isolation and characterization of catalytic and calmodulin-binding domains of bordetella pertussis...

Eur. J. Biochem. 196,469-474 (1991) 0 FEBS 1991

0014295691001652

Isolation and characterization of catalytic and calmodulin-binding domains of Bordetella pertussis adenylate cyclase Heline MUNIER’, Anne-Marie GILLES’, Philippe GLASER’, Evelyne KRIN2, Antoine DANCHIN’, Robert SARFATI and Octavian BARZU

Unite de Biochimie des Regulations Cellulaires, ’ Unite de Regulation de 1’Expression Genitique, and Unite de Chimie Organique, Institut Pasteur, Paris, France

(Received October 5, 1990) - EJB 90 1190

A truncated Bordetella pertussis cya gene product wds expressed in Escherichia coli and purified by affinity chromatography on calmodulin-agarose. Trypsin cleavage of the 432-residue recombinant protein ( M , = 46 659) generated two fragments of 28 kDa and 19 kDa. These fragments, each containing a single Trp residue, were purified and analyzed for their catalytic and calmodulin-binding properties. The 28-kDa peptide, corresponding to the N-terminal domain of the recombinant adenylate cyclase, exhibited very low catalytic activity, and was still able to bind calmodulin weakly, as evidenced by using a fluorescent derivative of the activator protein. The 19- kDa peptide, corresponding to the C-terminal domain of the recombinant adenylate cyclase, interacted only with calmodulin as indicated by a shift in its intrinsic fluorescence emission spectrum or by the enhancement of fluorescence of dansyl-calmodulin. T28 and T19 fragments exhibited an increased sensitivity to denaturation by urea as compared to uncleaved adenylate cyclase, suggesting that interactive contacts between ordered portions of T28 and T19 in the intact protein participate both in their own stabilization and in stabilization of the whole tertiary structure. The two fragments reassociated into a highly active calmodulin-dependent species. Reassociation was enhanced by calmodulin itself, which ‘trapped’ the two complementary peptides into a stable, native-like, ternary complex, which shows similar catalytic properties to intact adenylate cyclase.

Bordetella pertussis adenylate cyclase is synthesized as a large bifunctional precursor of 1706 amino acid residues [l]. The N-terminal segment of the protein (400 residues) has calmodulin-activated ATP-cyclizing activity, while the rest of the molecule is thought to be responsible for the haemolytic activity of the pathogen [l, 21. The large precursor, purified from extracts of B. pertussis as a 200-kDa protein [3 - 51, is found in culture media most often in low-molecular-mass forms of 50, 45 or 43 kDa [6-81. Tryptic cleavage of these low-molecular-mass forms of adenylate cyclase yields two complementary fragments of 25 kDa and 18 kDa [9]. By com- bining genetic and biochemical information it has been shown that the 25-kDa fragment harbors the active site, whereas the 18-kDa fragment corresponds mostly to the calmodulin- binding domain of the enzyme [lo, 111.

Correspondence to 0. Bgrzu, Unite de Biochimie des Regulations Cellulaires, Institut Pasteur, 28, rue du Docteur Roux, F-75724 Paris Cedex 15, France

Abbreviation. P235 -254 is a synthetic peptide corresponding to residues 235 - 254 of B. pertussis adenylate cyclase: Arg-Glu-Arg-Ile- Asp-Leu-Leu-Trp-Lys-Ile-Ala-Arg-Ala-Gly-Ala-Arg-Ser-Ala-Val- Gly.

Enzyme. Adenylate cyclase, ATP pyrophosphdte-lyase (cyclizing) (EC 4.6.1.1).

Note. The novel nucleotide sequence data published here has been deposited with the EMBL sequence data banks and is available under accession number Y00545.

The novel amino acid sequence data published here has been deposited with the NBRF sequence data bank.

