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An Extended Conformation of Calmodulin Induces Interactions between the Structural Domains of Adenylyl Cyclase from Bacillus anthracis to Promote Catalysis*
Recipient of an American Heart Association Established Investigator Award. To whom correspondence should be addressed: University of Chicago, Dept. of Neurobiology, Pharmacology, and Physiology, 947 E. 58th St., MC0926, Chicago, IL 60637. Tel.: 773-702-4331; Fax: 773-702-3774
* This research was supported in part by National Institutes of Health Grants GM53459, DA05778 (to C. D.), and AR41637 (to Z. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. § Recipient of a fellowship from the American Heart Association Chicago Affiliate and University of Chicago Committee on Cancer Biology. ** Recipient of a Robert A. Welch Foundation Grant C1119. ‡ Collaborative principal investigators who contributed equally to this work.
The edema factor exotoxin produced byBacillus anthracis is an adenylyl cyclase that is activated by calmodulin (CaM) at resting state calcium concentrations in infected cells. A C-terminal 60-kDa fragment corresponding to the catalytic domain of edema factor (EF3) was cloned, overexpressed inEscherichia coli, and purified. The N-terminal 43-kDa domain (EF3-N) of EF3, the sole domain of edema factor homologous to adenylyl cyclases from Bordetella pertussis andPseudomonas aeruginosa, is highly resistant to protease digestion. The C-terminal 160-amino acid domain (EF3-C) of EF3 is sensitive to proteolysis in the absence of CaM. The addition of CaM protects EF3-C from being digested by proteases. EF3-N and EF3-C were expressed separately, and both fragments were required to reconstitute full CaM-sensitive enzyme activity. Fluorescence resonance energy transfer experiments using a double-labeled CaM molecule were performed and indicated that CaM adopts an extended conformation upon binding to EF3. This contrasts sharply with the compact conformation adopted by CaM upon binding myosin light chain kinase and CaM-dependent protein kinase type II. Mutations in each of the four calcium binding sites of CaM were examined for their effect on EF3 activation. Sites 3 and 4 were found critical for the activation, and neither the N- nor the C-terminal domain of CaM alone was capable of activating EF3. A genetic screen probing loss-of-function mutations of EF3 and site-directed mutations based on the homology of the edema factor family revealed a conserved pair of aspartate residues and an arginine that are important for catalysis. Similar residues are essential for di-metal-mediated catalysis in mammalian adenylyl cyclases and a family of DNA polymerases and nucleotidyltransferases. This suggests that edema factor may utilize a similar catalytic mechanism.
G-protein
guanine nucleotide-binding regulatory protein
CaM
calmodulin
EF
edema factor
EF3
C-terminal 60-kDa edema factor
EF3-N
N-terminal 43-kDa of EF3 (aa 291–640)
EF3-C
C-terminal 19-kDa EF3 (aa 640–800)
MLCK
myosin light chain kinase
M13
synthetic peptide corresponding to the CaM-binding region of smooth muscle MLCK
Gsα
the α-subunit of G-protein that stimulates adenylyl cyclase
AC
adenylyl cyclase
PAGE
polyacrylamide gel electrophoresis
1
5-IAEDANS,N-iodoacetyl-N′-(5-sulfo-1-naphthyl)ethylenediamine (AEDANS when attached to a protein)
DAB
4-dimethyl-aminophenylazophenyl-4′-maleimide
aa
amino acids
Mes
4-morpholineethanesulfonic acid
cAMP is a key second messenger that modulates a particularly diverse set of physiological responses including sugar and lipid metabolism, cell differentiation, apoptosis, neuronal activity, and ion homeostasis. Certain pathogenic bacteria have evolved exotoxins that severely alter the internal cAMP levels of infected cells. These exotoxins work through two common mechanisms. The first is to ADP-ribosylate the α-subunits of heterotrimeric G-proteins.1 Cholera and pertussis toxin ADP-ribosylate Gs and Gi, respectively. In doing so the toxins increase the activity of endogenous adenylyl cyclases to pathogenic levels (
Moss J. Iglewski B. Vaughan M. Tu A.T. Handbook of Natural Toxins-Bacterial Toxins and Virulence Factors in Disease. 8. Marcel Dekker, Inc.,
New York, Basel, Hong Kong1995: 59-80
