Hydrophobic residues in the C-terminal region of S100A1 are essential for target protein binding butnot for dimerization

Cell Calcium (1998) 24(2). 137-l 51 0 Harcouri Brace 8 Co. Ltd 1998

Research

Hydrophobic residues in the C-terminal region of SlOOAl are essential for target protein binding but not for dimerization Dirk Osterlohl, Vasily V. Ivanenkov*, Volker Gerkel ‘Institute for Medical Biochemistry, ZMBE, University of Muenster, Muenster, Germany 2Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati,

Cincinnati,

Ohio, USA

Summary SlOO proteins are a family of small dimeric proteins characterized by two EF hand type Caz+ binding motifs which are flanked by unique N- and C-terminal regions. Although shown unequivocally in only a few cases SlOO proteins are thought to function by binding to, and thereby regulating, cellular target proteins in a Ca2+ dependent manner. To describe for one member of the family, SlOOAl , structural requirements underlying target protein binding, we generated specifically mutated SlOOAl derivatives and characterized their interaction with the a subunit of the actin capping protein CapZ shown here to represent a direct binding partner for Sl OOAl. Chemical cross-linking, ligand blotting and fluorescence emission spectroscopy reveal that removal of, or mutations within, the sequence encompassing residues 88-90 in the unique C-terminal region of SlOOAl interfere with binding to CapZa and to TRTK12, a synthetic CapZa peptide. The Sl OOAl sequence identified contains a cluster of three hydrophobic residues (Phe88, Phe-89 and Trp-90) at least one of which - as revealed by qualitative phenyl Sepharose binding and hydrophobic fluorescent probe spectroscopy - is exposed on the protein surface of Ca 2+ bound SlOOAl. As homologous hydrophobic residues in the closely related SlOOB protein were shown by NMR spectroscopy of Ca2+-free SlOOB dimers to provide intersubunit contacts [Kilby P.M., van Eldik L.J., Roberts G.C.K. The solution structure of the bovine SlOOB dimer in the calcium-free state. Structure 1996; 4: 1041-1052; Drohat A.C., Amburgey J.C., Abildgaard F., Starich MR., Baldisseri D., Weber D.J. Solution structure of rat apo-SlOOB (beta beta) as determined by NMR spectroscopy. Biochemistry 1996; 35: 11577-115881, we characterized the physical state of the various Sl OOAl derivatives. Analytical gel filtration and chemical cross-linking show that dimer formation is not compromised in Sl OOAl mutants lacking residues 88-90 or containing specific amino acid substitutions in this sequence. Thus a cluster of hydrophobic residues in the C-terminal region of SiOOAl is essential for target protein binding but dispensable for dimerization, a situation possibly met in other Sl 00 proteins as well.

Ca2+plays a pivotal role as a second messenger involved in the regulation of a variety of cellular processes ranging Received 11 May 1998 Revised 21 July 1998 Accepted 22 July 1998 Correspondence to: Volker Gerke, Institute for Medical University of Muenster, D-48149 Muenster, Germany Tel: +49 251 83 56722; Fax: +49 251 83 56748 E-mail: [email protected]

Biochemistry,

from muscle contraction to cell differentiation and cell death. Ca*+ signalling and intracellular Ca2+ levels are controlled by a number of energy-dependent Ca*+ transporters and Ca*+ channels located in the plasma membrane and in the membranes of intracellular organelles with high luminal Ca2+ concentrations. Within

ZMBE, Abbreviations

used:

BP, bis(sulfosuccinimidyI)suberate;

DTT, dithiothreitol;

EDC; 1 -ethyl-3-(3dimethylaminopropyl)-carbodiimide; NHS, Nhydroxysulfosuccinimide; TNS, 6-(ptoluidino)naphthalene-2-sulfonic

acid.

137

138

D Osterloh, V V Ivanenkov, V Gerke

the cytoplasm, transient increases in Ca2+ levels are mediated through a number of Ca2+binding proteins (for review see [ 11). One class of these Ca2+mediator proteins is characterized by a Ca2+ binding motif of the so-called EF-hand type, which consists of two c+helices (named after the E- and F-helix of parvalbumin; [Z]) and an interhelical loop providing carbonyl and carboxyl oxygen ligands for Ca2+coordination (for review see [3]). EF hand motifs have been identified in a large number of proteins making the EF hand superfamily the largest group of Ca2+ binding proteins known to date (for review see [4]). While some EF hand proteins have been implicated in intracellular Ca2+homeostasis, it is thought that most of these proteins transmit Ca2+ signals by binding to, and thereby regulating, specific target proteins when activated in their Ca2+ bound conformation. This has been shown unequivocally for EF hand proteins such as calmodulin, myosin light chain and troponin C (for review see [ 11). Within the EF hand superfamily, SIOO proteins form the largest subfamily with 17 members known to date (for review see [5,6]). S 100 proteins are small (lo- 12 kDa in molecular mass) dimeric polypeptides characterized by two EF hand motifs connected through a central hinge region and flanked by two relatively hydrophobic stretches (also called extensions) at the N- and Cterminal end, respectively. The N- and C-terminal EF hands differ in architecture with the loop of the latter showing the canonical EF hand length of 12 amino acid residues and the loop of the former being of unusual length (14 residues) and thus SlOO specific (for review see [7]). As a result, the N-terminal EF hand only binds to Ca2+ with low affinity (& approximately 200-500 I.1M), whereas the C-terminal EF hand is capable of high affinity Ca2+binding (Kr, approximately 1O-50 l&I). A number of targets thought to be regulated by SIOO proteins in their Ca2+ bound conformation have been identified by employing biochemical approaches. These include several members of the amrexin family of Ca2+/lipid binding proteins as well as glycolytic enzymes and cytoskeletal proteins such as vimentin, glial fibrillary acidic protein and tubulin (for review see [6]). However, in several cases the intracellular and/or tissue distribution of the target protein does not correlate with that of the respective SlOO protein casting doubts on the purely biochemical approaches. An example where the criterion of overlapping intracellular localization is met is that of the SlOOAlKapZ pair. CapZ is a heterodimeric actin capping protein which, in striated muscle, is present at the Z-lines of the sarcomers [8,9] thus showing a distribution comparable to that of SlOOAl [lo]. An interaction between SlOOAl and CapZ has been described recently and a synthetic peptide (TRTK-12) corresponding in sequence to a C-terminal region in the Cell Calcium

(1998)

24(2),

137-151

CapZa subunit has been shown to bind Ca2+dependently to SlOOAl and SlOOB, the closest homologue to SlOOAl within the SlOO family [l l-141. It has been shown for several SlOO proteins that Ca*+ binding induces conformational changes resulting in an increased hydrophobic nature of the protein (for review see [7]). Analysis of cysteine residues located in the unique C-terminal regions of a number of SlOO proteins, e.g. SlOOAl, SlOOB and SlOOP, revealed that these residues are exposed on the surface of the molecules upon Ca2+ binding [15-l 71. Thus, it was generally assumed that Ca2+ binding to SlOO proteins leads to an increased exposure of the C- and N-terminal regions flanking the two EF-hands. Direct evidence for such conformational changes, which also involve residues of the central hinge region, has been obtained recently by NMR and crystal structure analyses of S 1OOB[ 18-201. As a consequence, hydrophobic side chains present in these regions would be available for an interaction with target proteins. Site-directed mutagenesis approaches confirming this suggestion have been performed for S 1OOA6 and SlOOC and their interaction with the target proteins annexin XI and amrexin I, respectively [2 1,221. Moreover, S 1OOA10, the only S100 protein incapable of binding Ca2+ due to mutations in the EF hand loops was shown to bind to its target, annexin II, through hydrophobic protein-protein interactions and hydrophobic residues in the C-terminal region of the SIOO protein were implicated in mediating complex formation [23]. However, this view was challenged recently when the three-dimensional structures of Ca2+-free SlOOA6 and SlOOB were elucidated by NMR spectroscopy [24-261. Here, hydrophobic residues equivalent in a sequence alignment to those identified in SlOOAlO as being important for annexin II binding are involved in SIOO dimer formation. Therefore, it has been suggested that mutations in these positions lead to perturbations of the monomer-dimer equilibrium which, in turn, may contribute to a reduced amrexin II binding [24]. Here, we chose a mutagenesis approach to address this apparent controversy and selected SlOOAl, a close homologue of the structurally well characterized SlOOB, and its interaction with CapZ. We show by a gel overlay approach that SlOOAl binds directly and specifically to the cr-subunit of the heterodimeric CapZ. Analysis of the interaction of Sl OOAl mutants with CapZa and the CapZa peptide TRTK-12 reveals that the sequence encompassing residues 88-90 (FFW) in the C-terminal region of SlOOAl is of crucial importance for target protein binding but not for dimerization. Thus, our data show for the first time that hydrophobic residues most likely participating in dimer formation in Ca2+-free SIOO are involved in target protein binding when the SlOO protein is activated by Ca2+binding. 0 Harcourt