In order to gain more insight into the information transfer between the calmodulin-binding site and the catalytic center of adenylate cyclase, we examined the biochemical properties of these domains obtained by proteolytic cleavage of a truncated B. pertussis cya gene product expressed in Escherichia coli. We report that isolated fragments act as independent entities which can reassociate into a highly active calmodulin- dependent species resembling the native adenylate cyclase in many respects. The formation of the ternary active complex, involving the two separated domains of adenylate cyclase and calmodulin, is a unique phenomenon among calmodulin- activated enzymes and apparently has little analogy with any enzymatic system investigated so far.

MATERIALS AND METHODS Chemicals

Adenine nucleotides, and restriction enzymes were from Boehringer Mannheim. Bovine brain calmodulin, dansyl- calmodulin, calmodulin-agarose, trypsin (treated with tosyl- phenylalanylchloromethane) and soybean trypsin inhibitor were from Sigma. Urea (fluorimetrically pure) was a product of Schwartz/Mann. [u-~~P]ATP (3000 Ci/mmol), [32S]- dATP[aS] (> 1000 Ci/mmol), and [3H]cAMP (40 Ci/mmol) were obtained from Amersham International (Amershdm, UK). Oligonucleotides were synthesized according to the phosphamidinate method using a commercial DNA syn- thesizer (Applied Biosystems). Synthetic peptide corresponding to residues 235 - 254 of B. pertussis adenylate cyclase

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Fig. 1. Restriction i m p of plasmid pDIA5227 and deduced amino acid sequence of the recombinant udenylute cyclase. The first 399 codons of the cyclolysin gene fused to 33 codons of vector sequences were expressed under transcriptional and translational control of the lucZ gene. The amino acid rcsidues (represented by the standard one-letter code) of the chimeric protein ( M , = 46659) were numbered from -33 to 399 to keep numbering identical to equivalent residues in the wild-type adenylate cyclase of B. pertussis. Lower case letters from - 33 to - I represent the 'foreign' sequence. Tryptic cleavage of the recombinant protein at Arg224 generates two fragments, with theoretical M , of 27731 (T28) and 18946 (T19). T28 corresponds in thc wild-type enzyme to a 25-kDa peptide, called T25. The black box covers to the lac2 gene AUG start codon and the following 32 codons of the chimeric gene. The inserted B. pertussis chromosomal DNA is shaded. Restriction sites are represented as follows: E, EcoRI; V, EcoRV; S, SulI. Other abbreviations: amp, ampicillin resistance gene; OP lac, operator and promoter from the lactose opcron; cya, adenylate cyclase gene

(P235-254) was obtained by a solid-phase method on chloromethyl-substituted polystyrene resin cross-linked by 1 %O divinyl benzene.

Bacterial strains und growth conditions

The E. coli BMH7118 [12] strain was used for sequence analysis and site-directed mutagenesis. Production of recombinant protein was performed using the protease-deficient strain Y1083 BNN103 [I31 which harbors plasmid pDIA17 (this work). Cultures were grown in LB medium [I41 supplemented with 100 pg/ml ampicillin and 20 Fg/ml chlor- amphenicol. The lac promoter was induced by 1 mM isopro- pyl fl-D-thiogalactoside when cultures reached an absorbance of 0.5 at 600 nm. Bacteria were harvested by centrifugation 4 h after induction.

Plasmids

Plasmid PDIA5227 (Fig. 1), a derivative of plasmid pDIA5202 [lo], directs the synthesis of a 432-amino-acid- residue chimeric protein. The 33 N-terminal residues of this protein (numbered from - 33 to - 1) are encoded by vector sequences. They are followed by the first 399 residues of adenylate cyclase. Transcription and translation signals of the chimeric gene are those of the E. coli L a c 2 gene, carried by the expression vector pTZ19.

Plasmid pDIA17 expresses the lacI gene under the control of the tetracycline promoter. It was obtained by insertion of a BamHI fragment of plasmid EBR212-6 encompassing the lacl gene at the BanzHI site of plasmid pACYC184 [15]. Plasmids pDIA5227 and pDIAl7 are compatible replicons.

