as a control). The urea concentrations required to elute the individual p l l derivatives from this affinity matrix provided relative values for the affinities of the mutant proteins toward annexin 11. The results of the Acl-14 matrix assay are summarized in Tahle I. Four of the pll mutants (I54A, "A, L58A, 180A) yielded values similar to the wild-t-ype molecule indicating that the respective amino acid replacement did not markedly affect the annexin 11-binding site on p l l . Three other mutant proteins (F72L, L78A, V87A) showed only a slight to moderate decrease in binding activity. However, the remaining point mutants were either significantly impaired in their affinity toward Acl-14 (Y85A, and F86A, and to a lesser extent C82S and M90A) or did not hind to the Acl-14 matrix altogether (C82Q). The latter observation is in line with the finding that alkylation of Cys-82 also results in a p l l molecule which does not interact with annexin I1 (.Johns- son and Weber, 1990a). With the exception of Cys-82, which is probably not directly involved in hydrophohic interactions in the annexin 11-pll complex, all other p l l derivatives with a markedly reduced affinity for annexin TI, i . ~ . Y85A, FNA, and M90A, carried an alanine replacement for a large hydrophobic side chain. Interestingly, all 3 hydrophohic residues are located in the C-terminal extension and are deleted in the Y85Stop mutant. Probably as a consequence of this deletion Y85Stop has completely lost the ahilitv to hind annexin I1 (Fig. 3, Table I).

Among the hydrophohic amino acids which were mutated, the alanine replacement for Tyr-85 and Phe-86 led to the strongest defect in annexin I1 hinding. To evaluate in more detail the importance of the hydrophohic nature of the side chains in positions 85 and 86, we also introduced in these positions the amino acid tryptophan whose side chain has a similar hydrophohic character. Interestingly, the corresponding p l l mutants, i.e. Y85W and F86W, show an annexin TI affinity which is markedly increased when compared to the alanine mutants (YSSA, FRGA) and is almost comparahle to that of wild-type p l l (Tahle I). This shows that hydrophohic

50 ti0 70 Hr) ' V i ' Y;

WTpll P S O M E H A M E T M M F T F H K F A G D K G Y L T K E D L R V L M E K E F P G F L E N O K D P L A V D K I M K D L D 0 C R D G K V G F O S F F S L I A G L T I A C N ~ Y F V V ~ M K O K G K K * 1 5 4 A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A . . . . . . . . . . . .

M55A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

L 5 8 A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A " . . . . . . . . . . . . .

F 7 2 L . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . L . . . L 7 8 A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A

- A - 1 8 0 A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

C 8 2 0 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 . (.82S . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S . y 8 5 A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A

W y 8 5 w . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

F 8 6 A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A

VI F 8 6 W . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

" 8 7 A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A M 9 0 A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A

FIG. 4. Amino acid substitutions in the individual p l l point mutants. The wild-type sequence o f human pl1 is given in the u p p t row. Point mutations present in the individual proteins are indicated helow. The lines ahove the sequence of LV'TpI 1 mark the positions o f the EF-hand-like loops. The C termini are indicated hv asterisks.

14180 p l l -Annexin 11 Interaction TABLE I

Binding of different p l I mutant proteins to the AcI-14 affinity matrix

Total E. coli extracts containing different p l l derivatives were incubated with the Acl-14 affinity matrix as outlined under “Exper- imental Procedures.” After extensive washing the matrix was ali- quoted and incubated with stepwise increasing urea concentrations. The urea concentrations (in mol/liter) required for elution of the respective p l l derivative are listed. “ 0 indicates no binding.

Protein Binding to Acl-14 peptide matrix

WTpll 8

G94Stop 7 K91Stop 4 Y85Stop 0

I54A 7 M55A 7 L58A 7 F72L 6 L78A 6 I80A 7 C82Q 0 C82S 4 Y85A 314 Y85W 6 F86A 3 F86W 718 V87A 6 M90A 5

side chains in positions 85 and 86 are indispensable for high affinity annexin I1 binding. Thus, Tyr-85 and Phe-86 most likely represent direct contact points for the hydrophobic face of the amphiphilic Acl-14 helix.