Brace

& Co. Ltd

1998

139

S 1OOA 1-CapZ interaction

MATERIALS

AND

METHODS

Generation of Sl OOAl mutants

Mutations were introduced by PCR using as template human SlOOAl cDNA cloned into the pGEMEX-2 expression vector (kindly provided by Drs B. Schafer and C. Heizmann, University of Zurich, Switzerland). The oligonucleotides employed as PCR primers installed a N&I restriction enzyme site 5’ to the ATG start codon (sense primer) and different stop codons and an adjacent H&&I restriction enzyme site at the 3’ end of the cDNA (antisense primers). The sequences of the different PCR primers introducing the mutations indicated were as follows: Sense primer: [5’-ggagatatacatatgggctctgagctg-3’1 Antisense primers: (E91Stop) [5’-tcaactgttcaagcttactcaccagaagaaatt-3’1 (N87Stop) [5’-ccagaagaaaaagcttgatcagttacaggccac-3’1 (V83Stop) [5’-ccagaagaaattaagcttggctcatgtgagagcagc-3’1 (W9OI) [5’-gtggaaecttgctcaactgttctcaatgaagaaattgtt-37 (FFW88-9OAAI) [S-gtggaasxttgctcaactgttctcgattgctgcattgttacaggc-3’] PCR was carried out in a buffer containing 20 mM TrisHCl @H 8.4), 3 mM MgCl,, 50 mM KCl, 0.32 mM of each dNTP and 0.1 nmol of each PCR primer. After denaturation at 95°C for 5 min, 2.5 units Taq polymerase were added and 35 cycles of denaturation (95”C, 1 min), annealing (45”C, 1 min) and elongation (72”C, 1 min) were performed. The PCR products were gel-purified, digested with A&de1and Hi&III and cloned into the appropriately linearized pGEMEX-2 expression vector. Subsequently, all mutant cDNAs were characterized by DNA sequencing [27]. Bacterial expression of wild-type and mutant SlOOAl derivatives

Eschetichiu coli BL 21 (DE 3) pLysS cells (Novagen) expressing wild-type @VT) or the different mutant SlOOAl proteins were grown in 100 ml LB medium containing 0.2% glucose, 0.5 mM IPTG and 0.15 mg/ml ampicillin. After incubation at 37°C for 4 h, bacterial cells were harvested and the expression of SlOOAl was verified by analyzing an aliquot of the total bacterial lysate by SDS-PAGEusing 16% (w/v) polyacrylamide gels and the tricine buffer system [28]. For protein purification, the bacterial pellet was resuspended in 2.5 ml lysis buffer containing 20 mM Tris-HCl, pH 74, 300 mM NaCl, 10% glycerol, 1 mM EDTA, 2 mM D’IT, 1 mM PMSF and 5 kg/ml leupeptin. After sonification using a 0 Harcouti

Brace

& Co. Ltd 1998

Branson soniher (setting 3, 4x30 s), the suspension was clarified by centrifugation at 100 000 g for 1 h. The supematants containing WT SlOOAl or the different SlOOAl mutant derivatives were processed as described below for analytical phenyl Sepharose binding or large scale purification of S 1OOA1. Analytical binding of wild-type and mutant SlOOAl derivatives to phenyl Sepharose

120 ~tl of 50% phenyl Sepharose suspensions in buffer A (25 mM Tris-HCl, pH 75, 150 mM NaCl, 1 mM DTT) containing 5 mM CaCI, were placed into 1.5 ml tubes. Clarified bacterial extract containing 400 pg recombinant WI or mutant SlOOAl protein were adjusted to 300 ~1 with lysis buffer containing 6 mM CaCI, and added to the phenyl Sepharose suspension. The tubes were rotated end over end for 1 h at 4°C. After pelleting of the phenyl Sepharose beads by centrifugation at 50 gfor 3 min, the supematants were removed and saved and the beads were washed three times with 1 ml of buffer A containing 1 mM CaCl,. The final bead pellet was washed a fourth time with 140 ~1 of buffer A containing 1 mM CaCl, for 3 min. The phenyl Sepharose beads were pelleted again at 50 gfor 3 min and the supematants were collected and saved. To extract proteins bound Ca2+-dependently to the matrix, the beads were suspended in 140 ld buffer A containing 5 mM EGTA. Subsequently, the beads were pelleted and the EGTA supematants were collected and saved. Equivalent amounts of the different phenyl Sepharose supematants (unbound material, final Ca2+ wash, EGTA released proteins) were subjected to SDSPAGE in 16% (w/v) polyacrylamide gels using the tricine buffer systeme [28]. Purification of wild-type SlOOAl and the SlOOAl mutants E91 Stop and W9Ol

The clarified bacterial extracts containing the respective SlOOAl derivatives were adjusted to 6 mM CaCl, and applied to 1.5 ml phenyl Sepharose columns equilibrated in buffer A containing 0.25 mM PMSF and 1 mM CaCl,. The columns were washed with 200 ml buffer A containing 0.25 mM PMSF and 1 mM CaCl, and the bound proteins were eluted with buffer A containing 1 mM EGTA. As judged by SDS-PAGEthis protocol yielded SlOO proteins of a purity of more than 95%. Purification of the less hydrophobic SlOOAl mutants N87Stop, V83Stop and FFW88-90AAI

As the analytical phenyl Sepharose binding experiments indicated a less hydrophobic nature of the SlOOAl mutants N87Stop, V83Stop and FFW88-90AAI a different Cell Calcium

(1998)

24(2),

137-151

140

D Osterloh, V V Ivanenkov, V Gerke

approach was applied to purify these protein mutants. 5 ml of clarified extract from 250 ml bacterial cultures expressing the respective mutant were diluted 6 times with buffer C (20 mM Tris-HCl, pH 75, 1 mM EGTA, 1 mM DTI) and applied to a 1.5 ml DE-52 column equilibrated in the same buffer containing 50 mM NaCl. The column was then washed with 10 ml buffer C containing 50 mM NaCl and bound proteins were eluted in 60 ml linear gradients of 50 mM to 400 mM NaCl in buffer C. Fractions were analyzed by SDS-PAGEusing the tricine buffer system and those containing the recombinant SlOOAl protein were pooled, adjusted to 4 mM CaCI, and applied on a phenyl Sepharose column (inner diameter 1 cm, height of the packed matrix 12.5 cm) equilibrated in buffer A containing 1 mM CaCl,. After application, the column was washed with 40 ml buffer A containing 1 mM CaCI, and fractions were collected. Although the three SlOOAl mutants did not bind firmly to the column they were retarded in their migration through the phenyl Sepharose matrix and, thus, were separated from the bulk of the other bacterial proteins chromatographed on the phenyl Sepharose column in the presence of 1 mM CaCl,. Fractions containing the Sl OOAl mutants were identified by SDS PAGE and immunoblotting using an anti-SlOOAl antibody raised in rabbits (kindly provided by Drs B. Schafer and C. Heizmann). They were pooled and rechromatographed on the phenyl Sepharose column in buffer A containing 1 mM CaCI,. For concentration, fractions containing the pure SlOO derivatives were pooled, diluted with the same volume of buffer C and applied to a 1.5 ml DE-52 column in buffer C. Proteins were eluted by adding 3 x 1 ml buffer C containing 400 mM NaCl. Biotinylation derivatives

of wild-type and mutant SlOOAl

WT or mutant SlOOAl protein was dialyzed against sodium-carbonate buffer, pH 8.5. For biotinylation, 6 pg of SlOOAl in 24 ~1 of the same buffer were incubated with 4.2 pg/ml S-NHS-biotin (N-hydroxysulfosuccinimide, Pierce) for 1 h at 25°C. For the analysis of biotin incorporation, an aliquot of the biotinylation reaction was subjected to SDS-PAGE using the tricine buffer system [28] and then blotted onto PVDF membrane. After blocking in TBST (20 mM Tris-HCl, pH 75,150 mM NaCl, 0.2% Tween 20) containing 3% bovine serum albumin, the membrane was incubated in the same buffer containing 2 pg/ml avidin-HRP (Pierce) for 45 min at 25°C. Following extensive washing in TBST, biotinylated SlOOAl was detected by chemoluminiscence using the ECL-kit (Amersham) and Kodak X-Omat film. Cell Calcium

(1998)