Site-diiwtecl nzutagrne~ I S und sequence analysis

Oligonucleotide-directed mutagenesis was performed on the single-strand form of pDIA5227, using the Amersham system based on the method of Taylor et al. [I61 and following

the supplier's instructions. The glutamine (CAG) codon at position 400 was modified to a stop (UAG) codon using the oligonucleotide "GTG GAA CGC TAG GAT TCC G3'. The absence of any other mutation was verified by the dideo- xynucleotide sequencing method [17].

Purification of the truncated B. pertussis cya gene product expressed in E. coli and tryptic cleavage of culmodulin-agarowimmobilized protein

Bacteria suspended in 50 mM Tris/HCI pH 8 were disrupt- ed by ultrasonication. Adenylate cyclase in the pellet fraction after centrifugation was extracted with 8 M urea in the same buffer [9]. The 'urea' extract was diluted eightfold with 50 mM Tris/HCl pH 8 containing 0.1% Triton X-100 and 1 mM CaC12, then adsorbed onto calmodulin-agarose. The gel with bound adenylate cyclase was washed several times with 50 mM Tris/HCl pH 8 containing 0.5 M NaC1. Enzyme was then eluted with 8 M urea in 50 mM Tris/HCl pH 8. Urea was removed by chromatography on Sephadex G-25 columns equilibrated with 50 mM Tris/HCl. The specific activity of pure enzyme at saturating concentrations of calmodulin was approximately of 2800 units/mg protein, corresponding to a k,,, of 2100 s- l at 30'C and pH 8. Tryptic fragments of bacterial adenylate cyclase were obtained as follows. Calmodulin- bound enzyme was resuspended in Tris/HCl buffer to which trypsin (25 pg for each 1000 units adsorbed enzyme) was added. The mixture was rotary shaken for 10 min at 4 ' C, then proteolysis was stopped with an eightfold excess (by mass) of soybean trypsin inhibitor. After extensive washes with 50 mM Tris/HCl pH 8, the gel was allowed to sediment in a Bio- Rad column (1 x 20 cm) and proteins were eluted at room temperature with 8 M urea in Tris/HCl. Fractions absorbing at 280nm were analyzed by SDSIPAGE, then pooled. Samples were lyophilized after removal of urea by chromatography on Sephadex G-25 with 25 mM ammonium acetate pH 6.4 as solvent.

47 1

Fluorescence measurements

Binding of adenylate cyclase or of its tryptic fragments to dansyl-calmodulin was analyzed with a Perkin-Elmer LS-5B luminescence spectrometer thermostatted at 25 "C using ultra- violet-grade quartz cuvettes (1 x 1 cm; sample volume 2 ml). The dansyl moiety of calmodulin was excited at 340 nm. The titration of fluorescence enhancement by calmodulin-binding peptides was performed by recording the fluorescence emission at 480 nm, one data point corresponding to fluorescence intensities integrated over a total time of 8 s. The intrinsic fluorescence spectra (kcxc = 295 nm) of adenylate cyclase or of its tryptic fragments (I pM) in the absence or presence of calmodulin (1.2 pM) were recorded in 50 mM Tris/HCl pH 8, 100mM NaCl and 0.5mM CaCl, from 305nm to 400 nm.

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Analytical procedures

Adenylate cyclase activity was measured using the procedure of White [18] as described previously [8 - 111. One unit of adenylate cyclase activity is the amount catalyzing the formation of 1 pmol cAMP/min at 30°C and pH 8. Protein concentration was determined according to Bradford [19] using a Bio-Rad kit or according to the procedure of Smith et al. [20] with reagents purchased from Pierce Chemical Co. SDSjPAGE was performed as described by Laemmli [21]. Amino acid analysis was performed on a Biotronik amino acid analyzer LC 5001 using a single-column procedure [22]. The N-terminal amino acid sequence of T19 was determined using the manual 4-(dimethy1amino)azobenzene 4'-isothio- cyanatelphenyl isothiocyanate double-coupling technique. The resulting 4-(dimethy1amino)azobenzene - 4'-isothiocyanate derivatives were identified on polyacrylamide layers as described by Chang et al. [23].