The immobilized Acl-14 peptide also provided a highly selective affinity matrix for a rapid large scale purification of recombinant p l l . Wild-type p l l and mutant proteins with considerable affinity for Acl-14 (G94Stop, I54A, M55A, L58A, F72L, L78A, BOA, V87A) were obtained in more than 90% purity in a one-step procedure using the Acl-14 affinity matrix and total bacterial extracts (see “Experimental Pro- cedures”). This protocol yielded 50 mg of recombinant p l l from 1 liter of bacterial culture and could also be employed to isolate authentic p l l from mammalian cells (not shown). Thus, the immobilized Acl-14 efficiently competes with the pll-binding site on the annexin I1 present in mammalian cells.

Spectroscopic Analysis of the Annexin 11 Affinity of Some p l l Point Mutants-To obtain a set of independent data for the affinity of the different p l l mutants for annexin 11, we used Prodan-labeled Acl-14 in a spectroscopic assay. Cou- pling of the fluorophore Prodan to Cys-8 of the N-terminal annexin I1 peptide does not influence its ability to bind p l l but introduces a fluorescence probe that is sensitive to p l l binding (Johnsson et al., 1988; Becker et al., 1990). The fluorescence emission from the Prodan group is significantly altered upon addition of p l l . The emission maximum is shifted from 530 to 483 nm, and the fluorescence intensity is increased by a factor of 5 (Johnsson et al., 1988). Several recombinant p l l derivatives were purified and assayed spec- troscopically for their ability to interact with Prodan-labeled Acl-14. Fig. 5 shows three examples. The typical changes in the fluorescence emission of the Prodan group which are induced by p l l binding are also obtained with the recombinant wild-type molecule. When wild-type p l l a n d Prodan- labeled Acl-14 were mixed in equimolar amounts the blue- shift in the emission maximum, which indicates a more hydrophobic environment of the Prodan group, and the increase

Wavelength (nm)

FIG. 5. Fluorescence emission spectra of Prodan-labeled Acl-14 in the presence or absence of different p l l derivatives. The spectra of fluorescent Acl-14 (3 mM) were recorded in the absence or presence of equimolar amounts of WTpll, F86W, or F86A, respectively. The excitation wavelength was set at 380 nm. In each case the spectrum of free Prodan Acl-14 (with the emission maximum at 530 nm) is compared to that of the corresponding fluorescent Acl-14-pll complex. Note the characteristic blue-shift of the emission maximum and the 5-fold increase in fluorescence intensity which is observed upon binding of WTpll. The same changes in the fluorescence signal are induced upon binding of the F86W mutant, whereas addition of the F86A protein leads to very small effects.

.,, .............. .. ..,.......... .’ ’’

w ...........

I 1 4 I I

05 1 .o 1.5 2.0 25 Amount of protein (nmol)

FIG. 6. Complex formation of fluorescent Acl-14 with different p l 1 mutant proteins. 1 nmol of Prodan-labeled Acl-14 was incubated with increasing amounts of the different p l l derivatives (see inset) and subjected to fluorescence emission spectroscopy (excitation at 380 nm). Complex formation is accompanied by a characteristic increase in emission intensity. This is plotted against the amount of p l l derivative required to establish the increase. Fluores- cent intensities were normalized with respect to WTpll, i.e. the intensity of the fluorescence emission from the fully established complex between Prodan Acl-14 and WTpll arbitrarily received a value of 1.0.

in fluorescence intensity were indistinguishable from the effects obtained with authentic p l l purified from mammalian cells (Johnsson et al., 1988). Similarly, the F86W mutant induced the characteristic blue-shift and intensityincrease in fluorescence emission at an equimolar protein to peptide ratio (Fig. 5). The F86A protein, however, showed a marked differ- ence. Addition of an equimolar amount of F86A to Prodan- labeled Acl-14 provided only a slight blue-shift and a small intensity increase. Thus, annexin I1 binding by F86A is strongly impaired.

Several other purified p l l mutants were also analyzed in the spectroscopic assay. The results summarized in Fig. 6 generally confirm the data obtained in the Acl-14 matrix experiments. Mutants M55A, F86W, and G94Stop showed the same affinity for annexin I1 as the wild-type protein, i.e. the characteristic changes in the fluorescence emission spectrum of Prodan-labeled Acl-14 were induced by the addition of equimolar amounts of p l l derivative. Intermediate values, i.e. a saturable blue-shift and an intensity increase which does not reach the level of wild-type p l l were obtained in the case of K91Stop and Y85W. The most severe defects were again observed with Y85A and F86A. In both cases, even a sever- alfold excess of p l l mu tan t over Acl-14 did not saturate the fluorescence changes and resulted only in a relatively small increase in fluorescence intensity.