24(Z),

137-

15 1

Gel overlay analysis of the SlOOAl-Cap2 interaction

CapZcx or CapZB cDNAs from chicken muscle [29,30] were cloned into the pQE 60 expression vector (Qiagen) and the resulting plasmids were used to transform E. coli cells. Bacterial extracts containing the recombinantly expressed CapZ subunits were separated in SDS 12% polyacrylamide gels. The gels were fixed, renaturated and blocked as described [3 1,321. For detection of SlOOAlbinding proteins, the gels were then incubated for 12 h in gel overlay buffer (50 mM Tris-HCl, pH 25, 200 mM NaCl) containing 0.25 &ml equivalently biotinylated WT or mutant SlOOAl and 0.5 mM CaCl, or 0.5 mM EGTA. After extensive washing of the gels in gel overlay buffer containing either 0.1 mM CaCl, or 0.5 mM EGTA, biotinylated S lOOA specifically bound to gelimmobilized proteins was blotted onto PVDF membrane (Millipore) and detected by the avidin-HRP reaction described above. Chemical crosslinking the TRTK-12 peptide

of different SlOO proteins and

WT and mutant Sl OOAl proteins were dialysed against buffer B (50 mM HEPES, pH 7.5, 150 mM NaCl). Crosslinking reactions were carried out in 30 ~1 of the same buffer using 5 l.tg of SlOOprotein and a 5-fold molar excess of the TRTK12 peptide (sequence: TRTKIDWNKILS). Reactions were pre-incubated for 20 min at 25°C in the presence of 2 mM CaCl, or 2 mM EGTA, respectively Subsequently, the crosslinking was initiated by addition of 3 i.tg of the bifunctional crosslinking reagent BS3 [bis(sulfosuccinimidyl)suberate, Pierce] in buffer B. After 30 min incubation at 25°C the reactions were quenched by the addition of 3 ~1 of 200 mM 2-aminoethanol-HCl in buffer B and a subsequent incubation for 15 min at 25°C. 116 volume of 6x concentrated SDS sample buffer [33] was added and the samples were subjected to SDS-PAGE in 16% (w/v) polyacrylamide gels using the tricine buffer systeme. Dimer formation as revealed by chemical crosslinking

WT SlOOAl and the FFW88-90AAI mutant protein were dialyzed against crosslinking buffer (200 mM MES, pH 5.4, 300 mM NaCl and 1 mM DTI) and crosslinking reactions were then carried out in the presence of 0.5 mM Ca2+in 30 ~1of crosslinking buffer with 10 pg protein and 0.5 and 1.O pg of EDC [ 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, Serva) and SulfoNHS (N-hydroxysulfosuccinimide, Pierce), respectively. Reactions were incubated for 1 h at 25°C and terminated by the addition of l/6 volume of 6x concentrated SDS sample buffer (Laemmli) containing 3.0 mM 2-mercaptoethanol. Products were subjected to 0 Harcourt

Brace

& Co. Ltd 1998

S 1OOA1-CapZ interaction

SDS-PAGE in 16% (w/v) polyacrylamide tricine buffer systeme.

gels using the

Fluorescence emission spectroscopy

Fluorescence emission spectra were recorded on a Hitachi F-2000 or a FIuoromax (Spex) fluorescence spectrophotometer using 0.5 or 1 ml quartz cuvettes, respectively. In experiments with the hydrophobic fluorescent probe 6@toluidino)naphthaIene-2-suIfonic acid (INS), the sample was excited at 365 nm and spectra were recorded between 380 and 600 nm. Wild-type and mutant SlOOAl derivatives were dialyzed against 100 mM Tris-HCI, pH 8.3, 0.5 mM EDTA and 8 mM of the respective SlOOAl protein were mixed with 20 )LM TNS in 100 mM Tris-HCl, pH 8.3, 0.5 mM EDTA at 25°C. After recording of the emission spectrum, the Ca*+ concentration in the cuvettes was increased stepwise by the addition of CaCI, from a stock solution (0.5 M CaCl, in 100 mM Tris-HCI, pH 8.3) to a final concentration of 0.25,0.5,0.75 and 1 mM resulting in effective free Ca2+ concentrations of less than 1O-2, l-10, 250 and 500 pM, respectively (calculated by the WinMaxc ~1.7 program). Following adjustment of each Ca*+ concentration, the corresponding emission spectrum was recorded. Detailed Ca2+ titration experiments of the TNS interaction with WT and W901 SlOOAl, respectively, were carried out using 4 @vl of the respective SlOOAl protein and 20 @vl TNS in 100 mM Tris-HCl, pH 8.3, and 150 mM Na-citrate as Ca*+ chelator. The total Ca2+ concentration in the reaction mixture was increased stepwise by the addition of CaCl, from a stock solution to yield free Ca2+ concentrations of 0.75, 1.15, 1.92, 3.08,

141

chromatography system (Pharmacia). The hydrodynamic analysis was carried out with 5-25 pg of the respective protein. The column buffer was 25 mM Tris, pH 75, 150 mM NaCI, 3.5 mM 2-mercaptoethanol and 0.5 mM EGTA or 1 mM CaCI,. The column was operated at a flow rate of 40 $/min and protein was detected by absorbance of the peptide bond at 230 nm. RESULTS

SlOOAl binds directly and specifically to the CapZa polypeptide

A Ca2+ dependent interaction between SlOOAl and a synthetic peptide (TRTKIDWNKIIS, herein referred to as TRTK-12) corresponding to a sequence in the C-terminal region of the CapZcl subunit has been demonstrated recently by chemical cross-Iinking and NMR spectroscopy [ 12,141. Moreover, TRT’K12 binding has been shown to compete with a cross-linking of SlOOAl to the CapZu//p heterodimer indicating that within the CapZ complex the SlOOAl binding site is indeed located in the cl-subunit [ 121. To demonstrate this directly, we employed an in situ renaturation/gel overlay approach. Full-length CapZa and CapZp polypeptides were expressed recombinantly in

Coomassie

gel overlay

A kDa

5.04, 25, 10.2, 12.7, 15.0, 20.2, 24.7, 30, 45, 60, 75, 90, 120 and 248 pM (calculated by the WinMaxc ~1.7 program).

TNS emission spectra were recorded after the adjustment of each Ca2+concentration. The interaction of the tryptophan-less S 1OOAl mutants V83Stop, N87Stop, W901 and FFW88-90AAI with the TRTK-12 peptide was analyzed by recording the fluorescence emission of the sole tryptophan in the TRTK-12 peptide after excitation at 295 nm. The analysis was carried out in 100 mM Tris-HCI, pH 8.3, 0.5 mM EDTA using 8 @I TRTK- 12 peptide and an equimolar amount of the respective SlOOAl derivative. As described above for the TNS spectroscopy, the individual emission spectra were recorded in the presence of 0.5 mM EDTA and after adjusting the free Ca2+ concentration to less than 10-2, l-10 and 250 @l, respectively (calculated by the WinMaxc ~1.7 program). Gel filtration

Gel filtration was carried out on a Superose-12 column (PC 3.2/30, Pharmacia) connected to the SMART 0 Harcoutt

Brace

& Co. Ltd 1998

Fig. 1 Gel overlay analysis of the interaction of SlOOAl with fulllength CapZ subunits. Bacterial extracts containing recombinant chicken CapZo (lanes 1) or CapZj3 (lanes 2) were subjected to SDS-PAGE. Following fixation and renaturation of the separated polypeptides in situ, their interaction with biotinylated SlOOAl was analyzed as described in ‘Materials and methods’. (A) Shows a Coomassie Blue stain of the proteins. (B) (two separate experiments) and (C) show the result of the SlOOAl overlay carried out in the presence of 0.1 mM CaCI, or 0.5 mM EGTA, respectively.(D) Depicts the result of a control experiment in which the entire procedure was performed in the presence of 0.1 mM CaCI, but with omitting biotinylated SlOOAl in the gel overlay buffer. Note the specific and Ca*+ dependent binding of SlOOAl to CapZa which is not observed for CapZj3 or any other protein of the total bacterial extract.