RESULTS

Separation and characterization of peptides resulting f rom tryptic cleavage of truncated B. pertussis adenylate cyclase

Truncated B. pertussis adenylate cyclase, expressed in E. coli and representing up to 5% of bacterial protein, was recovered in the pelleted fraction after cell ultrasonication and centrifugation. Insoluble adenylate cyclase was extracted with 8 M urea in buffer solution, then adsorbed onto calmodulin- agarose. The adsorbed enzyme was cleaved with trypsin in this 'immobilized' form. The procedure has two advantages : (a) the affinity gel could be used many times without significant alteration in the binding of enzyme; (b) impurities non- specifically adsorbed were removed upon proteolysis. Two main fragments of 28 kDa (T28) and 19 kDa (T19) were eluted from calmodulin-agarose with 8 M urea. The adenylate cyclase and its tryptic fragments were separated by gel-permeation chromatography on Sephadex G-100 under de- naturing conditions (Fig. 2). The identity of fragments was controlled by several methods: (a) amino acid analysis of pure peptides; (b) N-terminal sequence analysis of purified T19 peptide which was found to be Ala-Ala-Ser-Glu-Ala. From these results, we deduced that T28 corresponds to residues -33 to 224 (Mr = 27731), whereas TI9 corresponds to residues 225 - 399 ( M , = 18946). In previous work, using tryptic and chemical cleavage of adenylate cyclase purified from B. pertussis and SDSjPAGE analysis of fragments, we tentatively

" 40 60 80 100

SAMPLE NUMBER

Fig. 2. Separation of complementary peptides T28 and T19 after cleavage of truncated B. pertussis adenylate cyclase with trypsin. Trypsin- cleaved recombinant adenylate cyclase (3.5 mg; see Materials and Methods) was dissolved in 0.5 ml 9% formic acid and loaded onto a Sephadex G-100 column (110 x 1 cm) equilibrated with the same solvent. Fractions of 1 ml were collected at a flow rate of 5 ml/h and analyzed for absorbance at 280 nm. Inset shows SDSjPAGE (10'/0) and Coomassie-blue staining of samples 59,61,63,73,76 and 80. The M , markers for SDSjPAGE were: (a) phosphorylase a (94000); (b) bovine serum albumin (67000); (c) ovalbumin (43000); (d) carbonic anhydrase (30000); (e) soybean trypsin inhibitor (20 300); (f) lysozyme (14400). Samples 57 - 65 and 72 - 76 were pooled and the formic acid removed by rotary evaporation at low pressure and lyophilization

assigned the site of tryptic cleavage to Arg235 or Arg237 [l l] . Purified T28 had a very low catalytic activity representing 0.1% of the activity of intact enzyme at saturating concentration of calmodulin, while T19 was found to be inactive. T28 still required calmodulin for full activity, although the curve of activation by calmodulin, had an unusual pattern extending over several orders of magnitude of calmodulin concentration.

Interaction of recombinant adenylate cylcase and its complementary peptides with calmodulin

The fluorescence spectrum of adenylate cyclase which has two tryptophan residues (Trp69 and Trp242), showed a maximum at 342 nm upon excitation at 295 nm. This maximum was shifted to 334 nm in the presence of equimolar concentrations of calmodulin and excess Ca2+ ions [24]. T19, which has a single tryptophan (Trp242), behaved similarly and the shift in the fluorescence spectrum upon addition of Ca2+-calmodulin from 345 nm to 332 nm was accompanied by a 1.4-fold increase in the maximum of emission (Fig. 3 A). Excess EGTA almost completely reversed the effect of calmodulin on the fluorescence spectrum of adenylate cyclase and of T19 fragment (not shown). The affinity of T19 for calmodulin (Kd = 20 nM), determined indirectly in competitive binding experiments [25] as the shift of the activation curve by calmodulin of native enzyme in the presence of fixed amounts of T19, was significantly lower than that of intact adenylate cyclase [9 - 1 I]. T28 exhibited a completely different fluorescence spectrum with a maximum at 338 nm for which the peak height represented about 40% of that of wild-type protein. Addition of Ca2+-calmodulin to T28 fragment of