). The second strategy is to secrete an adenylyl cyclase directly into the host. This strategy is employed by Bordetella pertussis, Bacillus anthracis, and Pseudomonas aeruginosa. To overcome the problem of unregulated cAMP production within the pathogen, these cyclases have evolved to become active only upon binding a factor endogenous to the host (
). Adenylyl cyclases from B. pertussis and B. anthracis are activated by calmodulin (CaM). The exotoxin from P. aeruginosa is activated by an unknown, soluble cellular factor (
). The adenylyl cyclase from B. anthracis, also known as edema factor, is secreted as an inactive enzyme by bacterial cells and associates with protective antigen, another B. anthracis exotoxin. Protective antigen is a pH-dependent protein transporter that upon proteolytic activation, endocytosis, and pH change, translocates edema factor into the cytoplasm of the target cells (
). After gaining access to the cytoplasm, edema factor binds CaM at the resting calcium concentration and is thus activated to produce unregulated levels of cAMP within the cytosol (
). In the course of anthrax pathogenesis, edema factor works in concert with another enzyme exotoxin, lethal factor. Lethal factor has been shown to proteolytically inactivate ERK kinase (MAP-kinase kinase) (
). The N-terminal 30-kDa domain bears strong homology to the N-terminal domain of lethal factor and is centrally involved in edema factor association with protective antigen and entrance into host cells. The C-terminal 60-kDa domain consists of CaM-sensitive adenylyl cyclase activity. The N-terminal portion of this 60-kDa region is similar to the adenylyl cyclase exotoxins from B. pertussisand P. aeruginosa, with 34 and 29% sequence identity, respectively. However, this region has no significant similarity to the adenylyl cyclases from enterobacteria and eukaryotes. In contrast to mammalian adenylyl cyclases that contain a catalytic core composed of two homologous domains, edema factor and its related exotoxins have no internal sequence repeat and are active as monomers (
). CaM is present in all eukaryotic cells, and it binds to and modulates, in a Ca2+-dependent manner, a number of enzymes including myosin light chain kinase, cyclic nucleotide phosphodiesterase, calcineurin, phosphorylase kinase, and adenylyl cyclase (
). There are two globular domains in CaM, each containing two Ca2+-binding EF-hand motifs. The two globular domains are connected by a continuous single α-helix that gives CaM its characteristic dumbbell shape. Binding of calcium ions has been shown to induce the movement of a pair of helices in a two EF-hand domain. This leads to an opening of the domain and an exposure of a hydrophobic pocket, which becomes a target interaction site. Blocking this opening by introducing a disulfide bond in either domain reversibly inactivates CaM (
CaM binding sites have been identified in a number of enzymes. With a few exceptions they are amphipathic helices capable of interacting with both domains of CaM in a 1:1 complex. The structures of the Ca2+/CaM complexes with the CaM-binding domain of MLCK and CaM-dependent kinase type II have been solved (
). In both structures, the two globular domains of CaM (residues 6–73 and 83–146) remain essentially unchanged upon complex formation. However, the long central helix (residues 65–93) of CaM, which connects the two domains in the crystal structure of calcium-bound CaM, is disrupted. This enables the C- and N-terminal domains to clamp the helical peptide, bringing the two domains of CaM close to each other.
In this study, we use biochemical, genetic, and spectroscopic methods to analyze the regulation of edema factor by CaM and gain insight into its catalytic mechanism. Our results show that whereas the mode of activation for edema factor is distinctly different from that seen in mammalian adenylyl cyclases, these two classes of enzymes are likely to share a common catalytic mechanism.
EXPERIMENTAL PROCEDURES
Materials
The gene encoding edema factor was kindly provided by Dr. S. Leppla (NIDR, National Institutes of Health). Restriction enzymes and Vent DNA polymerase were from New England BioLabs; QuikChange kit and XL-1 red competent cells were from Stratagene; Bradford reagent was from Bio-Rad; Ni2+-NTA resin and anti-H5 antisera were from Qiagen.N-iodoacetyl-N′-(5-sulfo-1-naphthyl)ethylenediamine (1,5-IAEDANS) andN-(dimethylamino-3,5-dinitrophenyl)-maleimide (DAB)-mal probes were from Molecular Probes.