Cell Calcium

(1998)

24(2),

137-151

142

D Osterloh, V V Ivanenkov, V Getke

1

10

20

30

40

50

60

70

80

90

93

GSELETAMETLINVFHAHSGKEGDKYKLSKKELKELLQTELSGFLDAQKDVS* GSELETAMETLINVFHAHSGKEGDKYKLSKKELKELLQTELSGFLDAQ~~A~~ELD~G~~FQE~V~L~AC~F~* GSELETAMETLINVFHAHSGKEGDKYKLSKKELKELLQTELSGFLDAQ~~A~K~KELD~G~E~FQE~LV~L~ACN' GSELETAMETLINVFHAHSGKEGDKYKLSKKELKELLQTELSGFLDAQ~~A~K~KELD~G~~FQE~V~LT~

WT SlOOAl E9 1STOP N87STOP V83STOP

Fig. 2 Amino acid sequences of different SlOOAl derivatives. Given are the amino acid sequences of wild-type (WT) human SlOOAl and the different Sl OOAl mutant proteins generated in this study. The C-terminal extension is progressively truncated in E91 Stop, N87Stop and V83Stop Sl OOAl , whereas W901 and FFW88-9OAAI SlOOAl contain single and triple amino acid substitutions, respectively. These are located within the highly hydrophobic sequence encompassing amino acids 88, 89 and 90.

bacteria and proteins present in a total extract from transformed bacteria were separated by SDS-PAGE.After fixation, polypeptides in the gel were subjected to a renaturation procedure and then probed with biotinylated S 1OOAl . Subsequently, bound S 1OOAl was blotted onto PVDF membrane and visualized by avidinHRP. This analysis reveals that S 1OOAl interacts with the 36 kDa CapZa polypeptide but not with CapZp or any other band of the total extract (Fig. 1). Moreover, this interaction is strictly Caz+ dependent (Fig. 1). Interestingly, no binding of biotinylated SlOOAl to CapZa is observed when the proteins of the bacterial extracts are first transferred to PVDF or nitrocellulose membrane and then subjected to the renaturation procedure (not shown). Thus immobilization of the SDSdenatured CapZa on the membrane hampers its renaturation at least as far as the SlOOAl binding site is concerned. The specific and direct binding of SlOOAl to the CapZa polypeptide indicates that the S 1OOAl binding to the CapZa peptide TRTK-12 mimics the interaction between the native full-length proteins. Structural requirements interaction

for the Sl OOAl-TRTK-12

Next we wanted to map the TRX-12 binding site within the SlOOAl molecule. Therefore, a series of SlOOAl mutant proteins were generated and analyzed with respect to their interaction with the CapZ peptide. We concentrated on sequences located in the unique Cterminal region flanking the second EF hand since this region was implicated previously in mediating the interaction between other SIOO proteins and their target proteins [22,23]. The mutagenesis either introduced premature stop codons resulting in S 100 derivatives with progressive truncations of the C-terminal extension (EglStop, N87Stop and V83Stop SlOOAl) or generated site-specific ammo acid replacements within a cluster of hydrophobic residues between positions 88 and 90 (FFW88-9OAAl and W901 SlOOAl). The amino acid sequences of the different S1OOAl mutants are given in Figure 2. Cell Calcium

(1998)

24(2),

137-151

B

A

20kDa _ 1OkDs -

123455 Fig. 3 Purification of recombinant SlOOAl (A) Recombinantly expressed SlOOAl was purified as outlined in ‘Materials and methods’, and fractions of the purification steps were analyzed by SDS-PAGE. Lane 2 shows a total protein extract from induced bacteria expressing WT SlOOAl whereas the equivalent extract from non-induced bacteria is shown in lane 1. High-speed centrifugation of the extract shown in lane 2 yields a soluble protein fraction containing SlOOAl (lane 3). This fraction was applied to a phenyl Sepharose column in the presence of Caz+. Unbound material and proteins which were removed from the column by extensive washing in the presence of Ca2+ are depicted in lanes 4 and 5, respectively. Bound material eluted by the addition of EGTA is shown in lane 6. Note that this single chromatographic step yields essentially pure protein. (B) SDS-PAGE analysis of purified WT SlOOAl and the mutant derivatives FFW88-9OAAI, W901, E91 Stop, N87Stop and V83Stop Sl OOAl . The mutant proteins were expressed recombinantly in E. coliand purified as shown for WT SlOOAl in Figure 3A, except for FFW88-9OAAI, N87Stop and V83Stop SlOOAl . These mutant proteins were purified by a combination of ion exchange chromatography on DE-52 and hydrophobic retardation chromatography on phenyl Sepharose (see Materials and methods). The SDS-PAGE analysis shows that both purification protocols yield proteins of comparable purity.

Wild-type and mutant SlOOAl derivatives were synthesized in a bacterial expression system and purified from the soluble protein fraction of the transformed bacteria by exploiting the ability of S 100 proteins to interact, Ca2+-dependently, with hydrophobic matrices such as phenyl Sepharose. As a representative example, Q Harcourt

Brace

& Co. Ltd 1998

SlOOAl-Capzinteraction

2 2 g

143

3000

5 g

2000

8 !i b 3 E

1000 I

320

340

360

360

400

wavelength

320

340

360

360

wavelength

420

440

I

320

(nm)

1

I

I

360

360

400

wavelength

(nm)

400

340

420

440

320

340

360

360

wavelength

I

,

420

440

(nm)

400

420

(nm)

440

320

340

360

360

wavelength

400

420

440

(nm)

Fig. 4: Sl OOAl -TRTK-12 interaction analyzed by fluorescence emission spectroscopy. Equimolar amounts of the TRTK-12 peptide, which contains a single tryptophan, and the tryptophan-less SlOOAl derivatives W901, FFW88-90AAI, N87Stop and V83Stop were mixed in the presence of 0.5 mM EDTA, and fluorescence emission of the peptide tryptophan was recorded after excitation at 295 nm (-). Subsequently CaCI, was added stepwise to final concentrations of 0.25 (-.-), 0.5 (--), 0.75 (- - -) and 1 mM (.-.,.).The individual emission spectra were recorded after each Ca2+ addition. Note the significant Ca*+ induced blueshift in the TRTK-12 tryptophan fluorescence in the presence of W9Ol but not in the presence of N87Stop, V83Stop and FFW88-90AAI SlOOAl

the purification of wild-type (WT) S 1OOAl is depicted in the SDS-PAGE analysis in Figure 3A. It reveals that a single affinity step on the phenyl Sepharose column yields essentially pure protein. The mutant proteins N87Stop, V83Stop and FFW88-90AAI SlOOAl proved to be less hydrophobic in nature in their Ca2+bound state as compared to WT, E91Stop and W901 SlOOAl (see also below). Therefore, these mutant derivatives were purified by a combination of ion exchange chromatography on DE-52 and hydrophobic retardation chromatography on phenyl Sepharose (see ‘Materials and methods’). As revealed by SDS-PAGE analysis of the purified SlOOAl derivatives, this protocol yields mutant proteins of comparable purity (Fig. 3B). The interaction of the different Sl OOAl mutants with the CapZ peptide was first analyzed by chemical crosslinking using the bifunctional reagent BS3. In these experiments, TRTK-12 is cross-linked to E9 1Stop and 0 Harcourt

Brace

& Co. Ltd 1998

W901 S lOOA with an efficiency comparable to that of the TRTK-12-WT SlOOAl crosslink (data not shown). Moreover, in all cases, the reaction is strictly dependent on the presence of Ca2+. On the other hand, N87Stop, V83Stop and FFW88-90AAI SlOOAl only show a very minor cross-link to the CapZ peptide (not shown). These data indicate that the region in SlOOAl encompassing residues 87-91 is required for establishing a firm interaction with TRTK-12 and that the two aromatic residues at positions 88 and 89 are of particular importance. To circumvent the use of a chemical cross-linking reagent which could stabilize rather weak and potentially non-meaningful interactions we next employed fluorescence spectroscopy to characterize the SlOOAl-TRTK12 interaction. Therefore, we made use of the W901 SlOOAl mutant protein in which the sole tryptophan was replaced by an isoleucine. This mutant was indistinguishable from Cell Calcium

(1998)

