Insight into the activation mechanism of Bordetella pertussis adenylate cyclase by calmodulin using fluorescence spectroscopy Jacques Gallay 1 , Michel Vincent 1 , Ine ` s M. Li de la Sierra 2, *, He ´ le ` ne Munier-Lehmann 2 , Madalena Renouard 1 , Hiroshi Sakamoto 2, †, Octavian Ba ˆ rzu 2 and Anne-Marie Gilles 2 1 Laboratoire pour l’Utilisation du Rayonnement Electromagne ´ tique, UMR 130 du CNRS, Universite ´ Paris-Sud, Orsay, France; 2 Laboratoire de Chimie Structurale des Macromole ´ cules, URA 2185 du CNRS, Institut Pasteur, Paris cedex, France The interaction of the adenylate cyclase catalytic domain (AC) of the Bordetella pertussis major exotoxin with its activator calmodulin (CaM) was studied by time-resolved fluorescence spectroscopy using three fluorescent groups located in different regions of AC: tryptophan residues (W69 and W242), a nucleotide analogue (3¢-anthraniloyl-2¢- deoxyadenosine 5¢-triphosphate, Ant-dATP) and a cysteine- specific probe (acrylodan). CaM binding elicited large changes in the dynamics of W242, which dominates the fluorescence emission of both AC and AC–CaM, similar to that observed for isolated CaM-binding sequences of dif- ferent lengths [Bouhss, A., Vincent, M., Munier, H., Gilles, A.M., Takahashi, M., Baˆ rzu, O., Danchin, A. & Gallay, J. (1996) Eur. J. Biochem. 237, 619–628]. In contrast, Ant- dATP remains completely immobile and inaccessible to the solvent in both the AC and AC–CaM nucleotide-binding sites. As AC contains no cysteine residue, a single-Cys mutant at position 75 was constructed which allowed labeling of the catalytic domain with acrylodan. Its environment is strongly apolar and rigid, and only slightly affected by CaM. The protein’s hydrodynamic properties were also studied by fluorescence anisotropy decay measurements. The average Brownian rotational correlation times of AC differed signi- ficantly according to the probe used (19 ns for W242, 25 ns for Ant-dATP, and 35 ns for acrylodan), suggesting an elongated protein shape (axial ratio of % 1.9). These values increased greatly with the addition of CaM (39 ns for W242, 60–70 ns for Ant-dATP and 56 ns for acrylodan). This suggests that (a) the orientation of the probes is altered with respect to the protein axes and (b) the protein becomes more elongated with an axial ratio of % 2.4. For comparison, the hydrodynamic properties of the anthrax AC exotoxin were computed by a mathematical approach ( HYDROPRO ), which usesthe3Dstructure[Drum,C.L.,Yan,S Z.,Bard,J., Shen, Y Q., Lu, D., Soelalman, S., Grabarek, Z., Bohm, A. & Tang, W J. (2002) Nature (London) 415, 396–402]. A change in axial ratio is also observed on CaM binding, but in the reverse direction from that for AC: from 1.7 to 1.3. The mechanisms of activation of the two proteins by CaM may therefore be different. Keywords: adenylate cyclase; Bordetella pertussis; calmodu- lin activation; fluorescent probe; hydrodynamic properties. cAMP is a key factor for the hormone-dependent control of important physiological functions such as sugar and lipid metabolism, cell differentiation, ion homoeostasis, and apoptosis. Some pathogenic agents have developed toxins, which interfere with this regulatory pathway by either altering the endogenous adenylate cyclase activity or injecting a protein capable of synthesizing cAMP in the target cell in such large quantities that it completely deregulates cell metabolism. This is the case for three pathogens: Bordetella pertussis, Bacillus anthracis and Pseudomonas aeruginosa. The major exotoxin of Bordetella pertussis, the causative agent of whooping cough [1,2], is a large bifunctional 1706-amino-acid protein called CyaA toxin. It harbors both adenylate cyclase (AC) and hemolytic activities. The toxin is responsible for the unregulated synthesis of cAMP [3] on activation by calmodulin (CaM) [4] present only in the target cells. The C-terminal part is responsible for the hemolytic phenotype of B. pertussis,and for the translocation of the catalytic domain into the target cells [5–7]. The N-terminal domain of about 400 amino acids contains the CaM-dependent AC [2]. This domain can be proteolytically split from the rest of the toxin without losing ATP-cyclizing activity [8–10]. Many molecular characteristics of the N-terminal cata- lytic domain have been investigated in great detail [11]. The AC domain of CyaA can be further cleaved by trypsin into two fragments or subdomains [12,13]. The N-terminal 224-amino-acid fragment possesses the catalytic site; the C-terminal fragment corresponds mainly to the CaM- binding subdomain. The catalytic activity of AC depends on Correspondence to J. Gallay, LURE baˆ timent 209D, PO Box 34, Universite ´ Paris-Sud, 91898 Orsay cedex, France. Fax/Tel.:+33164468082, E-mail: jacques.gallay@lure.u-psud.fr Abbreviations: AC, adenylate cyclase catalytic domain; CaM, cal- modulin; AC-Y75C, AC mutant in which Tyr75 is replaced by Cys; Ant-dATP, 3¢-anthraniloyl-2¢-deoxyadenosine 5¢-triphosphate; MEM, maximum entropy method. *Present address: CNRS FRC550, Institut de Biologie Physico- Chimique, 13 rue Pierre et Marie Curie, 75005 Paris, France. Present address: Laboratoire de Biologie et Ge ´ ne ´ tique du Paludisme, Institut Pasteur, 75724 Paris cedex 15, France. (Received 7 October 2003, revised 30 December 2003, accepted 9 January 2004) Eur. J. Biochem. 271, 821–833 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.03987.x three distinct segments common to that of the B. anthracis AC toxin [11] (Fig. 1). These segments include the P-loop, consisting of 24 amino acids (residues 54–77 in B. pertussis AC) and a stretch of 13 amino acids containing D188 and D190, essential for both catalysis and nucleotide binding [11,14]. These residues are situated in the catalytic sub- domain of the protein. The third segment, which is important for enzymatic activity, is located in the regulatory subdomain of the molecule and corresponds to the sequence comprising residues 294–314. The CaM-binding site of AC partially overlaps the N-terminal and C-terminal subdomains [13,15,16] (Fig. 1). A 72-amino-acid sequence located between amino acids 196 and 267 contributes 90% of the binding energy of CaM [11]. Further chemical or proteolytic cleavage of this fragment, solid-phase synthesis of peptides of various sizes, and site- directed mutagenesis experiments combined with spectro- scopic studies led to the conclusion that the amino-acid sequence around W242, which forms an amphiphilic helical structure, is the ÔcoreÕ of the CaM-binding site of the enzyme [9,16–21]. The mechanism of AC activation by CaM, and in particular the role of the conformational change resulting from CaM binding, remains obscure. The AC–CaM complex has a much higher affinity for the fluorescent ATP analog, 3¢-anthraniloyl-2¢-deoxyadenosine 5¢-triphos- phate (Ant-dATP), than the free enzyme [22], and therefore a CaM-induced conformational change in the