Expression of Edema Factor and CaM
To construct the plasmid for the expression of EF-cya62 (aa 262–800) and EF3 (aa 291–800) (pProExH6-EF-cya62 and pProExH6-EF3, respectively), restriction siteNotI was first introduced to the appropriate codons by site-directed mutagenesis (QuikChange kit, Stratagene). The resulting plasmid was then digested with NotI and KpnI, and the fragment that encoded EF-cya62 or EF3 was isolated and ligated to pProEx-H6, which was digested with the same enzymes to express a recombinant edema factor, hexahistidine-tagged at its N terminus. A similar strategy was employed to generate pProEx-CH6-EF3 that had a C-terminal hexahistidine tag. The plasmid for the expression of EF3-N (aa 291–640) was generated by introducing a termination codon at residue 641 using pProExH6-cya62 as a template. The plasmid for the expression of EF3-C (aa 641–800) was made by first introducing aNotI site at the codons for aa 638–640 of pProExCH6-EF3 and subsequent removal of the fragment for the coding region (aa 291 to 640).
To express the truncated forms of edema factor, the appropriate plasmids were transformed into Escherichia coli BL21(DE3) cells that harbored pUBS520, a plasmid that encodes tRNA for the AGA and AGG codons. The resulting cells were grown in a modified T7 medium with 50 μg/ml ampicillin and 25 μg/ml kanamycin at 25–30 °C toA600 = 0.4, induced by adding isopropyl-1-thiogalactopyranoside to a final concentration of 100 μm and harvested 12–19-h postinduction. All the subsequent procedures were performed in a 4 °C cold room. E. coli cells were lysed in T20β5P0.1 buffer (20 mm Tris-HCl, pH 8.0, at 4 °C; 5 mmβ-mercaptoethanol; 0.1 mm phenylmethylsulfonyl fluoride) with added lysozyme to 0.1 mg/ml final concentration and a 4-min sonication with a 1-s on and 3-s off cycle. The supernatant of E. coli lysate was applied directly onto a Ni2+-NTA column that was equilibrated with T20β5P0.1N100(T20β5P0.1 containing 100 mm NaCl). The Ni2+-NTA column was washed with T20β5P0.1N500 and then T20β5P0.1N100I20(T20β5P0.1N100containing 20 mm imidazole, pH 7.0. The column was then eluted with the high imidazole buffer, T20β5P0.1N100I150. The eluate was diluted 5-fold with M20E1D1P0.1 (30 mm Mes, pH 6.5, 1 mm EDTA, 1 mmdithiothreitol, 0.1 mm phenylmethylsulfonyl fluoride) and applied to an Amersham Pharmacia Biotech FPLC SP-Sepharose column that had been equilibrated with M20E1D1P0.1, pH 6.5. The column was washed with one-column volume of M20E1D1P0.1, pH 6.5, and absorbed protein was eluted at 2 ml/min with a linear NaCl gradient (500–550 mm, 350–400 mm, and 450–500 mm NaCl for purification of EF3, EF3-N, and EF3-C, respectively) in M20E1D1P0.1. The protein peaks of edema factor fragments were analysed for molecular size on SDS-PAGE and for EF3 by adenylyl cyclase activity. Purified EF3, EF3-N, and EF3-C were concentrated in a Centricon 10 microconcentrator (Amicon) and stored at −80 °C (protein concentration >5 mg/ml).
An alternative scheme was also used to purify EF3 based on the fact that little binding of EF3 to a DEAE column at pH 7.0 was observed. In this case, E. coli cells were lysed in M30E4N25P0.1, pH 7.0, as described above. Lysate was passed first through a DEAE column and then directly onto an Amersham Pharmacia Biotech FPLC SP-Sepharose column, and columns were washed with three volumes of M30E4P0.1, pH 7.0. EF3 protein was then eluted off with a NaCl gradient in M30P0.1as the major protein peak and identified by SDS-PAGE. The fractions containing EF3 were pooled and loaded onto a Ni2+-NTA column. The Ni2+-NTA column was washed with T20β5P0.1N100I20and eluted with T20β5P0.1N100I150. Purified EF3 protein was concentrated as described above. This procedure modestly increased purification of the protein.
