The structural organization of Bordetella pertussis adenylate cyclase was examined by limited proteolysis with trypsin and/or cross-linking with azido-calmod-.
THEJOURNAL OF BIOLOGICAL
CHEMISTRY
Vol. 263, No. 6, Issue of February 25, pp. 2612-2618.1988 Printed in U.S. A.
0 1988 by The American Society for Biochemistry and Molecular Biology, Inc.
Interaction of Bordetella pertussisAdenylate Cyclasewith Calmodulin IDENTIFICATIONOF
TWO SEPARATEDCALMODULIN-BINDING
DOMAINS*
(Received for publication, April 10, 1987, and in revised form, November 10, 1987)
Daniel Ladant From the DeDartement de Biochimie et Genetiaue MoEculaire, Unite de Biochimie des Regulations Cellulaires, Znstitut Pasteur, 28 rue duDdcteur Roux, 75724 Paris Ceder 15: France
The structural organization of Bordetella pertussis adenylate cyclase wasexamined by limited proteolysis with trypsin and/or cross-linking with azido-calmodulin a pbotoactivable derivative of its activator, calmodulin (CaM). Adenylate cyclase (which consists of three structurally related peptides of 50, 45, and 43 kDa as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) formed a 1:l complex with CaM or azido-CaM. CaM-boundadenylate cyclase was cleaved by trypsin into two separate trypsin-resistant fragments of 25 and 18 kDa which both interacted with CaM as judged by their ability to be cross-linked with azido-CaM. These two fragments remained associated with CaM in a catalytically activeconformation resembling that of the undigested complex. When proteolysis was carried out in the absence ofCaM, the adenylate cyclase was completely inactivated in less than 3 min. Sodium dodecyl sulfate-polyacrylamide gel revealed a single 24-kDa trypsin-resistant fragment. Since thisfragment cannot be cross-linked with azidoCaM we suggest that the CaM-binding site on the 25kDa moiety of the adenylate cyclase is located on a short segment of 1 kDa.
pertussis adenylate cyclase and CaM. This task was considerably facilitated by our recent purification to homogeneity of adenylate cyclase from culture supernatantsof B. pertussis (16). In this paperI examined the CaM-adenylatecyclase interaction using structural probes, such asa photoactivable derivative of CaM and limitedproteolysis by trypsin. EXPERIMENTALPROCEDURES
Chemicak-Blue-Sepharose, ATP, CAMP, TPCK-trypsin,and soybean trypsin inhibitor were from Sigma. Affi-Gel-CaM was from Bio-Rad. Urea (fluorimetrically pure) was aproduct of Schwarzl Mann. Methyl-4-azido-benzimidatewas purchased from Pierce Chemical Co. [m3'P]ATP (3000 Ci/mmol), and NalZ51(1000 Ci/ mmol) were obtained from the Radiochemical Center (Amersham Corp.). [3H]cAMP (40 Ci/mmol) was purchased from CEA (Saclay). Bovine brain CaM was a kind gift from Professor E. Carafoli (Swiss Federal Institute of Technology, Zurich). Microcrystalline cellulose precoated thin-layer plates (CEL400-10) were obtained from Macherey-Nagel. Ultrogel AcA-44 came from LKB. CaM was azidated essentially as described by Zurini et al. (17). To 1 mg of CaM dissolved in 0.5 ml of 60 mM sodium borate, pH 9.8, 0.2 mM CaCl,, 0.1 M NaC1,0.5mgof methyl-4-azido-benzimidatedissolved in 0.5 ml of the same buffer were added. After 2 h of stirring a t room temperature in the dark, the reaction mixture was desalted on a 10-ml SephadexG.25 column equilibrated with 25 mM Tris-HC1, p H 7.4,O.l M NaCl, 0.2 mM CaCl,. Incorporation of azido groups into Bordetellapertussis and Bacillus anthracis, causative agents CaM was estimated spectrophotometrically:a value of 1.3 mol of azido groups/mol of CaM was found. of whooping cough and anthrax,respectively, are theonly two Azido-CaM was stored a t -20 "C protected from light. prokaryotic organisms which are so far known to secrete an Purification and Assay of Adenylate Cyclase-Concentrated culture adenylate cyclase (1-3). B. anthracis adenylate cyclase, iden- supernatant of B. pertussis, 18323, phase I (type strain ATCC 9797) tified as the edema factor( 3 , 4), enters animal cells by means was obtained as described previously (16). The specific activity of concentrated culture supernatantwas between 100 