nucleotide- binding site was postulated. The recent resolution of the 3D structure of the anthrax AC exotoxin suggests, however, that the activation by CaM is the result of a large conformational change involving a major portion of the protein [23]. In the absence of 3D data describing the interaction of CaM with AC, because both AC and AC–CaM have proved difficult to crystallize, we explored the effect of CaM on the conformation, internal dynamics, and hydrodynamic properties of AC from B. pertussis in more detail by fluorescence spectroscopy. For this purpose, we used highly purified recombinant proteins. We used three types of probes. Two Trp residues are present in the protein: W69 in the catalytic domain and W242 in the CaM-binding sequence. The fluorescence signal of W69 is low relative to that of W242 [20]. Therefore, the Trp fluorescence emission mainly provides information on the CaM-binding domain (CaM does not contain a Trp residue). The fluorescent nucleotide derivative Ant-dATP wasusedasreporterofthelocalstructureanddynamics of the nucleotide-binding site. The anthraniloyl probe, introduced initially to label the nucleotide-binding site of cyclic nucleotide phosphodiesterase [24], is a small fluoro- phore relative to the nucleotide moiety, which provides a strongly enhanced signal when bound to proteins rather than buffer [25–27]. Taking advantage of the absence of cysteine residues in the wild-type protein, we constructed a mutant with a single Cys residue at position 75 in the catalytic subdomain (Fig. 1). This mutation has little effect on the enzymatic activity, i.e. less than 10% decrease in specific activity. The K d for CaM of the modified protein (0.3 n M ) was also not significantly different from that of thewild-typeenzyme(0.2n M ). We then used acrylodan, a probe sensitive to polarity [28,29], to label Cys75, to provide additional information on the conformation and dynamics of the catalytic subdomain of the protein. Equilibrium ultracentrifugation was also used to measure the molecular mass of both proteins. The results are discussed with respect to the structure of anthrax exotoxin and its changes on CaM binding [23]. Materials and methods Chemicals Ant-dATP was synthesized as described previously [22]. All the other chemicals were of the highest grade commercially available. Bacterial strains, plasmids and growth conditions A DNA fragment encoding the first 385 amino acids of B. pertussis AC was PCR-amplified, using oligonucleotides 5¢-GGGGCATATGCAGCAATCGCATCAGGCTGG TTA and RC3¢-CCCCAAGCTTCACGCCGGCACCGT TTCCAGTACATC. Genomic DNA from strain 18323 was used as the template. This PCR fragment was cloned into the expression vector pET24a (Novagen), between restriction sites NdeIandHindIII, resulting in plasmid pHSP247. To generate the mutant AC-Y75C, the template used was pDIA5311 (obtained by site-directed mutagenesis as described by Glaser et al. [14]; a gift from E. Krin, Pasteur Institute, Paris). The PCR fragment was cloned into pET24a as for the wild-type, resulting in plasmid pHL12-2. For the production of recombinant proteins, strain BL21(DE3)/pDIA17/pHSP247 or BL21(DE3)/pDIA17/ pHL12-2 was grown in a fermentor at 37 °Cin2YT medium containing kanamycin (100 lgÆmL )1 )andchlo- ramphenicol (30 lgÆmL )1 ), until D 600 reached a value of % 8. Addition of isopropyl b- D -thiogalactoside (1 m M final concentration) induced overproduction. Bacteria were har- vested by centrifugation after a further incubation of 3 h. Purification of the recombinant proteins AC purification. Bacteria were suspended in 50 m M Tris/ HCl, pH 8.0, and disrupted by sonication. After centri- fugation at 10 000 g for 30 min, the supernatant was discarded. The pellet was then washed three times in the same buffer and suspended in 8 M urea/50 m M Tris/HCl, pH 8.0. After centrifugation, the recovered supernatant was Fig. 1. AC domain of B. pertussis CyaA toxin. R224 is the site of trypsin cleavage of the protein in the two subdomains. I, II and IV correspond to those segments possessing high sequence identity with B. anthracis AC. These segments harbor amino-acid residues (K58, K65 or H63 in I, D188 and D190 in II and H298 and E301 in IV) critical for ATP cyclization. III corresponds to the segment responsible for tight binding of CaM to AC. In italics (Y75, W69 and W242) are indicated those residues used as fluorescent probes. Y75 was muta- genized to Y75C and labeled with acrylodan. 822 J. Gallay et al.(Eur. J. Biochem. 271) Ó FEBS 2004 diluted 10-fold with 50 m M Tris/HCl, pH 8.0, and loaded on to a DEAE-Sephacel column equilibrated at 4 °Cwith 50 m M Tris/HCl, pH 8.0. AC was eluted with 0.1 M NaCl in the same buffer. The purification by hydrophobic chromatography increased the specific activity of AC eluted with 0.1 M NaCl by a factor of 1.3 (from 138 UÆmg )1 to 185 UÆmg )1 ) and the percentage of CaM activation by a factor of 1.6. It produces a pure protein, as revealed by SDS/PAGE and gel filtration on a Shodex KW802.5, which shows a single elution peak. Fractions containing the enzyme were pooled and concentrated before being loaded on to a Sephacryl S-300 HR column equilibrated with 50 m M Tris/HCl, pH 8.0. CaM purification. Mouse brain CaM was cloned and expressed in Escherichia coli as described [30]. The protein was recovered in the supernatant after bacterial disruption by sonication. It was then kept in 50 m M Tris/HCl, pH 7.4, for further purification. The protein was purified by hydrophobic chromatography as follows: 146 mg unpuri- fied CaM in 50 m M Tris/HCl (pH 7.4)/5 m M CaCl 2 was loaded on to a Phenyl-Sepharose column equilibrated at room temperature with 50 m M Tris/HCl, pH 7.4, contain- ing 0.5 m M CaCl 2 and 1 m M dithiothreitol. The column was washed first with the equilibration buffer, secondly with 0.5 M NaCl in the equilibration buffer, and thirdly with 50 m M Tris/HCl, pH 7.4, containing 0.1 m M CaCl 2 and 1m M dithiothreitol. Finally, the CaM protein was eluted in 50 m M Tris/HCl, pH 7.4, containing 1 m M dithiothreitol and 1 m M EGTA. The CaM solution was dialysed against 50 m M Tris/HCl, pH 7.4. AC–CaM complex purification. For the formation of the AC–CaM complex, the urea extract containing AC was diluted eightfold (1 M urea final concentration) then incubated in % 1:1molarratiowithCaMfor2hin 50 m M Tris/HCl, pH 8.0. The AC–CaM mixture was loaded on to a DEAE-Sephacel column equilibrated with 50 m M Tris/HCl, pH 8.0 at 4 °C. The column was washed extensively (10 volumes) with 50 m M Tris/HCl, pH 8.0, and then with 0.1 M NaClinthesamebuffer.TheAC–CaM complex was eluted with 0.2 M NaCl in 50 m M Tris/HCl, pH 8.0. The AC–CaM complex obtained in this first step was concentrated and loaded in a second step on to a Sephacryl S-300 HR column equilibrated at 4 °Cwith 50 m M Tris/HCl, pH 8.0. Assay of AC Enzyme activity was monitored by ATP formation (reverse reaction), at 334 nm and 30 °C in 0.5 mL final