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Fig. 3. Fluorescence ernission spectra of T28 and T19 peptides in the absence ( A ) and presence ( B ) qf different concentrations of urea. (A) The intrinsic fluorescence spectra of proteins at 1 pM (Aex = 295 nm) in 50 mM Tris/HCl plI 8,100 mM NaCl and 0.1 mM CaCl, corrected for the buffer were recorded over 305 -400 nm. T28 in the absence ofcalmodulin (--); TI9 in the absence ofcalmodulin (----); TI9 in the presence of 1.2 pM calmodulin (.......) . (B) Protein solutions at 1 pM in 50 mM TrisIHCl pH 8,100 mM NaCI, 0.1 mM CaCI, and different concentrations of urea were prepared 2 h before measurements; occasionally spectra = 295 nm) were recorded after over- night incubation of samples in urea at 4°C with essentially identical results. ( 0 ) T28; (9) T19: (m) uncleaved adenylate cyclase

adenylate cyclase did not produce any variation in the fluorescence emission spectrum (not shown). Urea produces im- portant shifts in the spectrum of both adenylate cyclase and T28 and T19 fragments (Fig. 3B), indicating an increased sensitivity of isolated peptides to denaturation as compared to native, uncleaved, adenylate cyclase.

Since the absence of changes in the intrinsic fluorescence spectrum of T28 peptide upon addition of Ca2+-calmodulin does not necessarily exclude interactions between proteins, another experimental approach was considered. We examined the fluorescence properties of dansyl-calmodulin, a calmodulin derivative which binds and activates B. pertussis adenylate cyclase as well as native calmodulin. As shown in Fig. 4A, truncated B. pc2rtussis adenylate cyclase, T19 peptide, and a synthetic peptide derived from the calmodulin-binding site of adenylate cyclase (P235 ~ 254) enhanced the fluorescence emission of dansyl-calmodulin by a factor of 1.7 - 2.2 with shift of the maximum to a lower wavelength. Under identical experimental conditions, T28 determined only a slight, although constant, increase in the fluorescence emission spectrum of dansyl-calmodulin. Titration of fixed concentrations of dansyl-calmodulin with various concentrations of TI 9 (Fig. 4B) or P235 -254 peptide (not shown) and analysis of experimental data according to Scatchard allowed determi-

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Fig. 4. Fluorescence analysis of interaction between dansyl-calmodulin and B. pertussis adenylate cyclase or enzyme-derived peptides. (A) Emission spectrum (Aex = 340 nm) of dansyl-calmodulin (150 nM) in 50 mM Tris/HCl, 100 mM NaCl 0.5 mM CaCI,, alone (a) or in the presence of 1 pM of adenylate cyclase (c), T28 (b), TI9 (e) or P235 - 254 (d). (B) Titration of dansyl-calmodulin (50 nM) with adenylate cyclase (A), or TI9 peptide ( , 0 ) in the absence ( m) or presence ( 0 ) of 1 pM T28 peptide was performed in 50 mM Tris/HCl pH 8, 100 mM NaCl and 0.5 mM CaCl,. Excitation was performed at 340 nm, emission was recorded at 480 nm. One data point corresponds to fluorescence intensities integrated over a total time of 8 s. F, is the fluorescence intensity of dansyl-calmodulin alone, F is the fluorescence intensity of the complex, F,,, is the fluorescence intensity of dansyl-calmodulin at a saturating concentration of adenylate cyclase or T19 peptide. Inset shows a Scatchard plot analysis of TI9 binding to dansyl-calmodulin

nation of the Kd of complexes of dansyl-calmodulin with TI9 (25 nM) and with P235-254 peptide (570 nM) respectively. The values obtained agreed well with those obtained from competitive binding experiments with bovine brain calmodulin [Il l (and this paper). The Kd for the dansyl-calmodulin - adenylate-cyclase complex could not be determined accurately from these experiments, since the low Kd (0.2 nM) would have required subnanomolar concentrations of both peptides, which are below the detection limit. In addition, the very low affinity of T28 for dansyl-calmodulin did not allow determination of the Kd of the T28-dansyl-calmodulin complex. When fixed concentrations of dansyl-calmodulin mixed with a 20-fold molar excess of T28 were titrated with T19, the fluorescence enhancement curve of the calmodulin derivative was practically identical to that exhibited by the intact enzyme (Fig. 4B). This experiment indicated that the high-affinity calmodulin-binding property of intact adenylate cyclase was