24(2),

137-151

144

D Osterbh, V V Ivanenkov, V Gerke

WT SlOOAl in TRTK-12 cross-linking (described above) and CapZol binding (see below). However, since W901 SlOOAl did not contain a tryptophan we could employ this mutant in characterizing the TRTK-12 binding spectroscopically by analyzing the fluorescence emission of the unique tryptophan in the TRTK-12 peptide. Tryptophan was specifically excited at 295 mn and the emission spectra of TRTK-12 alone or a TRTK-12Mr901 SlOOAl mixture were recorded in the absence or presence of Ca *+. Figure 4 shows that the tryptophan in the free TRTK-12 peptide has an emission maximum at 350 nm which is not altered significantly by the addition of Caz+.Likewise, the position of the emission maximum is not changed when TRTK-12 is mixed with equimolar amounts of W901 SlOOAl in the absence of Ca*+ corroborating that the W901 mutant protein does not contribute to the fluorescence emission when the excitation wavelength is set at 295 nm. However, when the free Caz+concentration in the TRTK- 12/W901 S 1OOA1 mixture is raised to the low micromolar range, the fluorescence emission maximum is shifted to 335 mn. No additional changes are observed when the Ca2+ concentration is further increased to 250 j.th4 (Fig. 4) or higher values (not shown). The blue shift most likely results from a less aequeous environment of the TRTKI 2 tryptophan in the mixture containing TRTK12 and Caz+ bound W901 SlOOAl and indicates that the peptide-protein interaction is, at least in part, mediated through hydrophobic contacts. As fluorescence spectroscopy proved a valuable tool for analyzing the SlOOAl-TRTK12 interaction, we subjected all tryptophan-less S1OOAI mutants generated in this study to the same analysis. Figure 4 reveals that N87Stop and V83Stop SlOOAl fail to induce, and FFW889OAAI SlOOAl only induces a very minor, blue shift in the wavelength of the emission maximum of the TRTK12 ttyptophan upon addition of Ca2+. This is in sharp contrast to the effect observed with W901 SlOOAl . Albeit allowing only a qualitative description the fluorescence analyses indicate that residues in the C-terminal region of Ca2+-bound S 1OOAl, including Phe-88 and Phe-89, are in close proximity to the TRTK-12 tryptophan in the S 1OOA1-TRTK complex. SlOOAl mutations interfering with TRTK-12 binding also impair the interaction with CapZa

To verify that the characteristics of the SlOOAl/TRT&12 interaction can be extrapolated to the binding of SlOOAl to its target CapZa, we performed gel overlay analyses with different SlOOAl mutant proteins. Extracts from bacteria recombinantly expressing CapZcc were subjected to SDS-PAGE and in situ renaturation and then probed with biotinylated WT, W901, FFW88-90AAI or N87Stop Cell Calcium

(1998)

24(2),

137-151

WT

..---Caz+ + -

FFW68-SOAAI

W901

+

N%?Stop

+

+

5 6

7

kDa 66CapZa&=

31 -

s*c 12

a

3

4

Fig. 5 CapZa binding to mutant Sl OOAl proteins as revealed by gel overlay analysis. Bacterial extracts containing recombinant chicken CapZa were subjected to SDS-PAGE and subsequent in situ renaturation. Binding of equivalently biotinylated WT (lanes 1 and 2), W9Ol (lanes 3 and 4) FFW88-9OAAI (lanes 5 and 6) and N87Stop SlOOAl (lanes 7 and 8) was then analyzed as described in Materials and methods. The overlays were carried out in the presence of 0.1 mM CaCI, (lanes 1, 3, 5, and 7) or 0.5 mM EGTA (lanes 2, 4, 6, and 8) respectively.

SlOOAl, respectively. Figure 5 reveals that biotinylated WT and W901 SlOOAl bind specifically to the CapZa polypeptide of 36 kDa and that this binding requires the presence of Ca2+.In contrast, FFW88-90AAI and N87Stop SlOOAl fail to interact with CapZo. These data are in agreement with the TRTK-12 results and show that the C-terminaI residues of SlOOAl which appear to participate in TRTK-12 binding are also involved in the interaction with full-length CapZa. Hydrophobic side chains in the C-terminal region of SlOOAl are exposed upon CaZ+binding

To corroborate the view that the hydrophobic residues Phe-88 and Phe-89 of Ca2+ bound SlOOAl are in close contact to the TRTK-12 tryptophan and thus involved in TRTK12 binding, we analyzed the surface exposure of these residues in Ca2+ free and Ca2+ bound S1OOAl . The rationale behind these experiments was the assumption that surface exposure of hydrophobic contact points would be a prerequisite for target protein and, thus, TRTK-12 binding. In a first series of experiments, we characterized the ability of different SlOOAl mutants to interact with a hydrophobic matrix. Total lysates from transformed bacteria expressing either WT or different mutant SlOOAl derivatives were mixed with phenyl Sepharose beads in the presence of Ca2+.After washing in a Ca2+-containing buffer, Ca2+-dependently bound proteins were eluted by chelating Ca2+with EGTA. In this comparative analysis, WT SlOOAl and the E91Stop and W901 mutant proteins bind quantitatively and Ca2+dependently to the hydrophobic matrix. In contrast, only a small portion of N87Stop SlOOAl and FFW88-90AAI SlOOAl interacts with the matrix and is recovered by 8 Harcoutt

Brace

& Co. Ltd

1998

S 1OOA1-CapZ interaction

EQlSTOP

1234

1234

N87STOP

1234

VBJSTOP

1234

WQOI

1234

145

FFWBB-SoAAt

1234

Fig. 6 Analytical binding of wild-type and mutant SlOOAl derivatives to phenyl Sepharose. Bacterial extracts containing recombinantly expressed WT Sl OOAl or one of the mutant derivatives E91 Stop, N87Stop, V83Stop, W901 and FFW88-90AAI Sl OOAl were incubated with phenyl Sepharose beads in the presence of 5 mM Ca2+. After extensive washing in a buffer containing 1 mM Ca2+, Ca2+-dependently bound proteins were eluted by chelating Caz+ with EGTA. The individual fractions were then subjected to SDS-PAGE. In this comparative analysis, lanes 1 represent the bacterial extracts containing the respective recombinant SlOOAl protein. The unbound material is shown in lanes 2. Lanes 3 show the proteins released from the beads during the final CaZ+ wash, whereas lanes 4 represent the EGTA-extracted proteins. Note that WT, E91 Stop and W9Ol SlOOAl (polypeptides at -10 kDa) bind quantitatively to the beads whereas only a small fraction of N87Stop and FFW88-90AAI SlOOAl interacts with the hydrophobic matrix. The V83Stop SlOOAl mutant fails to interact with the matrix altogether.

EGTA elution. Finally, V83STOP SlOOAl fails to interact with phenyl Sepharose altogether (Fig. 6). We next employed a hydrophobic fluorescence probe, TNS, whose fluorescence emission is Significantly increased when interacting with hydrophobic amino acid side chains and which previously proved to be a valuable tool for analyzing the hydrophobic nature of SlOO proteins [ 151. TNS was mixed with different SlOOAl derivatives in the presence of increasing concentrations of Ca2+ and fluorescence emission spectra were recorded after TNS excitation at 365 mn. In line with previous analyses [ 151, Ca2+-bound WT S 1OOAl greatly enhances the fluorescence intensity of TNS emission indicative of TNS binding to hydrophobic sites of the protein (Fig. 7). The same intensity increase is observed in the case of Ca2+-bound E9lStop SlOOAl, whereas the W901 mutant protein only induces a small increase in TNS emission in the presence of Ca2+ (Fig. 7). This indicates that Tip-90 represents one of the hydrophobic TNS binding sites exposed in the Ca2+-bound conformation of SlOOAl and that replacement of this residue by isoleucine causes a less hydrophobic TNS environment in the W901 mutant. In contrast to WT, E91Stop and W901 SlOOAl, the other mutant derivatives (N87Stop, V83Stop and FFW88-90AAI SlOOAl) fail to induce significant changes in the TNS emission spectrum, although a sIightIy increased TNS fluorescence is observed in the case of N87Stop SlOOAl already in the absence of Ca2+(Fig. 7). Taken together, this analysis corroborates the qualitative phenyl Sepharose data (Fig. 6) and shows that Tip-90 and at least one of the phenylalanines at positions 88 and 89 represent the major hydrophobic binding sites for TNS in Ca2+-bound S1OOAl. Interestingly, in the case of W901 SlOOAl, the change in TNS fluorescence is already observed at free Ca2+ 0 Harcoufl

Brace

& Co. Ltd 7998

concentrations in the lower micromolar range as compared to higher values obtained for WT S 1OOAl . This suggests a somewhat increased affinity for Ca2+ of the CterminaI EF hand as a result of the W901 substitution. To test this hypothesis, we subjected WT and W901 SlOOAl to a more detailed TNS analysis by increasing the free Ca2+ concentrations in the protein-TNS mixtures in smaller increments. The results of the individual TNS emission reveal that the Ca2+ concentrations required to induce the half-maximal effect in the TNS fluorescence differ by a factor of 4 with W901 SlOOAl requiring only 10 i&l Ca2+ to elicit the half-maximal fluorescence increase as compared to 40 I.IM in the case of WT SlOOAl (not shown). Thus, the isoleucine for tryptophan substitution at position 90 induces a higher Ca*+ affinity of the C-terminal EF hand possibly by making the conformational change occurring upon Ca2+ binding energetically more favourable. SlOOAl dimerization is not affected in mutant proteins with impaired TRTK-WCapZa binding