volume in an Eppendorff photometer equipped with a temperature- controlled system. The reaction mixture contained 50 m M Tris/HCl, pH 7.4, 20 m M KCl, 1 m M glucose, 0.4 m M NADP, 5 m M cAMP, 6 m M MgCl 2 ,4m M sodium pyro- phosphate, 1 l M calmodulin, and 3 U each of hexokinase and glucose-6-phosphate dehydrogenase. The reaction was started with the AC sample. One unit of enzyme activity corresponds to 1 lmol product formed per min. The enzyme of highest purity exhibited % 500 UÆ(mg protein) )1 . This value is sixfold lower than that obtained in the forward reaction. Synthesis of the acrylodan conjugate of the AC-Y75C mutant A1mgÆmL )1 acrylodan stock solution (4.4 m M )indimeth- ylformamide was used for labeling. The AC-Y75C mutant (0.8 mgÆmL )1 ) was dialyzed twice against 500 mL 50 m M Hepes buffer, pH 7 for 2 h. It was then labeled by adding 4 lL of the acrylodan stock solution (35 l M final concen- tration) to 500 lL of the protein solution at a concentration of 25 l M . Incubation was performed in ice for 2 h. Free label was removed by gel filtration on a Shodex KW802.5 column equilibrated in 50 m M Hepes buffer, pH 7. A bound probe/protein molar ratio of % 1 was estimated using molar absorption coefficients for acrylodan of 16400 and 6200 M )1 Æcm )1 at 385 and 290 nm, respectively [28]. Steady-state and time-resolved fluorescence measurements Steady-state fluorescence emission spectra and anisotropy were recorded on an SLM 8000 spectrofluorimeter. Fluor- escence intensity and anisotropy decays were obtained by the time-correlated single-photon counting technique from the polarized components I vv (t) and I vh (t) on the experi- mental set-up installed on the SB 1 window of the synchro- tron radiation machine Super-ACO (Anneau de Collision d’Orsay) [31]. The excitation wavelength was selected by a double monochromator (Jobin Yvon UV-DH10, band- width 4 nm). A MCP-PMT Hamamatsu (model R3809U- 02) was used. Time resolution was % 20 ps, and the data were stored in 2048 channels. Automatic sampling cycles including 30 s accumulation time for the instrument response function and 90 s acquisition time for each polarized component were carried out so that a total of (2–4) · 10 6 counts was reached in the fluorescence intensity decay. Analyses of fluorescence intensity decay, I(t) recon- structed from the parallel I vv (t) and perpendicular I vh (t) polarized components, as sums of exponentials were performed by the maximum entropy method (MEM) as described in detail in previous publications [32–34]. The 1D model of anisotropy, in which each lifetime s i is coupled to any rotational correlation time h i , performed analyses of the polarized fluorescence decays. A 2D analysis, essential for describing the coupling between lifetimes and rotational correlation times, was also used. For fluorescence intensity and anisotropy decay analysis (with the 1D model), computations were performed on a DEC Vax station 4000/90. The 2D analyses were carried out on a DEC alpha computer Vax7620 with a set of 1600 independent variables (40 s and 40 h equally spaced in log scale). The programs including the MEMSYS 5 subroutines (MEDC Ltd, Cambridge, UK) were written in double precision FORTRAN 77. Other analytical procedures Protein concentration was determined as described by Bradford [35]. SDS/PAGE was performed as described by Laemmli [36], and native electrophoresis by the method of Bollag & Edelstein [37]. Gels were stained with Coomassie blue. Equilibrium sedimentation experiments were per- formed at 20 °C on a Beckmann XLA ultracentrifuge using Ó FEBS 2004 CaM-induced conformational transition in AC (Eur. J. Biochem. 271) 823 a double sector cell rotor AN60 equipped with a 12-mm opticalpathcell.Proteinsamplesin50m M Tris/HCl, pH 8, were centrifuged at 17 000 r.p.m. Radial scans of A 290 were taken at 2-h intervals. Equilibrium was achieved after 20 h centrifugation. Results Biochemical characterization of the AC, CaM and AC–CaM proteins Recombinant AC was isolated from inclusion bodies. Our aim was to obtain pure and homogeneous enzyme prepa- rations from the standpoint of activity. The purification procedure we described is based on the rapid re-activation of AC after denaturation by urea, and on the fact that AC and CaM differ largely in their isoelectric points, allowing them to be separated either in free form or in a complex, by ion-exchange chromatography. Thus, AC was purified as a free enzyme by elution in buffers close to neutrality and 0.1 M NaCl. CaM, which is more acidic, required 0.3 M NaCl to be eluted. The AC–CaM complex was isolated from the free proteins at 0.2 M NaCl. As the K d of the AC–CaM complex is 0.2 n M , dissociation of the complex under our experimental concentration conditions is insigni- ficant. This is shown in Fig. 2 in which AC, CaM and the AC–CaM complex were analyzed by gel electrophoresis under native or denaturing conditions. In each case, single bands of proteins were obtained, indicating purity and homogeneity of each molecular species. On the other hand, analytical ultracentrifugation studies indicated a homogen- eous AC–CaM complex with 1 : 1 stoichiometry (molecular mass of 53.7 kDa) and homogeneous uncomplexed AC (41.6 kDa). Dynamics of the AC nucleotide-binding site as probed by Ant-dATP To explore the dynamics of the catalytic domain of the protein, we used the fluorescent nucleotide Ant-dATP, a strong competitive inhibitor of AC activity [22]. Binding of Ant-dATP to the AC–CaM complex led to a large increase in the steady-state fluorescence intensity and a blue shift of the emission spectrum [22]. Time-resolved fluorescence of this kind of probe has proven useful for separating the free and bound nucleotides as they have very different lifetimes. This allows their dynamics and accessibility to the solvent in the nucleotide-binding site to be studied [27]. The time-resolved fluorescence intensity decay of Ant- dATP in solution was strongly modified in the presence of either AC or AC–CaM complex (Fig. 3). In the presence of AC (Fig. 4B) or AC–CaM complex (Fig. 4C), a population with a long lifetime (% 10 ns) appeared in the fluorescence decay curve, which was absent in the decay of the free probe (Fig. 4A), similar to that observed for Ant-dADP binding to CMP kinase from E. coli [27]. This long lifetime remained unchanged on CaM binding: only its amplitude increased as a result of the increased affinity of the complex for the ligand [22]. From this relative amplitude, the binding degree of Ant-dATP can be calculated. The K d values obtained in this way for AC and AC–CaM were 52 and 11 l M , respectively (Fig. 5). The last value is close to that measured by equilibrium dialysis (K d ¼ 6.8 l M ) or from kinetic measurements (K i ¼ 9 l M ) [22]. The K d value obtained for AC is, however, smaller than previously reported [22]. This is probably due to greater accuracy of the present measurements (Fig. 