and 120 units/mg of protectiveantigenandelevatesintracellularadenosine protein. Adenylate cyclase was purified in a single step by chrocAMP levels. Genetic and biochemical evidence incriminate of matography on Affi-Gel-CaM (16). Purified enzyme in buffer A (50 B. pertussis enzyme as playing a role in the pathogenesis of mM Tris-HC1, pH 8,0.1%Nonidet P-40,O.l mM CaC1,) with aspecific whooping cough (5-9). Adenylate cyclase from both organisms activity of 1600 units/mg of protein could be stored a t -80 "C for exhibits a striking property, namely activationby calmodulin several weeks with no loss of activity. SDS-polyacrylamide gel revealed three structurally related bands (see "Results") corresponding (CaM)' ( 3 , 10). Interaction of CaM with secreted adenylate cyclase of B. pertussis has been investigated mainly from a to 50,45, and43 kDa. Adenylate cyclase activity was measured using the procedure of kinetic standpoint using crude or partially purified enzyme White (18), as modified by Hanoune et al. (19). The reaction was preparations (10-15). Since CaM was shown to prevent entry performed a t 30 'C in 100 pl of a medium containing 50 mM Trisof the bacterialenzyme into targetcells (9), we were interested HCI, pH 8, 1 mM [w3'P]ATP (5 X IO5 cpm/assay), 6 mM MgCl,, 100 to learnmore about the mechanismof interaction between B. pg of bovine serum albumin, 0.13 mM [3H]cAMP (1.5 X 10' cpm/ assay), 0.12 mM CaCl,, and 0.1 p~ CaM (when added). One unit of adenylate cyclase corresponds to 1pmol of cAMP formed in 1 min a t *This work was supported by Grant UA 1129 from the Centre National de laRechercheScientifique, Grant831 025 from the 30 "C at pH 8. Zodinatiom-Five ml of concentrated culture supernatant containInstitut National de la Sante et de la Recherche Midicale, and the Fondation pour la Recherche Medicale. The costs of publication of ing about 30 units of adenylate cyclase were mixed with 70 pl of packed Affi-Gel-CaM and shaken overnight a t 4 "C. The gel which this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in retained more than 75% of enzyme activity was then washed several times with0.5 M NaCl in buffer A and further with 50 mM Tris-HC1, accordance with 18 U.S.C. Section 1734 solely to indicate thisfact. p H 8. Iodination of adenylate cyclase bound to Affi-Gel-CaM was The abbreviations used are: CaM, calmodulin; Blue-Sepharose, performed with chloramine T a t room temperature for 5 min with Cibacron Blue 3G-A-Sepharose CL-GB; azido-CaM, azido-calmodulin; SDS, sodium dodecyl sulfate; TPCK, L-1-tosylamido-2-phenyl- occasional shaking.The reaction was quenched by addition of sodium ethylchloromethyl ketone; EGTA, [ethylene bis(oxyethylenenitri1o)l metabisulfite, followed by addition of 0.7 ml of 8.8 M urea in buffer A. After 30 min of stirring a t room temperature, the mixture was tetraacetic acid.
2612
2613
B. pertussis AdenylateCyclase loaded onto a 10-ml Sephadex G-25 column equilibrated in buffer A, to remove both urea and free iodine. The iodinated adenylate cyclase (0.3-1 X lo' cpm/pg of protein corresponding to 0.08-0.25 mol of iodine/mol of adenylate cyclase) was fully active and stimulated by CaM 20-50-fold. Its specific activity was expressed in units per counts per minute of lZ5I. CaM and azido-CaM were iodinated by the chloramine-T method at room temperature to a specific activity of about 20 Ci/mmol (0.02 mol of iodine/mol of CaM) (20). Binding of Adenylate Cyclase toBlue-Sephurose-Adenylate cyclase was diluted in buffer B (50 mM Tris-HC1, pH 8, 0.1 mM Ca2+,0.1% Nonidet P-40, 20% glycerol) to 0.1 unitlml. T w o - h u n ~ e dpi of this solution were gently shaken at 4 "C in an Eppendorf tube with 10 pl of a Blue-Sepharose suspension corresponding to 2.5 pl of packed resin and with different concentrations of CaM or azido-CaM. After different times of incubation (between 30 min and 24 h), tubes were centrifuged and the activity of adenylate cyclase in the supernatant was measured. The percentage of enzyme bound to Blue-Sepharose was calculated by subtracting the activity remaining in the supernatant from the initial