Proteolytic Analysis of Edema Factor
Purified EF3 and CaM were exchanged into digest solution (10% glycerol, 50 mmTris-HCl, pH 7.5, 1 mm MgCl2, 1 mmCaCl2) by dialysis and two protein stock solutions were prepared for digestion. One contained only EF3, the other a 1:2 molar mixture of EF3 and CaM. The concentration of EF3 (0.5 mg/ml) was identical in both samples. Nine different protease stock solutions were prepared in the same digest solution. Digests with thermolysin, endoproteinase Lys-C, endoproteinase Glu-C (Roche Molecular Biochemicals), elastase, chymotrypsin, trypsin, protease type X, endoproteinase Arg-C (Sigma) and thrombin (heme Tech) were performed at two different protease concentrations, one 10-fold more dilute that the other. The protease concentrations were 1:100 and 1:1000 (w/w) for all proteases except for endoproteinase Lys-C, which was used at 0.03 and 0.003 units of protease/mg of protein. All digestions were held at 4 °C. Samples were taken after 10 min and 1, 4, and 14 h. All samples were transferred directly from the digestion mixture into hot SDS loading buffer and subjected to SDS-PAGE.
Adenylyl Cyclase Assay
Activity was assayed for 5–20 min at 30 °C in the presence of 10 mm MgCl2 (
). Full-length human and fruit fly CaM was expressed in bacteria and purified using a previously published protocol and yielded ∼5–10 mg of purified protein per liter of culture (
). When applicable, recombinant EF3 was premixed with CaM on ice for 5 min before the assay, and assays were done in the presence of 50 μmCaCl2.
Genetic Screen for Loss-of-function EF3 Mutants
This screen is based on the observation that overexpression of EF3 and CaM significantly reduces the viability of E. coli. The coding region encoding fruit fly CaM was generated by polymerase chain reaction using Vent DNA polymerase and cloned into pBB131, resulting in pBB131-dCaM. A library of plasmids that had mutations in the coding region of EF3 were constructed by propagating plasmid pProExH6-EF3 inE. coli mutator strain, XL-1 red (mutD5, mutS, mutT). Plasmid DNAs were isolated and transformed to E. coli Δcya strain, TP2000 that harbored pBB131-dCaM[TP2000(dCaM)]. The transformants were plated directly onto Luria broth plate containing 100 μmisopropyl-1-thiogalactopyranoside, 50 μg/ml ampicillin, and 25 μg/ml kanamycin). Plasmid DNAs from the resulting colonies were isolated and retransformed into TP2000(dCaM) to confirm the viability. The coding region of EF3 was then sequenced.
Measurements of Fluorescence Resonance Energy Transfer
Steady-state fluorescence emission spectra were recorded on a SPEX Fluorolog 2/2/2 photon-counting spectrofluorometer (Edison, NJ). Fluorescence lifetime measurements were carried out by the method of time-correlated single photon-counting on a modified ORTEC 9200 nanosecond fluorometer (
). The fluorescence donor was 1, 5-IAEDANS and the acceptor was DAB. The CaM mutant having cysteine at amino acid residues 34 and 110 was constructed and labeled randomly with donor and acceptor or with donor alone as described.
P. O'Hara, Y. Mabuchi, and Z. Grabarek, submitted for publication.
RESULTS
Expression and Characterization of the Catalytic Domain of Edema Factor
We have developed a novel purification scheme to facilitate large-scale production of the catalytic domain of edema factor. Our approach began with modifications of the edema factor gene that replaced the binding region of protective antigen with a hexahistidine tag at the N terminus. The expression of the truncated protein was monitored both by adenylyl cyclase assay and by immunoblot with anti-H5 antisera. We first constructed and analyzed the truncation mutant, EF-cya62 (aa 262–800) as previously reported (
). As expected, it had high adenylyl cyclase activity when stimulated by Ca2+/CaM. A progressive deletion of residues from the N and C termini of EF-cya62 was performed to search for an optimal construct for the expression of the functional, CaM-sensitive adenylyl cyclase. Deletion of 290 amino acids from the N terminus significantly improved the expression of a functional protein, and the product could be clearly visualized after the whole-cell lysate was run on a gel under the optimum induction conditions (100 μmisopropyl-1-thiogalactopyranoside over 12 h in enriched media at 25–30 °C). This construct is designated “EF3” (aa 291–800). Further deletion from either the N- or C-terminal end progressively reduced both protein expression and catalytic activity.