The experiments described above indicate that hydrophobic residues in the C-terminal region of SlOOA (Phe-88, Phe-89, Trp-90) become exposed upon Ca2+ binding and are involved in the interaction between Ca*+-bound SlOOA and the CapZ peptide TRTK-12. However, recent NMR spectroscopical analyses of other SlOO proteins, including the close homologue SlOOB, in their Ca2+-free conformation revealed that conserved hydrophobic residues corresponding in linear sequence alignments to Phe-89 of Sl OOAl provide intra- and interchain contact points in the SlOO dimer 124-261. To determine whether the mutations introduced in the CCell Calcium

(1998)

24(Z),

137-

15 1

146

D Osterloh, V V Ivanenkov, V Gerke

P ‘5

1200

ii % J

% 0 2

800

800

800

400

400

450

500

wavelength

550

600

400

450

500

wavelength

(nm)

550

600

I 400

(nm)

450

500

wavelength

800

550

800

(nm)

800

400

rl 400

450

500

wavelength

550 (nm)

60

400

450

500

wavelength

550 (nm)

600

400

450

500

wavelength

550

600

(nm)

Fig. 7 Surface exposure of hydrophobic residues as revealed by TNS fluorescence emission spectroscopy. The hydrophobic fluorescent probe TNS was mixed with WT Sl OOAl or one of the mutant derivatives (E91 Stop, N87Stop, V83Stop, W901 and FFW88-9OAAI Sl OOAl, respectively) in the presence of 0.5 mM EDTA and the fluorescence emission of TNS was recorded after excitation at 365 nm (-). Subsequently, CaCI, was added stepwise to a final concentration of 0.25 (-,.-), 0.5 (-.-) and 0.75 mM (- - -) and the individual emission spectra were recorded after each Ca*+ addition. Note that WT Sl OOAl and E91 Stop Sl OOAl induce a comparable increase in TNS fluorescence in the presence of 250 uM free Cap+. Only a minor Cap+-dependent increase in TNS-fluorescence is detectable in the presence of W9Ol Sl OOAl, whereas the other mutant derivatives N87Stop. V83Stop and FFWEE-9OAAI Sl OOAl fail to induce a significant change in the TNS emission spectrum.

terminal region of SlOOAl also affect dimer formation, we subjected WT SlOOAl and different mutant derivatives to chemical cross&king and analytical gel filtration. A comparative cross-linking analysis was performed with WT and FFW88-90AAI SlOOAl. The latter was chosen as an example of a mutant protein with a greatly reduced affinity for the CapZ peptide in which one potential dimer contact was replaced by a less hydrophobic amino acid. SDS-PAGE of the respective reaction products reveals that the amount of covalently cross-linked dimer formed under the conditions chosen is virtually identical in the case of WT and FFW88-90AAI SlOOAl, respectively (not shown). Moreover, the crosslinking efficiency is not affected by the absence or presence of Ca2+. Identical results were obtained with W901 and EBlStop SlOOAl, i.e. mutant derivatives capable of TRTK- 12 binding, and with N87Stop S 1OOAl, i.e. another mutant protein with an impaired TRTK-12 binding site (data not shown). Cell Calcium

(1998)

24(2),

137-151

Chemical cross-linking could stabilize dimers which only form transientIy in solutions. We thus chose analytical gel filtration to analyze the hydrodynamic parameters of different SlOOAl derivatives in solution. Results for WT SlOOAl and the different mutant derivatives are summarized in Figure 8. When compared to migration positions of a globular marker proteins, the migration of WT SlOOAl under the conditions chosen corresponds to that of a globular protein of approximately 35 kDa. This differs slightly from the 2 1 kDa position expected for a dimeric SlOO protein of perfectly globular shape and indicates that dimeric S1OOA1 shows some deviations from a globular shape already reported previously [34,35]. The dimeric nature of WT SlOOAl in the 35 kDa peak was verified by gel filtration of covalently cross-linked WT Sl OOAl dimers which showed a migration position indistinguishable from that of the non-cross-linked protein (not shown). When compared to WT SlOOAl, the C-terminalIy truncated 0 Harcouri

Brace

& Co. Ltd 1998

S 1OOA7-CapZ interaction

147

0.25,

kg o.m4(q 0.15. ii $

0.10.

jl

0.05-

.w I m

time/min

30

40

time/min

l.O-

0.6. 0.4 0.2 _ 0.0-r

h

I

30

20

40

timelmin 0.25 0.20 -

0.15. 0.10. 0.05 O.OO-m

I

30

20

30

40

timelmin 0.06 -

; 0.6 s: N 0.6

0.06. 0.04. 002. 0.00.

iii 8

0.4

1

0.2

:: 2

0.0

I 30

20

30

40

ttme/min Fig. 8 Dimerization of WT SlOOAl and mutant derivatives ESlStop, N87Stop, V83Stop, W901 and FFW88-90AAI SlOOAl analyzed by analytical gel filtration. Gel filtration of the purified proteins was carried out on a Superose S12 column in the absence of Ca2+. The respective protein peaks are given as a function of the elution time. Elution times of marker proteins gamma globulin (150 kDa) ovalbumin (45 kDa), myoglobin (18 kDa) and ubiquitin (8.5 kDa) are indicated for comparison. Note that WT, E91 Stop, N87Stop, W9Ol and FFW889OAAI SlOOAl show almost identical elution profiles with a single protein peak migrating at a position corresponding to a globular protein of -35 kDa. Gel filtration of covalently crosslinked WT Sl OOAl revealed that this corresponds to a dimeric Sl OOAl species. In contrast, the Cterminally truncated mutant V83Stop Sl OOAl elutes in two distinct peaks corresponding to globular proteins of 29 and 15 kDa, respectively, thus most likely representing a dimeric and a monomeric species, respectively.

mutants E9 1Stop and N87Stop S 1OOA1 as well as the W901 and the FFW88-90M SlOOAl mutants show almost identical migration properties, i.e. they elute in a single symmetrical peak at the dimer position. As expected from Q Harcoutt

Brace

& Co. Ltd 1998

the slightly lower molecular masses, progressive Cterminal truncations result in a small shift in the elution position towards longer column retardation times (Fig. 8). The dimeric nature of these SlOOAl derivatives is not Cell Calcium

(1998)

24(2),

137-151

148

D Osterioh, V V Ivanenkov, V Gerke

affected by the presence or absence of Caz+ indicating that dimerization occurs in the Ca*+-bound and the Caz+free conformation (not shown). The situation is significantly different in V83Stop SlOOAl. In this mutant derivative, two hydrophobic residues (Val-83 and Phe-89) corresponding to two amino acids providing dimer contacts in SlOOA6 [24] and SlOOB [25,26] have been removed. When analyzed by analytical gel filtration this mutant protein migrates in two distinct peaks corresponding to those of globular proteins of approximately 29 and 15 kDa, respectively (Fig. 8). The 29 kDa peak migrates at a position very similar to that of the other SlOO derivatives thus most likely representing a dimer. The elution position of the I5 kDa peak, on the other hand, would correspond to that of a monomeric species. This is supported by SDS-PAGEanalysis of the 15 kDa peak which only reveals the presence of the V83Stop S 1OOAl polypeptide with no indication of proteolysis and by the finding that a second gel filtration run of the isolated 15 kDa species again yields the two peaks at 29 and 15 kDa, respectively, indicative of a dynamic monomer-dimer equilibrium (not shown). The V83Stop SlOOAl elution profile indicates that, within this mutant protein population, more than 60% are monomeric under the conditions chosen. Collectively, the data show that substitution or removal of the three hydrophobic residues Phe-88, Phe-89, and Trp-90 in S 1OOA1 does not affect dimerization in our cross-linking and gel filtration analyses, whereas a removal of almost the entire Cterminal region in the V83Stop mutant significantly interferes with dimer formation. DISCUSSION

We have chosen different approaches to study in detail the structural requirements for the interaction between SlOOAl and TRTK-12, a peptide corresponding to a sequence in the CapZo subunit. This interaction mimics the binding of SlOOAl to the full-length CapZa polypeptide as: (i) Ca*+ bound SlOOAl directly and specifically binds to CapZa, i.e. the subunit harbouring the TRTK-12 sequence, in ligand blotting experiments (Figs 1 & 5); and (ii) TRTK12 effectively competes with the Capi%@ heterodimer for cross-linking to Ca2+bound SlOOAl [ 121. CapZ is a heterodimeric actin capping protein which binds to the barbed ends of actin filaments [30,36]. It was initially identified and purified from skeletal muscle and was shown to be a component of the sarcomeric Z-line [8,36]. Although homologous capping proteins have, meanwhile, been identified in other cell types, including yeasts [371, the localization of the muscle isoform at Z-lines and also at the fascia adherens of intercalated discs of cardiac myocytes clearly parallels that of SlOOAl [ 10,381 thus supporting the view that the Cell Calcium