5) than in the previous studies [22]. Quenching by water-soluble molecules permits assess- ment of the accessibility of the bound nucleotide to the aqueous solvent. Time-resolved acrylamide quenching measurements gave linear Stern–Volmer plots (Fig. 6), allowing determination of the bimolecular quenching con- stants (k q ) related to the accessibility of the fluorophore to the water-soluble quencher acrylamide (Table 1). The 10-ns lifetime assigned to Ant-dATP bound to either AC or AC–CaM is associated in both cases with a % 20 times lower k q value than that associated with the 2-ns lifetime (Table 1). The k q value for the latter is in turn similar to that for Ant-dATP in buffer, which is the value used as a Fig. 2. Gel electrophoresis under denaturing (A) (12.5% acrylamide) or native (B) (10% acrylamide) conditions of AC, CaM and AC–CaM complex separated by ion-exchange chromatography. Lane 1, AC (3 lg) eluted with 0.1 M NaCl;lane2,purifiedAC–CaMcomplex(3lg) eluted with 0.2 M NaCl; lane 3, CaM (3 lg) eluted with 0.3 M NaCl. Standard proteins: (a) phosphorylase a (94 000 Da); (b) BSA (66 200 Da); (c) ovalbumin (43 000 Da); (d) carbonic anhydrase (30 000 Da); (e) soybean trypsin inhibitor (21 000 Da); (f) lysozyme (14 000 Da). Fig. 3. Fluorescence intensity decay of Ant-dATP. (A) Instrumental response function; (B) Ant-dATP in water; (C) Ant-dATP with AC; (D) Ant-dATP with AC–CaM. 824 J. Gallay et al.(Eur. J. Biochem. 271) Ó FEBS 2004 reference for the fully solvent-accessible nucleotide (Table 1). This 2-ns lifetime present in the fluorescence decays of both AC and AC–CaM (in much smaller proportion in the latter case) is therefore probably due to free Ant-dATP in equilibrium with the bound nucleotide. The resulting k q ratio corresponds to a relative accessibility of the fluorescent moiety to the water-soluble quencher of less than 5% when the nucleotide is bound to either AC or AC–CaM [38]. We further explored the dynamics of the nucleotide bound to AC and AC–CaM by fluorescence anisotropy decay measurements. The fluorescence anisotropy decay of Ant-dATP in buffer declined smoothly and rapidly (Fig. 7A, curve 1). MEM analysis of the polarized decays using the 1D model of the anisotropy showed only one rotational correlation time of 0.27 ns (Fig. 7B), close to the value previously observed for Ant-dADP [27] correspond- ing to the Brownian rotation of the fluorescent nucleotide in solution. The initial anisotropy value, A t ¼ 0 , is however, 0.2, lower than the intrinsic anisotropy A 0 of 0.34 measured for the immobilized Ant-dATP (or Ant-dADP) in vitrified medium [27]. This suggests that faster motions are likely. In contrast, the fluorescence anisotropy decay of Ant-dATP in the presence of either AC or AC–CaM did not follow a smooth pattern. It began with a fast decrease (starting from an initial anisotropy A t ¼ 0 of 0.25–0.3) followed by a transient increase and a further slow decrease (Fig. 7A, curve 2). This type of switchback behavior, due to the additive rule of the anisotropy, is characteristic of systems presenting fluorescence heterogeneity with specific coupling Fig. 5. Binding of Ant-dATP to AC (d) and AC–CaM (m). The degree of binding of the ligand was calculated from the value of the ampli- tude of the long lifetime, which characterizes the bound fluorescent nucleotide. Fig. 6. Stern–Volmer plots of time-resolved acrylamide quenching of Ant-dATP. (d) 2 ns lifetime of Ant-dATP (2 l M )inwater;(s)2ns lifetime of Ant-dATP (2 l M ) in the presence of AC (50 l M )and AC–CaM (20 l M ); (j)10nslifetimeofAnt-dATP,(h) 10 ns lifetime of Ant-dATP (2 l M ) in the presence AC–CaM (20 l M ). Fig. 4. MEM-reconstructed excited-state lifetime distributions of Ant- dATP. The analyses were performed on the total fluorescence intensity S(t), reconstructed from the parallel and perpendicular polarized decay components I vv (t) and I vh (t) such as SðtÞ¼I vv ðtÞþ 2b corr I vh ðtÞ¼ R 1 0 aðsÞexpðÀt=sÞdt; where s is the excited-state life- time, a(s) its amplitude and b corr is a correction factor accounting for the difference in transmission of the I vv (t) and I vh (t) components by the monochromator [60]. Sets of 150 independent variables, equally spaced in log scale, were used for the analyses. The s i and a i values given in the legend are the center and relative area of each lifetime peak, respectively. (A) 4 l M Ant-dATP in 50 m M Tris/HCl buffer, pH 8, s 1 ¼ 0.14 ns, s 2 ¼ 2.1 ns, a 1 ¼ 0.21, a 2 ¼ 0.79. Excitation wave- length, 330 nm; emission wavelength, 430 nm. (B) 4 l M Ant-dATP in the presence of 26.3 l M AC, s 1 ¼ 0.39 ns, s 2 ¼ 2.3 ns, s 3 ¼ 10.5 ns, a 1 ¼ 0.12, a 2 ¼ 0.67; a 3 ¼ 0.21. Excitation wavelength, 340 nm; emission wavelength, 420 nm. (C) 5 l M Ant-dATP in the presence of 20 l M AC–CaM, s 1 ¼ 0.38 ns, s 2 ¼ 2.0 ns, s 3 ¼ 11 ns, a 1 ¼ 0.05, a 2 ¼ 0.58; a 3 ¼ 0.38. Excitation wavelength, 340 nm; emission wavelength, 420 nm. Ó FEBS 2004 CaM-induced conformational transition in AC (Eur. J. Biochem. 271) 825 between short lifetimes and short correlation times and conversely between long lifetimes and long correlation times [39]. The anisotropy at short times will thus decline rapidly, as the rotational motion of the short-lived emitter is fast. In contrast, the anisotropy at long times will decline more slowly, as the rotational motion of the long-lived emitter is slow. The anisotropy decay pattern at inter- mediate time will result from a combination of fast and slow decays of the free and bound probe, respectively. This type of behavior was originally observed in membranes [40], proteins [41,42] and nucleic acids [33]. The MEM analysis of the polarized fluorescence decays using the classical 1D model of anisotropy, which associates all lifetimes with all the correlation times, was unable to account for the fast initial decay as shown in Fig. 7C for AC–CaM. Only one long rotational correlation time (h > 100 ns) was obtained in this case. Visual inspection of the deviation function clearly showed that analysis at short times is not correct (Fig. 7C, insert). The two main lifetimes of Ant-dATP (2 and 10 ns) are therefore likely to be associated with different rotational dynamics. MEM allows analysis without apriorihypothesis on the association degree between lifetimes (s) and corre- lation times (h) [33]. In the present case, MEM analysis shows, as a result of the fit, a single association between the 2 ns excited-state population and a fast rotating component (% 200 ps). This is shown on the 2D (s, h) plots (Fig. 8A,B); the value is similar to that of the unbound nucleotide (Fig. 7B). Conversely, we show a single association of the long-lived excited-state population with a long rotational correlation time, which probably describes the Brownian rotational motion of the protein. The Brownian rotational correlation times increased greatly from % 25 ns for AC (Fig. 8A) to 60–70 ns for AC–CaM (Fig. 8B). No other