p l l -Annexin 11 Interaction 14181

The combined results stress the importance of hydrophobic amino acids in positions 85 and 86 for annexin I1 binding. Since the introduction of a tryptophan in these positions led to no or only a marginal reduction in affinity toward annexin I1 we studied the spectroscopic properties of these tryptophan residues in the presence and absence of Acl-14 peptide. Both proteins (Y85W and F86W) lack an additional tryptophan. Thus, the tryptophan introduced at either position 85 or 86 can be excited specifically at 295 nm (Fig. 7). Since tyrosine absorption was excluded under these conditions wild-type p l l yielded no fluorescence emission signal (Fig. 7, spectra e and f ) . Y85W and F86W, on the other hand, exhibited a typical tryptophan emission with the maxima residing at approximately 333 nm. In both mutant proteins the intensity of fluorescence emission was reduced about 20% upon addition of the peptide Acl-14 (Fig. 7). Interestingly, Acl-14 binding was also accompanied by shift of the position of the emission maxima from 333 to 328 nm. This shift indicates that the unique tryptophan in Y85W and F86W resides in a more hydrophobic environment after Acl-14 binding. Thus, the hydrophobic side chains at these positions (Tyr-85 and Phe- 86 in wild-type p l l ) are most likely in close contact to the hydrophobic side of the Acl-14 helix.

DISCUSSION

p l l binding modulates not only the physical state (mon- omer uersus heterotetramer) but also the biochemical properties displayed by annexin I1 (see Introduction). Hydropho- bicity is thought to be the major stabilizing factor of the annexin 11-pll complex since the pll-binding site on annexin I1 is restricted to a short amphiphilic a-helix of 10-14 amino acids where the hydrophobic face represents the major contact for p l l (Johnsson et al., 1988; Becker et al., 1990). In addition, other members of the superfamily of EF-hand proteins such as calmodulin and troponin C also interact via hydrophobic contacts with amphiphilic helices on their target proteins (Strynadka and James, 1990). We have now shown that certain hydrophobic side chains in p l l are indeed indispensable for high affinity annexin I1 binding.

Using site-directed mutagenesis and synthesis of recombinant proteins in E. coli, we have generated a collection of 17 p l l mutants and compared their affinities for annexin 11. In addition to Cys-82 previously identified by chemical modification experiments as a residue residing in close proximity to the annexin I1 contact site (Johnsson and Weber, 1990a), hydrophobic side chains of the C-terminal extension (residues

I 3 0 0 3 2 0 3 4 0 3 8 0 3 8 0 4 0 0 4 2

m b " * ("In)

FIG. 7. Fluorescence emission spectra of WTpll and tryptophan-containing mutant proteins in the presence and absence of the Acl-14 peptide. Emission spectra of WTpll ( e and f) and the mutant proteins Y85W ( b and d ) and F86W (a and c ) were recorded in the presence (c , d, and e ) or in the absence (a, b, and f ) of the annexin I1 peptide Acl-14. The excitation wavelength was set at 295 nm with excitation and emission band width of 4 nm. Note the absence of any signal from WTpll, which lacks a tryptophan, and the blue-shift in the emission maximum of Y85W and F86W which occurs upon Acl-14 binding.

78-96) were found to be important for high affinity annexin I1 binding. C terminally truncated p l l molecules revealed that amino acids 85-91 are required for binding. This portion of the molecule contains five hydrophobic amino acids (Tyr- 85, Phe-86, Val-87, Val-88, and Met-90) with side chains which represent possible contact points for the hydrophobic face of the amphiphilic annexin I1 helix. A comparison of p l l sequences from different vertebrate species reveals that Tyr- 85, Phe-86, Val-87, and Met-90 are highly conserved, whereas Val-88 is replaced by a lysine in Xenopus lueuis p l l (Kube et al., 1991). Since X. lueuis contains annexin I1 with a structur- ally and functionally conserved pll-binding site (Gerke et al., 1991) it seems that position 88 of p l l is not involved in a hydrophobic interaction with annexin 11. However, Tyr-85, Phe-86, and to a lesser extent Met-90 most likely represent some of the hydrophobic contact points predicted previously. Introduction of alanine in place of the large hydrophobic residues in these positions leads to a significantly reduced affinity toward annexin 11. The effect, which is most pro- nounced in the case of Tyr-85 and Phe-86, can be attributed to the hydrophobic nature of the side chain since the introduction of tryptophan, an amino acid with a similar hydrophobic character, results in no or only a marginal reduction in affinity. Thus, our mutational analysis corroborates the hypothesis that the annexin 11-p11 complex is stabilized by hydrophobic interactions and identifies two aromatic amino acids of p l l whose side chains contribute to this interaction.