EF3 can be effectively purified using Ni2+-NTA and SP-Sepharose, resulting in greater than 95% pure protein as judged by SDS-PAGE. This preparation yields approximately 42 mg/liter E. coli culture, roughly 10-fold better than that from cells expressing EF-cya62. The enzyme activity of EF3 is similar to that of EF-cya62. Both exhibit relatively low activity without CaM (0.5–1 μmol/mg/min) and can be stimulated by CaM by about 1000-fold. Because of its relatively basic pI (8.9), EF3 does not bind to the DEAE column at pH 7.0. Injecting the cell lysate through the DEAE column and then purifying it on SP-Sepharose and Ni2+-NTA columns, produced a slightly purer EF3 (∼97% pure) preparation. A C-terminal-tagged EF3 was also constructed and purified. Its yield was lower, 12 mg/liter culture, though the proteolytic contamination was mildly reduced. We analyzed the molecular weight of native EF3 by Superdex 200 gel permeation column and found that EF3 migrated as a 60-kDa protein. This is consistent with the notion that EF3 is a monomer.
Structural Insights from Proteolytic Digestion Experiments
We have probed the structural stability of EF3 with digestion by nine different proteases (thermolysin, endoproteinase Lys-C, elastase, chymotrypsin, trypsin, protease type X, endoproteinase Glu-C, endoproteinase Arg-C, and thrombin) in the presence or absence of Ca2+/CaM. The first six of these proteases showed a very similar digestion pattern (Fig. 1). In the presence of CaM (Fig. 1, bottom), little digestion of EF3 was observed. In the absence of CaM (Fig. 1, top), all six enzymes digested EF3 producing a 43-kDa fragment. Using N-terminal sequencing and mass spectrometry we found that the most stable proteolytic fragment (that generated by endoproteinase Lys-C) is a product of C-terminal truncation of 160 amino acids. These results suggest that the N-terminal 43-kDa fragment of EF3 constitutes an independent structural domain and that the 160-residue C-terminal of EF3 is structurally disordered in the absence of CaM.
Figure 1SDS-PAGE showing the proteolytic products of EF3 alone or in complex with CaM. All digestions were performed at 4 °C. The digestion conditions shown for the individual proteases are: thermolysin (1:1000 mass ratio of protease/EF3) 5 h, endoproteinase Lys-C (1:100) 3 h, elastase (1:100) 3 h, chymotrypsin (1:1000) 3 h, trypsin (1:1000) 14 h, and protease type X (1:100) 14 h.
To address the question of whether the catalytic domain of edema factor can indeed be further divided into two subdomains, we constructed and purified EF3-N (aa 291–640) and EF3-C (aa 641–800) (Fig.2A). The 43-kDa EF3-N was expressed and purified with a yield of 2 mg/liter. It had only one-tenth of the basal activity of the EF3 fragment and was only modestly activated by CaM (about 10–30-fold compared with more than 1000-fold stimulation for the EF3 fragment, Fig. 2B). The 19-kDa EF3-C fragment was also expressed and purified with a yield of only 0.5 mg/liter and a minor contamination of a 25-kDa protein (Fig.2A). By itself, EF3-C had no detectable catalytic activity (data not shown); however, with addition to EF3-N, EF3-C fully restored the CaM activation of EF3-N (Fig. 2B). Taken together with the proteolysis data, these results indicate that the 43-kDa EF3-N fragment indeed comprises the catalytic core of the edema factor. This domain appears to be structurally independent and capable of being regulated by Ca2+/CaM. It appears that the C-terminal 160-residue long EF3-C fragment has little structure on its own, but greatly enhances the Ca2+/CaM-dependent activation of EF3-N. In the presence of Ca2+/CaM, both segments of EF3 form a structurally stable catalytic-active entity. We further infer from these data that both EF3-N and EF3-C contain CaM binding sites.