cross-correlation peaks in the 200 ps time range or shorter were observed for this lifetime population for either AC or AC–CaM. This indicates that the bound Ant- dATP is immobile in the 100–200 ps time scale. Supporting this hypothesis, the initial anisotropy at t ¼ 0 when the probe is in the presence of AC or AC–CaM is 0.25–0.3 (Fig. 7A curve 2). This value is higher than that observed for free Ant-dATP and closer to 0.34, the intrinsic A 0 value measured for the immobile probe in vitrified medium [27]. Taking into account the additive rule of the anisotropies, we calculated the initial anisotropy value for the bound probe, knowing its partial intensity, as A t ¼ 0 % 0.31–0.34. There- fore, bound Ant-dATP may be subjected to motions of small amplitude occurring in a faster time scale than 200 ps, but they are not detectable in the 2D analyses even though we imposed an A 0 value of 0.34. These results confirm that the 2 ns lifetime corresponds to free Ant-dATP, which is fully accessible to and moving rapidly in the solvent, whereas the 10 ns lifetime character- izes the bound nucleotide, which is weakly accessible to the solvent and immobilized in its binding site in both the uncomplexed AC and the AC–CaM complex. Dynamics of the AC catalytic domain as probed by acrylodan Taking advantage of the absence of Cys residues in the native AC protein, the insertion of a Cys in a defined position allowed specific and unique labeling so that information on the conformational changes induced by CaM in different domains of the protein could be obtained. Table 1. Time-resolved dynamic acrylamide quenching constants for Ant-dATP in solution and in the presence of AC or AC–CaM. The bimolecular quenching constant k q was calculated as k q ¼ K sv /s. Standard deviations for 3 (Ant-dATP), 10 (Ant-dATP/AC–CaM) and 17 (Ant-dATP/AC) measurements are given. Sample s (ns) K sv ( M )1 ) k q · 10 9 ( M )1 Æs )1 ) Ant-dATP in buffer 2.07 ± 0.02 3.74 1.81 ± 0.02 Ant-dATP/AC 10.37 ± 0.39 1.09 0.11 ± 0.01 2.22 ± 0.04 4.14 1.86 ± 0.04 Ant-dATP/AC–CaM 10.75 ± 0.19 1.04 0.10 ± 0.01 2.02 ± 0.05 3.78 1.87 ± 0.05 Fig. 7. Fluorescence anisotropy decay of Ant-dATP in buffer and bound to AC. (A) Experimental fluorescence anisotropy decay AðtÞ¼ I vv ðtÞÀb corr I vh ðtÞ I vv ðtÞþ2b corr I vh ðtÞ of Ant-dATP in buffer (curve 1) and in the presence of AC–CaM (curve 2). (B) Rotational correlation time dis- tribution of Ant-dATP in buffer (insert, deviation function). (C) Rotational correlation time distribution of 1 l M Ant-dATP in the presence of 20 l M AC–CaM; v 2 ¼ 1.349 (insert, deviation function). 826 J. Gallay et al.(Eur. J. Biochem. 271) Ó FEBS 2004 Cys was inserted at position 75 (replacing the native Tyr residue) to label the catalytic domain, as W69 is almost silent. The mutation did not result in any alteration in enzymatic activity. Acrylodan was chosen because it is specific for SH groups and is extremely sensitive to polarity changes [28,29]. The maximum of the steady-state fluorescence emission spectrum of acrylodan conjugated to AC-Y75C was at 448 nm (Fig. 9A), revealing a local environment of very low polarity. In comparison, the maximum of the emission spectrum of the probe is at 462 nm in the aprotic solvent dimethylformamide, 485 nm in isobutanol, 490 nm in ethanol, 504 nm in glycerol, and 540 nm in water [28]. Binding of CaM led to a red shift of the emission spectrum to 466 nm (Fig. 9A), indicating that the local polarity and accessibility to the solvent is slightly increased in the AC- Y75C–CaM complex. It remains, however, of the order of that found in aprotic solvents. The fluorescence intensity Fig. 8. MEM-reconstructed G(s, h) distributions of Ant-dATP. (A) 4 l M Ant-dATP in the presence of 56 l M AC; v 2 ¼ 1.018; (B) 1 l M Ant-dATP in the presence of 20 l M AC–CaM; v 2 ¼ 1.006. The fit was performed on the polarized fluorescence intensity decays I vv (t) and I vh (t) using their classical expressions: I vv ðtÞ¼ 1 3 R 1 0 R 1 0 Cðs; hÞe Àt=s ð1 þ 2A 0 e Àt=h Þdsdh and I vh ðtÞ¼ 1 3 R 1 0 R 1 0 Cðs; hÞ Â e Àt=s ð1 À A 0 e Àt=h Þdsdh. G(s, h) is the relative proportion of emitter with lifetime s and correlation time h, A 0 is the intrinsic anisotropy. This analysis starts with an initial model of the G(s, h) distribution as a ÔflatÕ mapwhereallthe(s, h) are equiprobable. A value of the intrinsic anisotropy of 0.34 was used [27]. Fig. 9. Fluorescence characteristics of acrylodan bound to AC-Y75C mutant. (A) Fluorescence emission spectra of acrylodan bound to AC- Y75C (––) and AC-Y75C–CaM (- - -). (B) MEM-reconstructed exci- ted-state lifetime distributions of acrylodan bound to AC-Y75C (––) and to AC-Y75C–CaM (- - -) measured at the maximum of the fluorescence emission spectrum. (C) MEM-reconstructed rotational correlation time distribution of acrylodan bound to AC-Y75C (––), and to AC-Y75C–CaM (- - -). –– (AC): b 1 ¼ 0.050, b 2 ¼ 0.318, h 1 ¼ 9ns, h 2 ¼ 36 ns, A t ¼ 0 ¼ 0.368. - - - (AC–CaM): b 1 ¼ 0.030, b 2 ¼ 0.329, h 1 ¼ 8ns,h 2 ¼ 56 ns, A t ¼ 0 ¼ 0.359. Excitation wave- length, 400 nm; emission wavelength, 460 nm. Ó FEBS 2004 CaM-induced conformational transition in AC (Eur. J. Biochem. 271) 827 decayofacrylodanmeasuredinbothAC-Y75CandAC- Y75C–CaM did not show a large change: a major lifetime population, centered at 4.1 ns for the former and at 3.9 ns for the latter, characterized the decay, which accounted for 92% of the fluorescence intensity in both cases (Fig. 9B). The local mobility was weak and remained unaffected by CaM binding as shown by fluorescence anisotropy decay measurements. In both cases the decays show two rotational correlation times in the nanosecond range (Fig. 9C). The shortest is probably due to the existence of a slow local flexibility of weak amplitude. The initial anisotropy values A t ¼ 0 were close to that measured in vitrified medium (A 0 ¼ 0.370), therefore no subnanosecond motion of signi- ficant amplitude was present. The longest correlation time describing the average Brownian rotation of the protein displayed a large increase, however, from 36 ns for AC- Y75C to 56 ns for AC-Y75C–CaM. Dynamics of the CaM-binding domain probed by W242 W242 dominates the intrinsic fluorescence emission of the protein [20]. It is situated in the middle of the 72-amino-acid segment responsible for 90% of the AC–CaM binding energy (P196–267) and therefore provides information mainly on the dynamics of this region of the protein. This residue is probably widely accessible to the solvent in AC as shown by the maximum wavelength of the fluorescence emission spectrum of 350 nm as previously reported [18]. The fluorescence intensity decay was multiexponential as usually found in proteins, with four lifetime populations describing the decay (Table 2). Such large fluorescence heterogeneity is probably due to fast local dynamics and flexibility detected by fluorescence anisotropy measurements (Table 3). Two rotational correlation times