activity. Appropriate runs were made in the absence of Blue-Sepharose to account for enzyme inactivation oradsorption to Eppendorf tubes. It should be noted that in thepresence of 20% glycerol adsorption was less than 2% in 3 h. Isolation of the 43-, 45; and 50-kDa Polypeptides on SDS-Polyacrylamide Gel ~ ~ c t r o ~ ~ r e s ~ iodinated - T h e adenylate cyclase preparation was run on a 7.5% SDS-polyacrylamide gel (21), and the proteins were fixed in 10% acetic acid; the gel was further washed with 25% isopropyl alcohol, 10% acetic acid and then exposed for autoradiography a t 4 "C. The bands corresponding to the 50-, 45-,
loo
lr"--
and 43-kDa peptides were sliced from the gel with a razor blade. The slices were placed in siliconized tubes and washed extensively with 25% isopropyl alcohol, then with 10% methanol to remove SDS, and at last dried under a heat lamp. The slices, in Eppendorf tubes, were then soaked in 0.8 ml of 8 M urea in 50 mM Tris-HC1,pH 8, containing 0.1 mM Ca2+and 1%Nonidet P-40, for 18h a t 37 "C. More than 70% of the iodinated peptides were extracted from the gel slices by this procedure. The urea solutions containing the iodinated peptides were supplemented with CaM (final concentration, 3 pM) and dialyzed extensively against buffer A. Each peptide was assayed for adenylate cyclase activity as described above. Maps of Adenylate Cyclase Frag~ w f f - ~ ~ m ~ Tryptic ~ ~ f f nPeptide al ments-The iodinated tryptic peptides T25, T18, and T24 were separated on a 12.5% SDS-polyacrylamide gel and then treated as described above. Iodinated gel slices were digested with 50pg of TPCK-trypsin in 0.6 ml of 50 mM ammonium bicarbonate, pH 8.0, for 20 b at 37 "C.T h e s u ~ r n a t asolutions nt containing the solubilized peptides were lyophilized. The lyophilized samples were dissolved in 10 p1 o f buffer and spotted on cellulose thin-layer precoated plates 3.7 in (20 X 20 cm). The first dimension was el~trophoresis at pH pyridine/acetic acid/water (3:30867), run at 400 V for 1 h in a thin layer chromatography chamber (Desaga). The plates were dried at room temperature, and the chromatograms were developed for the second dimension in a system of pyridine/n-butyl alcohol/water/ acetic acid (2030:240.6). The plates were dried and exposed for autoradiography on X-Omat AR films a t -70 'C. Limited Proteolysis with Trypsin-Free or CaM-bound 1251-adenylate cyclase in buffer A was mixed with TPCK-trypsin (w/w ratio; 12). After different times of digestion a t 4 "C, aliquots were withdrawn and diluted in buffer A containing soybean trypsin inhibitor in a 20-fold molar excess over TPCK-trypsin. Adenylate cyclase activity was determined in the presence of 0.1 p~ CaM, and the remaining sample was run on a 12.5% SDS-polyacrylamide gel, as described by Laemmli (21). The gels werethen dried and exposed on X-Omat AR films at -70 "C for autoradiography. Photoaffinity Labeling-Photoaffinity labeling experiments were performed a t 4 'C for 1.5min. The reaction mixture containing either native or proteolyzed adenylate cyclase-azido-CaM complex was irradiated with a "long-wave'' mercury lamp (mineral. light UVSL 58 without screen) positioned at 5 cm from the samples.
RESULTS Interaction of B. pertussis Adenylate Cyclase with CaM and
2
3
25
incubation time ( h r s )
FIG. 1. Binding of adenylatecyclase toBlue-Sepharose. Binding experiments were done as described under "Experimental of 100 Procedures" either in the absence (0)or in the presence (e) nM CaM. Binding in the absence of both Caz+ and CaM (0)was measured in buffer B supplemented with 2 mM EGTA. Release of gel-bound adenylate cyclase by 100 nM CaM (arrow) was performed at 4 or at 30 "C.
2. Determination of dissociation constant (Kn) of the adenylate cyclase/CaM complex. Percentage of enzyme bound to Blue-Sepharose either in the presence of Caz+(buffer B) or in the presence of EGTA (buffer B supplemented with 2 mM EGTA) was determined after 3 h of incubation as described under "Experimental Procedures." The partition coefficient kd was plotted uersus the concentration of CaM (0)or azido-CaM (e).