(1998)

24(2),

137-

15 1

biochemically described binding of SlOOAl to CapZa represents a physiologically meaningful interaction. Recently, a second SlOOAl target protein has been identified in skeletal muscle. The Aplysiu twitchin kinase, a member of a family of giant myosin-associated protein kinases located in the sarcomeric A band, was found to be markedly activated by mammalian SlOOAl in a Ca2+ and Zn2+ dependent manner [39]. Although the twitchin kinase domain containing the SlOOAl binding site is localized towards the sarcomeric M-line, i.e. opposite to the SlOOAl localization at the Z-line, it remains possible that two different SlOO target proteins are expressed in myofibrils, a scenario somewhat reminiscent of the large number of intracellular calmodulin targets identified. Biochemical and spectroscopical studies have shown that SlOO proteins display an increased hydrophobic@ in their Ca2+ bound conformation and it has been suggested that this is a prerequisite for target protein binding (for review see [5,51). Chemical modification of cysteine residues which proved to be differently accessible in Ca2+bound and Ca2+ free S 100 proteins, the analysis of Ca2+ induced changes in the fluorescence emission of unique aromatic residues as well as NMR and crystal structure analyses indicated that the major conformational changes induced upon Ca2+ binding occur in the N- and/or C-terminal regions as well as in the central hinge region of SlOO proteins [15-20,401. Thus, it has been assumed that hydrophobic residues in these regions are exposed on the surfaces of Ca2+ bound SlOO proteins where they can interact with their respective target. Hydrophobic residues which become exposed in the Ca2+bound conformation of SlOOAl have now been mapped directly through the mutagenesis approach chosen in this study. Using both Ca2+ dependent phenyl Sepharose binding assays and TNS fluorescence spectroscopy, we show that at least one in a cluster of three hydrophobic residues encompassing positions 88-90 in SlOOAl becomes phenyl Sepharose and TNS accessible upon Ca2+ binding to the SIOO protein. Ca2+ titration experiments reveal that TNS binding to WT SlOOAl is completed at 100 @I free Ca*+ (data not shown). This indicates that the exposure of residues Trp-90 and Phe-88 and/or Phe-89 is coupled to Ca2+ binding to the C-terminal EF hand and that full occupation of the N-terminal EF hand occurring at Caz+ concentrations exceeding 100 @vl does not contribute to the exposure of hydrophobic TNS binding sites. The increase in TNS fluorescence is less pronounced in the case of W901 SlOOAl (Fig. 7) suggesting that Trp-90 represents a TNS binding site in Ca2+bound SlOOAl and that the Ile for Trp replacement markedly reduces the hydrophobic nature in the vicinity of bound TNS. Interestingly, our Ca2+titration also reveals that the Ca2+ concentration required to elicit the half-maximal TNS 0 liarcourt

Brace

& Co. Ltd 1998

SlOOAl-CapZinteraction

fluorescence increase is reduced by a factor of 4 in W901 as compared to WT S 1OOAl . Thus the affinity of the Cterminal EF hand for Ca*+ is most likely affected by mutations in the C-terminal extension. Mutagenesis approaches had implicated hydrophobic residues located in the C-terminal regions of two other SlOO proteins, SlOOAlO and SlOOC, in target protein binding. In both cases, complex formation with the respective target, annexin II and I, appeared to be stabilized through contacts between hydrophobic residues in the SlOO proteins and hydrophobic side chains in the N-terminal regions of the armexins [22,23,41]. Interestingly, the latter were shown to lie on the hydrophobic side of an amphiphilic a-helix [42] indicating that the SlOO-annexin interactions resemble, to some extent, the binding of other EF-hand proteins, e.g. calmodulin and troponin C, to amphiphilic helices in their target proteins [43]. This view was, however, challenged recently when the solution structures of Ca*+ free SlOOA6 [24] and SlOOB [25,26] were solved by NMR spectroscopy. In both cases, hydrophobic amino acids corresponding in a sequence alignment to hydrophobic residues of Sl OOAlO and Sl OOC implicated in target protein binding were localized to the hydrophobic core of the dimeric SlOO protein providing sites for intra- and interchain contacts. Thus, it has been proposed that removal (through deletion or site-specific amino acid replacement) of these hydrophobic residues, in particular those conserved in the position corresponding to Phe-89 of S 1OOAl, leads to perturbations in the monomer-dimer equilibrium resulting, in turn, in a reduced afftity for the target protein [24]. Our analysis of the SlOOAlCapZa interaction now addresses this point directly. In the N87Stop and FFW88-90AAI SlOOAl mutants, deletion or substitution of Phe-88 and the conserved Phe-89, corresponding to two of the dimer contacts in the closely related SlOOBin its Ca2+free form [25,26], has no effect on SlOOAl dimerization in our cross-linking and gel filtration analyses but significantly interferes with target protein binding. We propose to explain this apparently dual function of Phe-88 and Phe-89 and conserved hydrophobic side chains at corresponding positions of other SlOO proteins by a Ca*+-induced conformational change. Whereas these residues are buried in the hydrophobic core of the SlOO dimer in the absence of Ca2+, Ca2+ binding leads to an increased accessibility of these residues on the protein surface. An altered subunit contact in Ca*+ bound S100 is in line with the findings by Baudier and Gerard [ 161 who observed in S 1OOAl/S 1OOB heterodimers an increased inter-subunit exchange in the presence of Ca2+. Moreover, and more directly, we now show that the Ca2+-induced exposure of hydrophobic residues 88-90 of SlOOAl correlates very well with the binding of the protein to TRTK-12 and fullQ Harcourt

Brace

& Co. Ltd 7998

149

length CapZa. Mutants lacking the characteristic hydrophobic&y increase in the presence of Ca2+, like N87Stop SlOOAl, fail to bind TRTK-12KapZa whereas WT S lOOA and the derivatives capable of binding CapZa display a Ca*+-induced increase in hydrophobicity. However, although there is no such indication in our biochemical analyses (purification, cross-linking, gel filtration), we cannot exclude the possibility that indirect conformational effects are elicited by the mutations introduced in SlOOAl . Moreover, it appears likely that in addition to Phe-88 and/or Phe-89, other residues of S lOOA are also involved in TRTK- 12 binding as the peptide interaction with the closely related SlOOB protein has recently been demonstrated by NMR spectroscopy to elicit chemical shifts also in residues of the N- and C-terminal EF hand helices [ 141. Even though Ca2+binding most likely alters the surface exposure of Phe-88 and Phe-89 and their accessibility for inter-subunit contacts, our cross-linking and gel filtration experiments show that dimers of SlOOAl mutant derivatives lacking a large hydrophobic side chain at positions 88 and 89 also form in the absence of Ca2+. Dimer formation is, however, critically affected in the V83Stop SlOOAl mutant (Fig. 8), i.e. upon removal of an additional hydrophobic residue, Val-83, which corresponds to another subunit contact in the Ca2+ free SlOOA6 dimer [24]. On the other hand, a single glutamine for valine substitution in position 83 does not affect dimerization in the resulting V83Q S1OOAl mutant derivative (data not shown). Collectively our findings show for the first time that, while the SlOO dimer is stabilized by a number of hydrophobic inter-chain contacts, several of these contacts need to be loosened to induce significant monomerization. As a consequence, the most C-terminal contact point, Phe-89 in SlOOAl, could be available for target protein interaction without disrupting the Sl 00 dimer, a situation most likely realized in the Ca2+ conformation.