were observed in the nanosecond range, the shorter probably describing a local flexibility, the longer related to the Brownian rotation of the protein. The initial anisotropy value (A t ¼ 0 )was, however, significantly lower than the A 0 value measured in vitrified medium [43]. Therefore faster motions are probably present, leading to a substantial wobbling-in-cone angle x max (Table 3). We noticed that the dynamics of W242 in the protein is almost as large as that of W242 in the isolated peptide sequence P196–267 [16]. This strongly suggests that this part of the protein is highly dynamic, showing nanosecond flexibility. CaM binding reduces the dynamics of this region greatly. Two rotational correlation times in the nanosecond range were observed in the AC–CaM complex (Table 3). Some subnanosecond motions still remain possible, but they are very weak in amplitude as the initial anisotropy (A t ¼ 0 ) value was almost equal to that expected for an immobile Trp [43]. The wobbling-in-cone angle x max is therefore greatly reduced (Table 3). This observation is similar to that of the isolated peptide complexed with CaM [16]. In addition to the fast motions, a long rotational correlation time, which probably describes an average Brownian Table 2. Fluorescence intensity decay parameters of the Trp emission of AC and AC–CaM complex recovered by MEM. Excitation wavelength, 295 nm; emission wavelengths, 350 nm for AC and 335 nm for AC–CaM. Standard deviations for three measurements are given. MEM analysis was performed on the fluorescence intensity S(t) as described in the legend of Fig. 3. Sample s 1 (ns) a a b 1 I 1 c s 2 (ns) a 2 I 2 s 3 (ns) a 3 I 3 s 4 (ns) a 4 I 4 <s> (ns) d AC 0.34 ± 0.07 0.88 ± 0.01 3.24 ± 0.34 6.50 ± 0.59 1.73 ± 0.12 0.21 ± 0.03 0.56 ± 0.02 0.10 ± 0.03 0.13 ± 0.03 0.04 0.28 0.19 0.49 AC–CaM 0.49 ± 0.03 1.12 ± 0.10 4.18 ± 0.17 – 1.65 ± 0.02 0.27 ± 0.03 0.50 ± 0.01 0.23 ± 0.02 – 0.08 0.34 0.58 – a s i and b a i are, respectively, the values of the center and the normalized amplitude of each lifetime peak. c I i are the values of the partial intensity of the i th component I i ¼ a i s i /<s>. d The mean lifetime <s> is calculated as: hsi¼ P i a i s i . Table 3. Fluorescence anisotropy decay parameters of the Trp emission of AC and AC–CaM complex obtained by MEM analysis of the fluorescence polarized decays, using a 1D model of the anisotropy where all lifetimes s are coupled to all correlation times h. The fluorescence anisotropy decay is described in this model by a sum of exponential terms: AðtÞ¼ I vv ðtÞÀb corr I vh ðtÞ I vv ðtÞþ2b corr I vh ðtÞ ¼ R 1 0 bðhÞexpðÀt=hÞd h,withA 0 ¼ R 1 0 b(h)d(h). b(h) is the anisotropy associated with the rotational correlation time h, b corr is the correction factor defined in the legend of Fig. 3. The fit was simultaneously performed on the vertically I vv ðtÞ¼1=3 R 1 0 aðsÞe Àt=s ds½1 þ 2A 0 R 1 0 b(h)d(h)] and on the horizontally I vh ðtÞ¼1=3 R 1 0 aðsÞe Àt=s ds½1 À A 0 R 1 0 b(h)d(h)] emitted fluorescence decays, s is the excited state lifetime and a(s) its amplitude. The a(s) profile is obtained from a first analysis of I(t) by MEM and is held constant in a subsequent and global analysis of I vv (t) and I vh (t) which provides the distribution b(h) of correlation times [34]. Sets of 100 independent variables, equally spaced in log scale, were used for the analyses. The semiangle of the wobbling-in-cone motion was calculated as: b 2 A 0 ¼½1=2cosx max ð1 þ cosx max Þ 2 [59] with an intrinsic anisotropy value A 0 of 0.197 [43]. Experimental conditions as in Table 2. Sample h 1 (ns) h 2 (ns) b 1 b 2 A 0 x max (°) AC 2.3 ± 0.5 19 ± 5 0.094 ± 0.010 0.077 ± 0.013 0.171 ± 0.010 44 AC–CaM 2.5 ± 0.9 39 ± 2 0.035 ± 0.013 0.157 ± 0.027 0.192 ± 0.017 22 828 J. Gallay et al.(Eur. J. Biochem. 271) Ó FEBS 2004 rotation, is present in the decays of both AC and AC–CaM (Table 3). Its value was greatly increased in the AC–CaM complex relative to AC. Hydrodynamic properties of the AC and of the AC–CaM complex Hydrodynamic properties of proteins can be studied using fluorescence anisotropy decays [44]. The fluorescence anisotropy decay data for all the three probes used in this work showed the existence of a single long correlation time (‡ 20 ns), either for AC or AC–CaM, which probably describes average Brownian rotation. This long rotational correlation time differs, however, according to the fluoro- phore used. For AC, the values ranged from 19 ns for W242 (Table 3) to 25 ns for Ant-dATP (Fig. 8A) and up to 35 ns for acrylodan (Fig. 9C). This observation strongly suggests that AC is not spherical. For the AC–CaM complex, the values ranged from 39 ns for W242, 56 ns for acrylodan, and 60–70 ns for Ant-dATP (Table 3, Figs 9C and 8B), suggesting that the shape of the AC–CaM complex also diverges significantly from that of a sphere. These average Brownian rotational correlation times for AC–CaM were significantly larger than that for AC, suggesting that the former is more elongated than the latter. This will be discussed in greater detail in the discussion. Discussion Several mechanisms of CaM-mediated activation of differ- ent biological systems have been proposed. The molecular characteristics of the AC protein, however, are difficult to reconcile with any of them. Neither the pseudo-substrate mechanism, which involves an auto-inhibitory sequence [45], nor the flip-flop mechanism, which involves the existence of a CaM-like binding site in addition to the true CaM-binding site [46], can be applied to AC. The mosaic distribution of its different functional modules (Fig. 1), i.e. the ATP-binding site, the CaM-binding sequence, the residues of the catalytic site, favors a new activation mechanism. Recently, the resolution of the 3D structure of the AC exotoxin of B. anthracis andofitscomplexwithCaMledtotheproposal of a different CaM-dependent regulatory mechanism, in which two large protein segments, situated between the catalytic and the regulatory domains, undergo a large-scale conformational transition [23]. This system is closely related to that of B. pertussis, although the sequence alignment does not show much similarity, especially in the CaM-binding domain. Most of the crucial amino-acid residues responsible for catalysis and ATP binding are, however, conserved in both proteins [23]. With respect to the 3D structure of the B. anthracis exotoxin, all of the residues of the catalytic mechanism are present in the neighborhood of the catalytic site, but they are not ordered correctly in the exotoxin alone. CaM binding folds parts of the regulatory domain in such a way that these crucial amino acids are put together to build the binding/active site of the active protein. The purpose of this study was to attempt to observe such large conformational changes in AC, which might explain its activation by CaM. In the absence of any 3D structure of either AC or AC–CaM, we have used fluorescence