A z ~ - C a M - P r e l i m i n a ~experiments showed that free adenylate cyclase, but not the CaM-complexed enzyme, bound reversibly to Blue-Sepharose at neutral pH. Blue-Sepharosebound adenylate cyclase was released into the medium by excess CaM, with a half-time varying between several minutes a t 30 "C and several hours at 4 "C (Fig. 1). These differences in affinity for Blue-Sepharose between free and CaM-bound adenylate cyclase prompted me to investigate the CaM-adenylate cyclase interaction by a gel competition method. This method is similar in many respects to that reported by Schubert (22) for determination of the metal ions-nucleotide affinity constant.
+ Ca2*
FIG.
4 -
4 -
kd
1
2
3
4
5
100
CaM or azido-CaM
200
, nM
300
400
Adenylate B. pertussis
2614
Cyclase TABLE I Adenylate cyclase activity of the 50-, 45-, and 43-kDa polypeptides Polypeptides"
kDa Native preparation" 50 45 1.33 43
Total '1 radioactivity x103 cprn
90 16.5 40.0 19.5
Adenylate cyclase activity
unitsf IO7 cpm of
lZ5I
2.83 1.53 2.07
" T h e three polypeptides (50, 45, and 43 kDa) were separated on SDS-polyacrylamide gel and renatured as described under "Experimental Procedures." " Iodinated adenylate cyclase preparation before SDS-polyacrylamide gel. 0
2
5
10
50
CaM, nM
FIG. 3. Stability of the adenylate cyclase-CaM complex in the presence of EGTA. Binding of adenylate cyclase to BlueSepharose was measured in buffer B, in the presence of 0.1 mM Ca2+ ( 0 )or 2 mM EGTA (W) and 2-50 nM CaM, as described under "Experimental Procedures." After 3 h of incubation, percentage of enzyme remaining in the supernatant was determined and plotted versus the concentration of CaM. Then, 2 mM EGTA were added to the samples which were previously incubated in thepresence of Ca2+. After another incubation of 3 h the percentage of enzyme remaining in the supernatantwas determined and plotted versus the concentration of CaM (+).
Mr
- 67 adenylate cy c Iase
- -
-50 45 43
38
- 20 FIG. 4. Autoradiography of the iodinated adenylate cyclase preparation resolved on a 10%SDS-polyacrylamide gel. The M , markers were bovine serum albumin (67 kDa), glyceraldehyde-3phosphate dehydrogenase (38 kDa), and soybean trypsin inhibitor (20 kDa).
If we take into consideration thefollowing two equilibria: adenylate cyclase
+ Blue-Sepharose
(1)
e (adenylate cyclase) (Blue-Sepharose) adenylate cyclase
+ n(CaM) e (adenylate cyclase) (CaM),
(2)
thepartition coefficient, i.e. theratio betweenenzyme in solution and enzyme bound to the gel, in the presence( k d )or absence ( k s )of CaM will be defined bythe following equation:
KDbeing the dissociation constant of the adenylate cyclase-
CaM complex. As shown in Fig. 2, kd is a linear function of CaMconcentration,eitherinthe presence or absence of calcium ions, which is consistent with a stoichiometry of 1:1 for adenylate cyclase-CaM interaction. Dissociation constants calculated from data obtained in several experiments range from 0.09 to 0.17 nM in the presence of Ca2+and from 13 to 23 nM in the presence of alarge excess of EGTA. When dissociation constants have been calculated from CaM-adenylate cyclase dose-response curves, the values obtained were similar (not shown). Despite these differences in affinity of B. pertussis adenylate cyclase for CaM, once the complex was formed in the presence of Ca2+, additionof excess EGTA did not promote its dissociation (Fig. 3). This is consistent with our previous observationthatadenylate cyclase bound to CaM-agarose cannot be eluted by EGTA. Azido-CaM, a photoactivable derivative of CaM (23), behaves almost identically to the parent compound, both in activating the bacterial adenylate cyclase (not shown) and bindingtothe enzyme (Fig. 2). Thus,the half-maximum activating concentrationsof azido-CaM are the same as those of CaM either in the presence of Caz+ or EGTA. KO values calculated fromdata shown in Fig. 2 indicated a similar affinity of CaM andazido-CaM for adenylate cyclase. Solid-phase Iodination of Pure Adenylate Cyclase-Solidphase iodination of pure adenylatecyclase bound toAffi-GelCaM yielded an active'ZsII-adenylate cyclase preparation which was activated up to 50-fold by 100 nM CaM. Autoradiography after SDS-polyacrylamide gel of the iodinated enzyme revealed the same three peptidesof 50, 