413

Table 1. Reussociation of T28 und T19 into cutulytically active species T28 (0.5 pM) and T19 (1.25 pM) were incubated in 50 mM Tris/ HC1 pH 8 and 0.1 YO Triton X-100 supplemented or not with 10 pM calmodulin. After 30 min of incubation at 30”C, 1-pI aliquots were assayed for adenylate cyclase activity, then samples were diluted 50- fold with Tris/Triton buffer. After another 30 min of incubation at 30”C, adenylate cyclase activity was assayed again. The final concentrations of ATP, Ca2+ and calmodulin in the enzyme assay medium were 5 mM, 0.1 mM and 1 pM, respectively. k,,, was calculated as- suming in all cases a KkTp of 0.6 mM and molecular masses of 27.7 kDa (T28) or 46.66 kDa (adenylate cyclase or reconstituted protein)

Experimental conditions kcat

S K 1

T28 1.3 Adenylate cyclase 2200 T28 + T19 202 T28 + T19, 50-fold diluted 22 T28 + T19 + calmodulin 863 T28 + TI9 + cdlmoduhn, 50-fold dilution 513

restored when complementary tryptic peptides and calmodulin were present simultaneously in the test cuvette.

Reassociation of T28 and TlYpeptides into catalytically active, calmodulin-dependent species

Isolated peptides possess little (T28), if any (T19), catalytic activity when compared to intact adenylate cyclase. Mixtures of purified fragments showed significant recovery of activity which increased with their increased concentration in the incubation medium. A major difficulty encountered in analyzing the kinetics of reassociation of isolated fragments, as well as the role of different ligands, is the fact that this process was expressed in terms of recovered catalytic activity and was always determined with excess Ca2 + and calmodulin. Two observations were, however, made from these experiments. (a) When mixtures of T28 and T19, preincubated at 30”C, were analyzed for catalytic activity, then diluted with buffer and re- analyzed for ATP-cyclizing activity, a significant decrease in k,,, was observed (Table 1). Since reassociation of T28 and T19 into catalytically active species requires only several mi- nutes,the limiting step in the formation of the T28-Tl9 complex is the thermodynamic factor, i. e. the equilibrium constant of the reaction T28 + T19ctT28 . T19. (b) Calmodulin, when present in the incubation medium of isolated fragments, caused a fourfold increase of the adenylate cyclase activtity. Subsequent dilution of medium containing T28, T19 and calmodulin had much less effect on ATP-cyclizing activity of fragments (Table 1). This was due most probably to the fact that calmodulin ‘trapped’ T28 and T19 into a stable, native- like, ternary complex. Gel permeation chromatography of such a ‘reconstituted’ calmodulin-activated complex revealed that it had hydrodynamic properties identical to those of uncleaved adenylate-cyclase-activator protein complex, in agreement with our previous data on enzyme secreted by virulent strains of B. pertussis [ll].

DISCUSSION

Calmodulin-dependent enzymes represent a heteroge- neous class of catalysts (for reviews see [26, 271). Although

many calmodulin-modulated enzymes have been identified, the mechanism of activation by calmodulin is still poorly understood, as detailed structural information on the target enzymes is scarce. In several cases, limited proteolysis released active calmodulin -independent forms of enzymes [28, 291. It was, therefore, proposed that the calmodulin-binding domains of these enzymes block the access of substrates to the active site [30, 311 and activation results by removal of this ‘autoinhibitory’ domain with proteases or following binding of calmodulin. Nucleotide-binding experiments on Bacillus anthracis adenylate cyclase, another calmodulin-dependent enzyme, showing that enzyme binds 3‘dATP only (or preferen- tially) in the presence of calmodulin [32], support the mechanism of catalytic site blocking by the calmodulin-binding site. The calmodulin-dependent conformational change of adenylate cyclase seems therefore to be equally critical for binding of the nucleotide and catalysis.