spectro- scopy with several specific labels distributed in different regions of the protein, to explore the structural and dynamic perturbations induced by CaM binding to AC. Biochemical studies of AC led to the proposal of a model in which it exists as an ÔopenÕ inactive structure in the absence of CaM and as a ÔclosedÕ active structure in its presence. In the inactive structure, the active site would not be completely shaped. An observation supporting this model was the large increase in the affinity of the AC–CaM complex for Ant-dATP compared with AC alone (this work and [22]), a property shared by the B. anthracis exotoxin [22,47,48]. Another observation in support of this model is the fact that the catalytic and regulatory domains could be split by trypsin cleavage at Arg224 in the absence of CaM, and re-associated by CaM [13], leading to a fully active protein, a property not shared by the B. anthracis exotoxin. This suggests that the two AC domains (catalytic and CaM-binding domains) are linked by a flexible amino- acid sequence. Activation would occur by folding of this sequence, which in turn would bring together in the correct 3D arrangement the important amino acids for catalysis, which are located along the AC sequence in the catalytic subdomain, in the central region and in the C-terminal segment, thereby forming the active site. Several structural and dynamic consequences can be suggested in the frame of this model of activation that can be tested by time-resolved fluorescence studies. If the ATP- binding site were not shaped in the uncomplexed AC, the mobility of Ant-dATP and its accessibility to the solvent would probably be higher in AC than in AC–CaM complex. Time-resolved measurements have proven very useful in this respect. Beside the fact that they explained the increase in Ant-dATP fluorescence intensity by a factor of 4 on binding to AC–CaM [22], they allowed the signals of the free and bound probe to be separated, which could not be easily done in steady-state measurements because the shift in their emission spectra is not large. Therefore, they allowed their accessibility to the solvent and their respective rotation motions to be measured separately. CaM binding would probably make the flexible amino- acid sequence between the catalytic and CaM-binding domains rigid, an effect that can be checked by studying the dynamics of W242 by fluorescence anisotropy decay measurements. It may also change the overall shape of the protein: a more compact complex would be obtained, with observable consequences on the Brownian rotation motion, which can be measured by fluorescence anisotropy decay. With respect to the mobility and solvent accessibility of Ant-dATP bound to the active site of the protein, it is remarkable that it remained immobile and shielded from the solvent in both AC and the AC–CaM complex. The fluorescence excited-state lifetime of the anthraniloyl ring is extremely sensitive to changes in its environment, partic- ularly to the presence of water [24,25,27,49] and from the absence of changes in this variable, we can conclude that the local interactions of the fluorescent inhibitor with its environment in the binding site remain undisturbed in AC–CaM compared with AC. One very obvious difference between AC and AC–CaM is the increased ratio of bound/ free Ant-dATP in the latter as detected by the CaM-induced increase in the relative amplitude of the long lifetime characteristic of the bound fluorescent nucleotide, caused by its affinity increase [14,22]. In fact, using this relative Ó FEBS 2004 CaM-induced conformational transition in AC (Eur. J. Biochem. 271) 829 amplitude, we estimated a K d very similar to that reported previously for AC–CaM. This difference in amplitude also suggests that a small proportion of AC is competent for ATP binding in equilibrium with a majority of incompetent protein. Binding to CaM shifts this equilibrium to the bioactive competent protein. The region of the catalytic subdomain, where a single Cys residue C75 was introduced (instead of a Tyr) in the conserved sequence A 73 GYIP 77 (AC numbering) in both B. pertussis and B. anthracis proteins, undergoes some conformational change on CaM binding. The local mobility of the acrylodan probe, attached to the C75 residue, remains slow and weak but the red shift of its fluorescence emission spectrum shows some increase in local polarity. A small conformational change was also observed in this region of the anthrax exotoxin on CaM addition [23]. In contrast with the relatively low sensitivity of the catalytic subdomain to CaM binding, at least indicated using the two probes acrylodan and Ant-dATP, the highly flexible CaM-binding sequence of AC is strongly rigidified on CaM binding, as shown by fluorescence anisotropy decays of W242 in the AC–CaM complex compared with AC. The W242 mobility is almost as large in AC as that observed for the peptide segment 196–267 [16] and the rigidifying effect of CaM binding is almost as strong too. This indicates that this segment in the protein behaves rather independently from the rest of the molecule. It is tempting to speculate that it adopts a similar conformation in AC–CaM to that in the isolated peptide, CaM producing stabilization of two potential a-helices in this sequence [13,15,16]. A turn-like geometry has been proposed, bring- ing the two a-helices closer in a helix–turn–helix motif [21]. Moreover, the complexes of CaM with these peptides exhibit elongated ellipsoidal shapes, by virtue of their much larger Brownian rotational correlation times than expected for hydrated spheres of equivalent mass [16], in contrast with the complexes of CaM with a-helical peptides derived from the myosin light chain kinase [50]. In the latter complexes, the CaM molecule wraps around the peptides, undergoing a conformational collapse achieved by bending of the interconnecting helix region and bringing the two Ca 2+ lobes in close contact [51,52]. An extended confor- mation of CaM may also prevail in the AC–CaM complex, as recently observed for the exotoxin from B. anthracis in solution [53] and confirmed in the 3D structure [23]. This large conformational change in the CaM-binding sequence may change the hydrodynamic properties of the AC–CaM with respect to AC. These hydrodynamic prop- erties of globular proteins can be studied by time-resolved fluorescence anisotropy decay of either intrinsic or extrinsic probes [44]. According to Perrin [54], the Brownian rotational correlation time (h) for a sphere is proportional to the hydrated volume of the particle (V h )andtothe viscosity/temperature factor (g/T)suchas: h ¼ V h g RT Proteins, however, are usually not spherical. When approxi- mated by ellipsoids of revolution, the fluorescence aniso- tropy decay can be described by three exponential terms [44,55], in the absence of internal motion. The time constants (rotational correlation times h i ) are related