45, and 43 kDa (Fig. 4) which were also detected by Coomassie Blue staining of the CaM-Affi-Gel-purified enzyme (16). Peptide mapping of these three bandsgave similar patterns (not shown). Inordertodeterminewhether all threepeptides were endowed with adenylate cyclase activity, they were separated bySDS-polyacrylamide gel andthecorrespondingbands, revealed by autoradiography, excised; the iodinated polypeptides were extracted with 8 M urea, 1%Nonidet P-40 (see "Experimental Procedures"). Upon dialysis in thepresence of CaM, each polypeptide recovered adenylate cyclase activity (Table I). Moreover, as will be shown below, the 50- and 45-kDa peptides can be converted to the 43-kDa species by limited proteolysis. These results suggest that the three peptides are structurally related. Since adenylate cyclase is released extracellularly, it seemslikely that the three peptides arose from a differential processing of a common precursor during secretion. Limited Proteolysis of Purified '2sI-Adenylate Cyclase by Trypsin-Incubation of free I21-adenylatecyclase with trypsin at 4 "C (at a 1:l (w/w) ratio) resulted in complete inactivation of the enzyme within 3 min (Fig. 5A); the 50-, 45-, and 43-kDa polypeptides were converted to a 24-kDa fragment
B. pertussis Adenylate Cyclase
A
AC
I
2615
1
AC-CaM
I
i
Mr 67 -
38 -
FIG.5. Limited proteolysis of purified 1261-adenylate cyclase by trypsin. A, 0.1 units of ’2sI-adenylate cyclase in 0.1ml of buffer A supplemented (AC-CUM)or not (AC) with 0.4 PM CaM were submitted to trypsin proteolysis a t 4 “C as described under “Experimental Procedures.” After different times of digestion (indicated at thebottom of the figures), aliquots were withdrawn and diluted inbuffer A containing soybean trypsininhibitorina 20-fold molar excess over TPCK-trypsin. Adenylate cyclase activity (expressed in percentage of initialactivity) was determined; the different samples corresponding to the different times of digestion were then run on a 12.5% SDS-polyacrylamide gel and the gel was autoradiographed. Molecular mass standards: bovine serum albumin (67 kDa), glyceraldehyde-3-phosphate dehydrogenase (38 kDa); soybean trypsin inhibitor (20 kDa), and lysozyme (14 kDa). The autoradiograph was overexposed for better visualization of T18. B, after autoradiography, the bands corresponding to the native polypeptides (50,45, and43 kDa) (0) or tothe T25 (W), and T18 (e) fragments were sliced from the dried gel and theradioactivity was measured.
b
Lr”
0 20
-
14
-
II)-T24 -.
..-.
.A n.
min of digestion activity
o
1
3
IO
0
100
4
1.5
0
100
1
2
83
5
79
8
63 60
1
2
20
58
54
B
h
? 0 E
n V
Y
v)
e
0
min
of
20
digestion
T 24
T 18
15
10
5
T 25
0
0
f d
te
r
0
0
-
electrophoresis
+
-
electrophoresis
0
+
-
FIG.6. Autoradiograms of two-dimensional tryptic peptide maps of T18,
electrophoresis
T25, and T24. The two-
dimensional tryptic peptide maps were performed as described under “Experimental Procedures.” 0 designates the origin of migration.
B. pertussis AdenylateCyclase
2616 A
-
50K45K 43K-
m
Ill).-
0
B
A
B
67K-
-
C
D
U
- -
I-AC
725
-
T18
38 K-
- azido-'"l- CaM
22K-
a
b
c
d
e
f
g
h
i
j
k l
d
h l
FIG. 7. Limited proteolysis of theisolated 50-, 45-, and 43kDa iodinated polypeptides. The 50-, 45-, and 43-kDa polypeptides were separated on SDS-polyacrylamide gel and renatured as described under "Experimental Procedures." Each peptide (15 X lo-'' units/ml in buffer A containing 3 p~ of CaM) was digested with 0.5 pg/ml of trypsin a t 4 "C. At the indicated times a 100-excess of soybean trypsininhibitor wasaddedover trypsinandadenylate cyclase activity was assayed; then the corresponding samples were run on a 9% ( A ) or a 12.5% ( B )SDS-polyacrylamide gel and autoradiographed. Lanes a-d: the 50-kDa polypeptide was digested for 0, 0.5,5, and 10min, respectively (corresponding % of adenylate cyclase activity: 100, 94, 59, and 43, respectively). Lanes e-h: the 45-kDa polypeptide was digested for 0, 0.5,5, and 10 min, respectively (% of adenylate cyclase activity: 100, 86, 56, 46, respectively). Lanes i-1: the 43-kDa polypeptide was digested for 0,0.5,5, and10 min, respectively (% of adenylate cyclase activity: 100, 92, 71, and 44, respectively). Note that in A the T18 fragment cannot be seenbecause it had migrated with the front of the gel.