Unlike other calmodulin-activated enzymes such as myo- sin light-chain kinase, erythrocyte Ca2+ pump and cyclic nucleotide phosphodiesterase, limited proteolysis generated only a ‘low-activity’ form of adenylate cyclase. Highly active forms of enzyme were obtained when the calmodulin-binding domain was reassociated with the catalytic domain in the presence of calmodulin itself. The fact that the two isolated domains when reassociated could recover the activity of the native protein is unique among calmodulin-activated enzymes and suggests that both T28 and T19 possess well defined, three-dimensional structures enabling them to recognize each other and their respective ligands. The higher sensitivity of isolated fragments to denaturation by urea compared to that of uncleaved adenylate cyclase suggests that interactive contacts between ordered portions of T28 and T19 in intact protein are essential not only for full catalytic activity, but also participate both in their own stabilization and in stabilization of the whole tertiary structure.

The affinity of T19 for calmodulin is significantly lower than that of the intact enzyme indicating that, indeed, T19 represents only part of the high-affinity calmodulin-binding domain. Although biochemical and genetic experiments have established unambiguously that the sequence situated around Trp242 in B. pertussis adenylate cyclase is involved in binding of calmodulin [lo, 11, 241, the exact extent of the calmodulin- binding sequence in the intact protein has not yet been firmly established. It is possible that this sequence spans the C- terminal end of T28 and the first 30 residues of T19. One might speculate that interactions with T28-calmodulin in the intact protein, though weak, are able to ‘reorient’ the calmodulin molecule once the latter is fixed to the corresponding T39 domain, and thereby reconstitute the high-affinity calmodulin-binding site. Therefore, it was of interest to know if upon reassociation of T28 and T19 into active species, the affinity of reconstituted protein for calmodulin increased. This was indeed the case, as shown by titration of dansyl- calmodulin with T19 in the absence and in the presence of T28.

A last point which we would like to discuss concerns the ‘absolute’ versus ‘relative’ requirement of B. pertussis adenylate cyclase for calmodulin. Previous work in different laboratories including ours has shown that the ‘basal’ activity (i. e. the absence of calmodulin) of crude or pure preparations of B. pertussis adenylate cyclase is variable and represents up to 5 % of the maximal activity at saturating concentrations of calmodulin. Moreover, upon storage at 4°C crude enzyme preparations lost the ability to be stimulated by calmodulin with concomitant increase of basal activity, a phenomenon

474

which was considered to be due to limited proteolysis by analogy with other calmodulin-regulated enzymes [33]. The results with recombinant adenylate cyclase, as well as with isolated tryptic fragments, do not support such an expla- nation, unless cleavage of B. pertussis adenylate cyclase by endogeneous proteases generates fragments with sizes and properties totally different from those obtained by trypsin digestion. The amphiphilic-mediated activation of B. pertussis adenylate cyclase, first described by Wolff and Hope Cook [34], most probably results from hydrophobic interactions between protein and lipids and mimicks to some extent the effect induced by calmodulin. This possibility is currently under investigation in our laboratory.

We are grateful to A. Ullmann, S. Michelson, B. Laoide and D. Ladant for fruitful discussion and helpful comments on the manu- script and to M. Ferrand for excellent secretarial help. This work was supported bq grants from the Centre Nutionul de In Recherche Srientifique (URA 1129), the Ministsre de la Recherche et de la Tech- nologie (grant 88T023), the Fondution pour la Recherche Midicule, and Pasteur Vuccins (DL/IEG-89/288). H.M. received a fellowship from the Fondution MPrieux.

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