to the Brownian principal rotational diffusion coefficients of the ellipsoid, whereas the pre-exponential terms are related to the relative orientation of the fluorophore transition moment with respect to the principal axes of the ellipsoid [55]. Experimentally, however, and in particular because of the intrinsic Poissonian noise, fluorescence anisotropy decay measurements are not accurate enough to permit the separation of these different rotational correlation times, especially if fast depolarization caused by internal motions occurs, which is usually the case. The fit of the polarized decays in most cases shows a single long correlation time, describing the average tumbling motion of the molecule. This approximation holds rather well in fact as shown by the linear relation between the experimental average Brow- nian correlation time of some 20 proteins, determined in our laboratory in recent years by fluorescence anisotropy decay (for most of them obtained with tryptophan) and their molecular masses (Fig. 10). We compared these data with the results of calculations of their hydrodynamic properties performed from their atomic structure (when available) using the downloadable version of the HYDROPRO program [56–58]. This program models the surface of proteins as joined beads including the water hydration layer. Fig. 10. Variation in the average Brownian rotational correlation time of proteins as a function of their molecular mass. (d)Valuescalculated with HYDROPRO [57]; (h) values experimentally measured from the fluorescence anisotropy decays. The peptides or proteins concerned are (sorted by increasing molecular mass; PDB code in parentheses when needed): adrenocorticotropic hormone (5–10) d,h; adrenocortico- tropic hormone-(1–24) d; glucagon (1gcn) h, bovine pancreatic trypsin inhibitor (4pti) d; black mamba dendrotoxin K (1dtk) d,h; black mamba dendrotoxin I (1dem) d,h; Aspergillus Orizae ribo- nuclease T1 (9rnt) d,h; human epidermal growth factor (1jl9) d,h; bovine ribonuclease A (1rbx) d; porcine pancreatic phospholipase A 2 (1p2p) d,h; FKBP59-I (1rot) d,h; sperm whale myoglobin (1mbo) d; E. coli CMP kinase (2cmk) d,h; bovine pancreatic chymotrypsi- nogen A (2cga) d; human recombinant annexin V (anx5) d,h; hen egg ovalbumin (1ova) d; Mycobacterium tuberculosis thymidine mono- phosphate kinase (dimeric form, 1g3u) d; pig heart citrate synthase (1cts) d; human serum albumin (1bmo) d,h; B. anthracis AC–CaM complex (1k93) d. 830 J. Gallay et al.(Eur. J. Biochem. 271) Ó FEBS 2004 [...]... compact shape Therefore, although the B pertussis and B anthracis toxins exhibit similar features with respect to the activation by CaM, differences in the CaM-induced conformational change are likely This does not permit a direct exploitation of the structural results of the former to explain the mechanism of CaM activation of the latter Crystallization and resolution of the 3D structures of AC and AC–CaM,... transitions within the calmodulin- binding site of Bordetella pertussis adenylate cyclase studied by time-resolved fluorescence of Trp242 and circular dichroism Eur J Biochem 237, 619–628 Glaser, P., Elmaoglou-Lazaridou, A., Krin, E., Ladant, D., Barzu, ˆ O & Danchin, A (1989) Identification of residues essential for catalysis and binding of calmodulin in Bordetella pertussis adenylate cyclase by site-directed... (1989) Characterization of the calmodulin- binding ˆ and of the catalytic domains of Bordetella pertussis adenylate cyclase J Biol Chem 264, 4015–4020 10 Haiech, J., Predeleanu, R., Watterson, D.M., Ladant, D., Bellalou, J., Ullmann, A & Barzu, O (1988) Affinity-based chromaˆ tography utilizing genetically engineered proteins Interaction of Bordetella pertussis adenylate cyclase with calmodulin J Biol Chem... & Barzu, O (1990) Intrinsic fluorescence of a ˆ truncated Bordetella pertussis adenylate cyclase expressed in Escherichia coli Biochemistry 29, 8126–8130 Precheur, B., Siffert, O., Barzu, O & Craescu, C.T (1991) NMR and circular dichroic studies on the solution conformation of a synthetic peptide derived from the calmodulin- binding domain of Bordetella pertussis adenylate cyclase Eur J Biochem 196, 67–72... binding ˆ and activation of Bordetella pertussis adenylate cyclase by calmodulin J Biol Chem 268, 1690–1694 Munier, H., Bouhss, A., Gilles, A.M., Palibroda, N., Barzu, O., ˆ Mispelter, J & Craescu, C.T (1995) Structural characterization by nuclear magnetic resonance spectroscopy of a genetically engineered high-affinity calmodulin- binding peptide derived from Bordetella pertussis adenylate cyclase Arch... compare the hydrodynamic properties of B anthracis exotoxin, based on its atomic structure and using the HYDROPRO software, with that of B pertussis AC obtained in this study by fluorescence anisotropy decay The expected averaged Brownian rotation correlation time for the exotoxin molecule, which has a molecular mass of 59 kDa, is 38 ns according to data of Fig 10 The calculated value obtained by HYDROPRO... Sakamoto, H., Bellalou, J., Ullmann, A & Danchin, A (1988) Secretion of cyclolysin, the calmodulin- sensitive adenylate cyclase- haemolysin bifunctional protein of Bordetella pertussis EMBO J 7, 3997–4004 6 Glaser, P., Ladant, D., Sezer, O., Pichot, F., Ullmann, A & Danchin, A (1988) The calmodulin- sensitive adenylate cyclase of Bordetella pertussis: cloning and expression in Escherichia coli Mol Microbiol... consequences of single amino acid substitutions in calmodulinactivated adenylate cyclase of Bordetella pertussis EMBO J 10, 1683–1688 Craescu, C.T., Bouhss, A., Mispelter, J., Diesis, E., Popescu, A., Chiriac, M & Barzu, O (1995) Calmodulin binding of a peptide ˆ derived from the regulatory domain of Bordetella pertussis adenylate cyclase J Biol Chem 270, 7088–7096 Bouhss, A., Vincent, M., Munier, H.,... D.P (2002) Determinants of DNA mismatch recognition within the polymerase domain of the Klenow fragment Biochemistry 41, 713–722 42 Bailey, M.F., Thompson, E.H & Millar, D.P (2001) Probing DNA polymerase fidelity mechanisms using time-resolved fluorescence anisotropy Methods 25, 62–77 43 Valeur, B & Weber, G (1977) Resolution of the fluorescence excitation spectrum of indole into the 1La and 1Lb excitation... apply as these probes are either immobilized for the former (Fig 8) or only slowly mobile for the latter (Fig 9C) This suggests that the acrylodan emission transition moment is oriented close to the long protein axis, as the anisotropy measures rotational motion around an axis perpendicular to the emission moment of the fluorophore [55] The 35-ns rotational correlation time may correspond to the long . Insight into the activation mechanism of Bordetella pertussis adenylate cyclase by calmodulin using fluorescence spectroscopy Jacques. exploitation of the structural results of the former to explain the mechanism of CaM activation of the latter. Crystallization and resolution of the 3D structures of