70K
- +- +
-+
-+
FIG. 9. Photoaffinity labeling of adenylate cyclasewith azido-CaM. 100 nM azido-'"I-CaM and 0.4 units of adenylate cyclase (lanes A and B corresponding to pure enzyme, and lane C to concentrated culture supernatant) in 50 pl of buffer A were incubated for 30 min a t 4 "C. An aliquot was photolysed as described under "Experimental Procedures" (+) and was run in parallel with a nonphotolysed aliquot (-) onto a10%SDS-polyacrylamide gel andautoradiographed. Sample B was identical to sample A , the only exception being that 2.5 p M of cold CaM was present. In lane D,400 nM azidoCaM and 0.2 units of pure "'I-labeled adenylate cyclase in 50 pl of buffer A were incubated for 30 min a t 4 "C, photolyzed (+) or not (-) for 1.5 min, then run on a 10% SDS-polyacrylamide gel, and autoradiographed. 12'Z-AC is 1251-adenylatecyclase. Molecular mass standards: bovine serum albumin (67 kDa); glyceraldehyde-3-phosphate dehydrogenase (38 kDa), and Escherichia coli catabolite activator protein (22 kDa).
conversion of the 50- and 45-kDa peptides to the 43-kDa species which was progressively cleaved to a major, trypsinAC insensitive fragment of 25 kDa (T25), and a minor one of c d about 18 kDa (T18) (Fig. 5A). The T24 fragment obtained in the absence of CaM appeared to be structurally related to T25 as shown by their two-dimensional tryptic peptide map I ' 8 (Fig. 6). The generation of the minor iodinated peptide T18, obtained only inthe presence of CaM, was highly reproducible with the same '"I-adenylate cyclase preparation. The apparent yield of this peptide varied with different preparationsof g 4 '"I-adenylate cyclase; for instance in a previous report it was m, not detected (16). It is likely that differences in intensity 0 2 could be accounted for by differencesin iodination of the x of exposed regions that yield T25 andT18 due to the number tyrosines in each fragment. T o elucidate the origin of T25 and T18, the bands corresample no sponding to these peptides (Fig. 5A) have been excised for FIG. 8. Gel filtration chromatography of trypsin cleaved quantification. As shown in Fig. 5B, the kineticsof appearance adenylate cyclase/CaM complex. 3 X 10" cpm of lZ5I adenylate cyclase (9 X lo-? units) in 50 p1 of buffer A containing 0.5 pg of CaM of the two tryptic peptides was similar and in addition, their is unlikely that T18 is were digested 10 min with 75 ng of trypsin a t 4 "C; the digestion was ratioremainedconstant.Thus,it T25. Moreover, the two-dimensional tryptic stopped by addition of 10 pg of soybean trypsin inhibitor (remaining derivedfrom activity: 6 X 10" units). The sample was then diluted in 100 p1 of peptidemap of T25 wassignificantly different from that bufferA containing 200 pg of bovine serumalbumin, 30 pg of obtained for T18 (Fig. 6), suggesting that T25 is not related adenylate kinase, 1pg of CaM, and 20 pg ofsoybean trypsin inhibitor to T18. Thus, T25 and T18 might represent two different and loaded onto anUltrogel AcA-44 column (0.6X 26 cm) equilibrated domains of adenylate cyclase. in buffer A containing 20 pg/ml of soybean trypsin inhibitor. FracTo determine further whetherall three native polypeptides tions of 0.1 ml were collected a t a flow rate of 0.5 ml/h and analyzed for activity (0)and radioactivity ( 0 )Insert . shows an autoradiograph (50, 45, and 43 kDa) contain both T25 and T18 domains, of a 12.5% SDS-polyacrylamide gel of fractions corresponding to the limited proteolysis was performed on the separated iodinated peak of activity. The M, markers for the gel filtration experiments polypeptides complexed with CaM. As shown in Fig. 7, upon were: bovine serum albumin, 67,000 ( a ) ;ovalbumin, 45,000 (b);adeexposure to trypsin, the 50- and 45-kDa bands were rapidly nylate kinase, 23,500 (c); soybean trypsin inhibitor, 20,000 ( d ) . AC converted into a 43 kDa one, which was further cleaved into designates the native adenylatecyclase-CaM complex. T25 and T18. These results demonstrate that each polypep(T24) which was largely resistant to furtherproteolysis, sug- tide of 50, 45, and 43 kDa contains a common region of 43 gesting a compact structure. When"'I-adenylate cyclase was kDa which can be proteolytically cleaved into two peptides of complexed with CaM prior toexposure to trypsin,more than 25 and 18 kDa. Gel Filtration Chromatography of Trypsin-cleaved Adenylate 50% of theenzymaticactivityremainedafter 20 min of incubation. SDS-polyacrylamidegel analysis revealed a rapid Cyclase-CaM Complex-Analysis of the trypsin-cleaved ade-