ACKNOWLEDGEMENTS

We would like to thank Kirsten Remmert and Horst Hinssen (University of Bielefeld, Germany) for E. coli extracts containing recombinant CapZ subunits and Beat Schafer and Claus Heizmarm (University of Zikich, Switzerland) for providing the S lOOA cDNA clone. Thanks also to Klaus Weber (Max Planck Institute for Biophysical Chemistry, Giittingen, Germany) for sharing some equipment with us and to Anja Rosengarth and Martin Tepel (LJniversity of Mtinster) for help with fluorescence spectroscopy. This study has been supported by a grant from the Deutsche Forschungsgemeinschaft to VG. Cell Calcium

(1998)

24(2),

137-151

150

D Osterloh, V V Ivanenkov, V Gerke

REFERENCES 1. Celia M.R. Guidebook to the calcium-binding proteins. Oxford, Oxford University Press, 1996. 2. Kretsinger R.H. Structure and evolution of calcium modulated proteins. CRC Crit Rev Biochem 1980; 8: 119-l 74. 3. Kawasaki H., Kretsinger R.H. Calcium-binding proteins 1: EFhands. Protein Fro>le 1995; 2: 297-490. 4. Nakayama S., Kretsinger R.H. Evolution of the EF-hand family of proteins. Annu Reu Biophys Biomol Struct 1994; 23: 473-507 5. Zimmer D.B., Cornwall E.H., Landar A., Song W. The SIOO protein family: history, function, and expression. Brain Res Bull 1995; 37: 417-429. 6. Schafer B.W., Heizmarm C.W. The SlOO family of EF-hand calcium-binding proteins: functions and pathology. Trends Biochem Sci 1996; 21: 134-140. 7 Hilt D.C., Kligman D. The S-100 protein family: a biochemical and functional overview. In: Heizmann C.W. (Ed) Novel calcium-binding proteins. Berlin, Springer, 1991: 65-103. 8. CaseBa J.F.,Craig S.W., Maack D J., Brown A.E. Cap Z (36/32), a barbed end a&r-capping protein, is a component of the Z-line of skeletal musc1e.J Cell Bioll987; 105: 371-379. 9. Schafer D.A., Korshunova Y.O.,Schroer TA., Cooper J.A.Differential localization and sequence ana&sis of capping protein betasubunit isofomrs of vertebrates. J Cell Bioll994; 127: 453-465. 10. Haimoto H., Kato K. S 1OOaOalpha alpha) protein in cardiac muscle. Isolation from human cardiac muscle and ultrastructural localization. EurJBiochem 1988; 171: 409-415. 11. Ivanenkov V.V.,Jamieson Jr G.A., Gruenstein E., Dimlich R.V. Characterization of S-100b binding epitopes. Identification of a novel target, the actin capping protein, CapZ. j Biol Chem 1995; 270: 14651-14658. 12. Ivanenkov V.V., Dimlich R.V., Jamieson Jr G.A. Interaction of S1OOaOprotein with the active capping protein, CapZ: characterization of a putative S1OOaObinding site in CapZ alpha-subunit. Biochem Biophys Res Commun 1996; 221: 46-50. 13. Bianchi R., Garbuglia M., Verzini M. et al. S- 100 (alpha and beta) binding peptide (TRTK-12) blocks S-lOO/GFAP interaction: identification of a putative S-100 target epitope within the head domain of GFAP. Biochim Biophys A& 1996; 1313: 258-267 14. Kirby P.M., van Eldik LJ., Roberts G.C.K. Identification of the binding site on SlOOB protein for the actin capping protein CapZ. Protein Sn’ 1997; 6: 2494-2503. 15. Baudier J., Gerard D. Ions binding to SlOO proteins: structural changes induced by calcium and zinc on S1OOaand S1OOb proteins. Biochemistry 1983; 22: 3360-3369. 16. Baudier J., Gerard D. Ions binding to SlOO proteins. II. Conformational studies and calcium-induced conformational changes in SlOO alpha alpha protein: the effect of acidic pH and calcium incubation on subunit exchange in S1OOa (alpha beta) protein. J Biol Chem 1986; 261: 8204-82 12. 17 Becker T., Gerke V., Kube E. Weber K. S 1OOP,a novel Cat*+)binding protein from human placenta. cDNA cloning, recombinant protein expression and Ca*+ binding properties. EurJBiochem 1992; 207: 541-542 18. Drohat A.C., Baldisseri D.M., Rustandi R.R., Weber DJ. Solution structure of calcium-bound S100 @eta beta) as determined by nuclear magnetic resonance spectroscopy. Biochemistry 1998; 37: 2729-2740. 19. Matsumura H., Shiba T., Inoue T., Harada S., Kai Y. A novel mode of target recognition suggested by the 2.0 angstrom

Cell Calcium

(1998)

24(2),

137-151

20.

21.

22.

23.

24.

25.

26.

27

28.

structure of holo S1OOB from bovine brain. S&JU%~~ 1998; 6: 233-241. Smith S.P.,Shaw G.S. A novel calcium-sensitive switch revealed by the structure of human SlOOB in the calcium-bound form. Sttxhre 1998; 6: 21 l-222. Watanabe M., Ando Y., Tokumitsu H., Hidaka H. Binding site of annexin XI on the calcyclin molecule. Biochem Biophys Res Commun 1993; 196: 1376-1382. Seemann J., Weber K., Gerke V Structural requirements for annexin I-S 1OOC complex formation. Biochem J 1996; 319: 123-129. Kube E., Becker T., Weber K., Gerke V. Protein-protein interaction studied by site-directed mutagenesis. Characterization of the annexin II-binding site on pl 1, a member of the S100 protein family. J Biol Chem 1992; 267: 14175-14182. Potts B.C., Smith J., Akke M. et al. The structure of calcyclin reveals a novel homodimeric fold for S100 Cat*+)-binding proteins. Nat Struct Bioll995; 2: 790-796. Drohat A.C., Amburgey J.C., Abiidgaard F., Starich M.R., Bakdisseri D., Weber D J. Solution structure of rat apo-SIOOB (beta beta) as determined by NMR spectroscopy. Biochemistry 1996; 35: 11577-l 1588. Kirby P.M., van E.ldik LJ., Roberts G.C.K. The solution structure of the bovine S1OOB dimer in the calcium-free state. Shrcccure 1996; 4: 1041-1052. Sanger F., Nicklen S., Coulson A.R. DNA-sequencing with chainterminating inhibitors. Proc Nat1 Acad Sci USA 1977; 74: 5463-5467 Schagger H., von Jagow G. Tricine-sodium dodecyl sulfatepolyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 1987; 166: 368-379.

29. CaselkaJ.F.,CaseBa SJ., HoBands J.A., Caldwell J.E., Cooper JA. Isolation and characterization of cDNA encoding the alpha subunit of Cap Z(36/32), an a&n-capping protein from the Z line of skeletal muscle. Pmc Natl Acad Sci USA 1989; 86: 5800-5804. 30. Caldwell J.E.,Waddle J.A., Cooper J.A., HoBands J.A., CaseBa SJ., Casella J.F. cDNAs encoding the beta subunit of cap Z, the a&n-capping protein of the Z line of muscle. J Biol Chem 1989; 264: 12648-12652. 3 1. Burgess W.H., Watterson D.M., van Eldik L J. Identification of cahnodulin-binding proteins in chicken embryo fibroblasts. J Cell Bioll984; 99: 550-557 32. Hertzberg E.L., van Eldik L J. Interaction of cahnodulin and other calcium-modulated proteins with gap junctions. Methods Enzymol1987;

139: 445-454.

33. Laemmli U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-685. 34. Mani RJ., Kay C.M. Hydrodynamic properties of bovine brain SlOO proteins. FEBS Lett 1984; 166: 258-262. 35. Kato K., Kimura S.,Haimoto H., Suzuki F. SlOOao (alpha alpha) protein: distribution in muscle tissues of various animals and purification from human pectoral muscle. J Neumchem 1986; 46: 1555-1560. 36. Casella J.F.,Maack DJ., Lin S. Purification and initial characterization of a protein from skeletal muscle that caps the barbed ends of actin filaments. J Biol Ckem 1986; 261: 10915-10921.

0 Harcourt

Brace

8 Co. Ltd 7998

SlOOA I-CapZ interaction

37 Sizonenko G.I., Karpova TX, Gattermeir D.J., Cooper J.A. Mutational analysis of capping protein function in Smchuromyces cerwisiue. Mol Biol Cell 1996; 7: l-l 5. 38. Kato K., Kimura S. SlOOao (alpha alpha) protein is mainly located in the heart and striated muscles. Biochim Biophys Acfu 1985; 842: 146-150. 39. Heierhorst J., Kobe B., Feil S.C. et al. Ca2+/S100 regulation of giant protein kinases. Nature 1996; 380: 636-639. 40. Moore B.W. Conformational and hydrophobic properties of rat and bovine S-100 proteins. Neurochem Res 1988; 13: 539-545.

Q Harcourt Brace & Co. Lid 1998

151

4 1. Becker T., Weber K., Johnsson N. Protein-protein recognition via short amphiphilic helices; a mutational analysis of the binding site of annexin II for p Il. EMBO J 1990; 9: 4207-42 13. 42. Johnsson N., Marriott G., Weber K. ~36, the major cytoplasmic substrate of src tyrosine protein kinase, binds to its p 11 subunit via a short amino-terminal amphiphatic helix. EMBO J 1988; 7: 2435-3442. 43.

Strynadka N.C., James M.N. Model for the interaction of amphiphilic helices with troponin C and calmodulin. Proteins 1990; 7: 234-248.

Cell Calcium (1998) 24(2), 137-151