The most severe defect in binding is observed with a p l l mutant in which Cys-82 is replaced by glutamine. This finding is line with the fact that alkylation of Cys-82 also results in a loss of annexin I1 binding. Since Cys-82 is not involved in disulfide bond formation (the pll-annexin I1 complex is formed even more readily under reducing conditions, Johns- son and Weber, 199Ob) the introduction of a bulkier side chain (in the C82Q mutant or the alkylated derivative) could create spatial constraints and thereby block a direct contact between hydrophobic residues in annexin I1 and p l l . Alter- natively, the Cys-82 modification might cause a conforma- tional change in the p l l molecule which inhibits annexin I1 binding. A simple spatial interference seems unlikely since the replacement of Cys-82 by serine, i.e. the introduction of a side chain with very similar dimensions, also leads to a significantly reduced affinity for annexin I1 (in the C82S mutant) although the effect is much less severe than in the C82Q mutant. Thus, a sulfhydryl group in position 82 may be essential for high affinity binding probably due to a specific electronic environment which differs from that of a hydroxyl group in the serine side chain.

Despite the identification of several hydrophobic amino acids which probably serve as direct contact points the precise architecture of the annexin 11-binding site on p l l is not known. In analogy with calmodulin and troponin C, other EF- hand proteins which interact with amphiphilic helices (Stry- nadka and James, 1990), the hydrophobic side of the annexin 11-helix might fit into a hydrophobic pocket on p l l . The aromatic side chains of residues Tyr-85 and Phe-86 probably reside on the surface of this putative pocket. This hypothesis is supported by the spectroscopic analysis of the Y85W and F86W mutant proteins. In both cases, the fluorescence emission from the unique tryptophan in position 85 or 86, respectively, is altered on Acl-14 binding. Since the emission maximum is shifted to shorter wavelengths a more hydrophobic environment of the respective tryptophan in the Acl-14 bound form is indicated. Structural studies, e.g. x-ray crystal- lography or nuclear magnetic resonance spectroscopy, may reveal whether a hydrophobic surface containing the side

14182 p l l -Annexin 11 Interaction

chains of Tyr-85 and Phe-86 is already present on the folded pl l molecule or becomes exposed only on formation of the annexin 11-pll complex, i.e. on contact with the amphiphilic Acl-14 helix.

Putative cellular protein ligands for S-100 proteins other than p l l have only rarely been indicated. The S-100 dimer seems to bind to microtubule-associated proteins and other cytoskeleton associated proteins, e.g. caldesmon, in a Ca2+- dependent manner (Baudier et al., 1989; Fujii et al., 1990). In addition, an interaction of S-100 with the glycolytic enzyme aldolase has been reported (Zimmer and Van Eldik, 1986) while calcyclin binds to glyceraldehyde-3-phosphate dehydro- genase under special conditions (Filipek et al., 1991). The structural principles which underly these interactions have not been determined. However, due to the sequence homolo- gies between S-100, calcyclin, and pll , which predict a similar three-dimensional folding, the mode of interaction might re- semble the annexin 11-pll complex formation. This hypothesis is particularly tempting in the case of S-100 since both, S-1OOa and S-lOOP (but not calcyclin), contain aromatic amino acids in positions of the C-terminal extension that correspond in the linear sequences to Tyr-85 and Phe-86 in pl l (for sequence comparison see Gerke, 1991). Thus, these residues might participate in hydrophobic interactions with yet to be identified amphiphilic helices in the putative target proteins. In addition to p l l and S-100, 18A2 and S-1OOL are the only members of the S-100 family which contain 2 consecutive aromatic residues in corresponding positions (see sequence comparison in Gerke, 1991). However, in each of these proteins (even in S-1OOa and S-loop) the immediate surrounding of the two aromatic amino acids is less hydrophobic than in p l l . Thus, future experiments have to show whether the mode of protein-protein interaction in the annexin 11-pll complex serves as a blueprint for yet to be characterized interactions between other S-100 proteins and cellular targets.

Acknourledgrnents-We thank Dr. N. Johnsson for materials and

helpful discussions and Dr. M. Osborn for the H21 antibody and for critical reading of the manuscript.

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Protein-Protein Interaction Studied by Site-directed Mutagenesis