fi
.I-
Adenylate B. pertussis
Cyclase
A
Mr
2617
1
2
-+
-- ++
3
-
67K-
38K-
FIG.10. Cross-linking of azidoCaM to tryptic fragments of adenylate cyclase. A, 0.1 units of pure '"122 Kadenylate cyclase in 50 $1 of buffer A supplemented with (lanes 1 and 2) or without ( l a n e 3) 0.4 p M of azido-CaM were incubated with 50 ng of trypsin a t 4 "C for the indicated time. Proteolysis was stopped by addition of 5 pg of soybean trypsin inhibitor,and adenylate cy- min of digestion clase activity was determined (expressed in % of initial activity). Sample 3 was % of initial activity supplemented with 0.4 p~ azido-CaM, and thenall samples were photolysed (+) or not (-), run on a 10% SDS-polyacrylamide gel, and the dried gel was autoradiographed. E, 0.1 units of pure adenylB ate cyclase in 50 $1 of buffer A supplemented with (samples 1 and 2) or without (sample 3) 100 nM azido-'2'II-CaMwere Mr submitted to trypsin proteolysis as above. Proteolysis was stopped with an excess of soybean trypsin inhibitor, and 67 Kadenylate cyclase activity was determined. To sample 3 100 nM azido-"'ICaM were added, and then all samples were treated as described in A. 38 K -
22K-
min of digestion
% of initial activity T24 r--
." I I
Tl8
1 1
T25
I
"-J
CdM
FIG.11. Proposed model of the structural organization of adenylate cyclase/CaM complex. T18,T24, and T25 are the tryptic fragments defined in the text. The dashed boxes a t the end of T18 and T25 represent additional peptides of the intact 50- and 45kDa polypeptides which are cleaved earlier in proteolysis to yield the 43-kDa species. nylate cyclase-CaM complex on AcA-44 gel filtration chromatography revealed a unique peak of adenylate cyclase activity comigrating with the main peak of radioactivity (Fig. 8). Its apparent M, was identical to that of the undigested adenylate cyclase-CaMcomplex (60 kDa).SDS-polyacrylamide gel analysis of the fractions corresponding to the peak of activity revealed the same ratio between T18 and T25 as shown in Fig. 5A. This indicates that after proteolysis, T25,
0
10
2
100
69
2
- + a
45 38
3
2
1
70 63
- +
- +
-
-
*
m
-
0
10
2
100
59
3
70 63 45
38
a~ido-'*~l-CaM
T18, and CaM remained associated by noncovalent interactions in anactive, native-like structure. Identification of CaM-binding Sites in Adenylate Cyclase by Photoaffinity Labeling with Azido-CaM-Preliminary experiments revealed that photolysis of a mixture of azido-"'I-CaM and pure or crude preparationsof adenylate cyclase resulted in one major cross-linked product exhibiting a M, of 70 kDa on SDS-polyacrylamide gel (Fig. 9) which was not observed when photolysis was carried out in the presence of a 25-fold molar excess of unmodified CaM. In theabsence of photolysis no cross-linked product could be revealed. In a similar way, photolysis of a mixture of ""I-adenylate cyclase and azidoCaM resulted, again, in one major cross-linked product with a M , of 70 kDa on SDS-polyacrylamidegel (Fig. 9). Since the apparent M, of CaM or azido-CaM on SDS-polyacrylamide gel was 20 kDa, the 70-kDa cross-linked product corresponds most likely to covalent attachment of one adenylate cyclase polypeptide to one azido-CaM molecule. Whenpure'TI-adenylate cyclase was complexed with
Adenylate B. pertussis
2618