Figure 2Characterization of the edema factor fragments expressed in E. coli.A, Coomassie Blue-stained SDS-polyacrylamide gels of purified edema factor fragments: EF3, EF3-N, and EF3-C. B, catalytic activity of EF3-N in the presence or absence of CaM, as a function of increasing concentrations of EF3-C. Adenylyl cyclase activity was measured with 10 ng of EF3-N (+CaM) or 500 ng of EF3-N (−CaM). The assay was performed for 5 min at 30 °C. Barsindicating mean ± S.E. are representative of at least two experiments.
To determine the conformation of CaM in the complex with EF3, we employed a fluorescence energy transfer technique. With CaM34/110, a two-cysteine CaM mutant in which Cys residues had been substituted for Thr-34 and Thr-110 in the N- and C-terminal domain, respectively.2 The Cα atoms of Thr-34 and Thr-110 are 54 Å apart in the crystal structure of Ca2+-bound CaM and ∼16 Å apart in CaM complexes with the synthetic peptides corresponding to the CaM-binding sites of myosin light chain kinase (M13) and CaM-kinase II. Thus, when labeled with a resonance energy transfer pair of probes, CaM34/110 is an extremely sensitive indicator of the global conformation of CaM.2 CaM34/110 was randomly labeled with 1,5-IAEDANS as a donor and DAB as an acceptor. For the control experiments, the protein was labeled with the donor alone. The fluorescence signal and the lifetime of IAEDANS alone attached to CaM34/110 are virtually insensitive to Ca2+ and to the addition of EF3 or M13 (Figs. 3 and 4), indicating that the immediate environment of the probes attached to CaM34/110 is not affected. Addition of Ca2+ to CaM34/110-IAEDANS/DAB causes a small decrease in fluorescence, which apparently reflects an increased dynamic of CaM and consequently a decrease in the average distance between the two domains of CaM in solution. An addition of M13 causes a large decrease in fluorescence intensity and a decrease in the lifetime to 3 ns, consistent with the compact conformation of the CaM·M13 complex (Fig. 3).2 In contrast to the M13 peptide, EF3 did not cause any decrease in fluorescence of CaM34/110-AEDANS, DAB. On the contrary, an addition of EF3 causes a reversal of the Ca2+ effect, and the fluorescence intensity of the complex exceeds that of CaM34/110-AEDANS, DAB alone in the absence of Ca2+ (Figs. 3 and 4). Because the CaM34/110 labeled only with the donor showed little change in fluorescence upon EF3 binding, we conclude that the change observed for the donor/acceptor-labeled CaM34/110 results from an increase in the distance between the N- and C-terminal domains of CaM upon interaction with EF3. The fluorescence decay of CaM34/110-AEDANS, DAB in the presence of CaCl2 could be fitted with two lifetimes (τ1 = 10.8 ns and τ2 = 6.9 ns) with relative contributions of 0.63 and 0.34, respectively, consistent with the dynamic nature of CaM in solution.2 In contrast, the fluorescence decay of EF3 complexes with singly and doubly labeled CaM34/110 were strictly monoexponential with lifetimes of 12.9 ns and 10.3 ns, respectively (Fig. 4). The distance between the probes calculated from these values assuming the orientation factor κ2 = 2/3 was 49.6 Å. We conclude that CaM adopts an extended conformation in its complex with EF3, approximately similar to the conformation in the crystal structure of the Ca2+-bound free CaM.
Figure 3Fluorescence emission spectra of CaM34/110 labeled with a resonance energy transfer donor only (A) or with a donor-acceptor pair (B). The concentrations of CaM34/110 and EF3 were 0.5 and 2 μm, respectively. λexc = 335 nm. M13, the CaM-binding fragment of myosin light chain kinase inB is shown for comparison as an example of a CaM target inducing a compact conformation. The fluorescence spectra for both EF3 and M13 complexes are recorded in the presence of 1 mmCaCl2. Note that the probe (AEDANS) attached to CaM34/110 is sensitive to Ca2+ and target-binding only in the presence of the acceptor (DAB) indicating that the change in fluorescence intensity reflects the change in the distance between the probes. An increase in fluorescence upon EF3 binding indicates an elongated structure whereas a dramatic decrease in fluorescence upon M13 binding results from CaM adopting a compact structure.