azido-CaM, then submitted to trypsin proteolysis for 10 min, 69% of enzymatic activity was retained while the enzyme was entirely converted into T25 and"18 polypeptides. When the digested complexwas subsequently photolyzed, three new cross-linked species of 63,45, and 38 kDa were evidenced (Fig. 1OA, lune 2). The 45- and 38-kDa bands might correspond to covalent attachment of azido-CaM to T25 and T18, respectively, whereas the 63-kDa band might result from covalent attachment of azido-CaM to both T25 and T18. Digestion of '"I-adenylate cyclase with trypsin prior to photolysis in the presence of azido-CaM did not yield any crosslinked product (Fig. lOA, lane 3). Similar experiments using unlabeled adenylate cyclase complexed with azido-lZ5I-CaMgave essentially the same results: after 10 min of trypsin digestion, the same cross-linked peptides of 63, 45, and 38 kDa were detected upon photolysis (Fig. 10B, lane 2). Again, trypsin digestion of adenylate cyclase for 2 min prior to addition of azido-lZ5I-CaMdid not lead to any cross-linked species upon photolysis (Fig. 10B, lane 3 ) . DISCUSSION
CaM stimulation of B.pertussis adenylate cyclase has been widely documented (10-15,24). The present results confirmed that CaM binds strongly to adenylate cyclase even in the absence of Ca2+albeit with less affinity than in the presence of the divalent cation. The 1:l CaM-adenylate cyclase complex did not dissociate upon addition of EGTA or in media of high ionic strength. This is not an unprecedented case since it has been shown that CaM (the 6 subunit) remained associated with muscle phosphorylase kinase in the absence of Ca2+(25). Controlled proteolysis of adenylate cyclase by trypsin and/ or photoaffinity labeling with azido-CaM suggested that adenylate cyclase is composed of two separate domains of 25 kDa (T25) and 18 kDa (T18)which both interact with CaM. After exposure of the adenylate cyclase-CaM complex to trypsin, the CaM molecule wouldbridge the two cleaved domains in a structure resembling the native complex. T25 and T18 associated with CaM appeared as very resistant species toward further proteolysis; in contrast, when proteolysis was carried out on adenylate cyclase in the absence of CaM, only a 24kDa trypsin-resistant fragment (T24) was detected. The specific radioactivity of this fragment as well as its tryptic map were similar to thatof T25, suggesting that it corresponds to the same region of the native polypeptide. Since this 24-kDa fragment could not be cross-linked with azido-CaM, it islikely that theextra 1-kDapeptide present in T25is involvedin the binding of CaM. Edelman et al. (26) have already described differences in affinity for CaM of chymotryptic fragments of myosin light chain kinase differing by only 2 kDa at their C terminus. These results suggest that adenylate cyclase containsa compact domain of about 24 kDa largely resistant to trypsin proteolysis whether CaM was present or not. In contrast the T18 fragment displays resistance toward protelysis only in the presence of CaM. This suggests that binding of CaM to adenylate cyclase could induce a conformational change in the T18 domain rendering it more resistant to proteolysis; alternatively, CaM could mask potential cleavage sites on T18 thus making them inaccessible to trypsin. In both cases, the protective effect involves a multisite interaction between CaM and T18 as depicted in the model presented in Fig. 11. The structure proposed for B.pertussis adenylate cyclase differs significantly from those known for other CaM-dependent enzymes which can be modified by proteolytic cleavage
Cyclase to yield active CaM-independent forms (17,26-30). Attempts to identify a CaM-independent catalytic domain of B.pertussis adenylate cyclase have failed thus far. It remains to determine whether this could be achieved using other approaches or if it represents aprofound structural difference between B. pertussis adenylate cyclase andother CaM-dependent enzymes. Acknowledgments-I am grateful to Dr. A. Ullmann for her constant interest, guidance, and help in the preparation of this manuscript and to Dr. 0. Birzu for helpful advice and critical comments. I thank Drs. J. M. Alonso, C. Brizin, and N. Guiso for providing me culture supernatant of B. pertussis and for helpful suggestions,Dr. A. M. Gilles for assistance in two-dimensional tryptic peptide mapping, and Dr. S. Cole for critical reading of this manuscript. I also thank M. Ferrand for expert secretarial help.
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