Figure 4Effect of EF3 on the fluorescence emission decay of CaM34/110 labeled with AEDANS and AEDANS, DAB. Fluorescence decay is shown for the EF3 complex with CaM34/110 labeled with AEDANS only (dots) and AEDANS, DAB (solid line), as well as for CaM34/110-AEDANS, DAB in the absence of EF3 (dashed line). The measurements were performed in the presence of 1 mm CaCl2, 0.1 m NaCl, 20 mm Tris-HCl, pH 7.5. Protein concentrations were 1 and 2 μm for CaM34/110 and EF3, respectively.
Equilibrium Binding Constant for the CaM34/110-AEDANS, DAB Interaction with EF3
We have utilized the change in fluorescence signal of CaM34/110, AEDANS, DAB upon binding EF3 to estimate the apparent equilibrium binding constant for the complex formation. Fig.5 shows a fluorescence titration of CaM34/110, AEDANS, DAB with a concentrated solution of EF3 in the presence of Ca2+. Fitting of the data gaveKd = 2.9 × 10−8, which is in excellent agreement with the Kd = 2.3 × 10−8 value obtained by Labruyẽre et al.(8) from the EC50 of the EF-cya62 fragment activation by bovine brain calmodulin. Thus, neither the mutations introduced into CaM34/110, nor the attached spectroscopic probes affect in any measurable way the interaction between CaM and edema factor.
Figure 5Equilibrium titration of CaM34/110-AEDANS, DAB with EF3. The solid line represents the best fit with the equation F = Fo + ΔFν. Where Fo is the initial fluorescence, ΔF is the maximal change in fluorescence, and ν is the fraction of the CaM34/110-AEDANS, DAB bound to EF3. ν is a root of the quadratic equation: nAν2 − (Kd + nA + B)ν + B = 0; where A and B are the total concentrations of CaM34/110-AEDANS, DAB and EF3, respectively; n is the apparent stoichiometry and Kd is the apparent equilibrium dissociation constant. The parameters of the fit of the above titration data were that Kd = 2.9 × 10−8 and n = 0.85. Note that non-integer values for the fitted stoichiometry (n) result mainly from imprecise estimation of protein concentration.
Two sets of CaM mutants (Q and K series) that have single point mutations at each of four Ca2+-binding sites have been used to probe the individual contributions of the four calcium binding sites in modulating the activity of various enzymes regulated by CaM (
). The conserved glutamate at position 12 of the Ca2+-binding site was mutated to either glutamine (B1Q to B4Q) or lysine (B1K to B4K). In isolated CaM, each mutation effectively eliminates Ca2+-binding of the affected site and the associated Ca2+-induced conformational changes (
). We found that mutations at either site 3 or 4 drastically reduced the ability of CaM to activate the EF3 fragment of edema factor whereas mutations at either site 1 or 2 had little effect (Fig.6). Thus we hypothesize that calcium binding at the CaM C-terminal domain plays a particularly important role in edema factor activation. This is a very different pattern than that seen in activity assays using the same mutants to stimulate smooth and skeletal muscle MLCKs and type I mammalian adenylyl cyclase (
). The order of potency for a mutation within a given calcium-binding site to decrease the ability of CaM to activate the two MLCKs or AC1, followed the series 4,2,3,1 in decreasing potency.
Figure 6Activation of edema factor by wild-type CaM and two series of Ca2+ binding site mutants. EF3 (10 ng) was used for an adenylyl cyclase assay for 5 min at 30 °C.Bars indicating mean ± S.E. are representative of at least two experiments.
In view of the above results we tested the possibility that the C-terminal domain of CaM is not only necessary but also sufficient for the activation of EF3. We have expressed the N- and C-terminal domains of CaM that contained only Ca2+ binding sites 1/2 and sites 3/4, respectively, and tested their ability to activate EF3. We observed no activation of EF3 by the individual CaM domains (data not shown). This suggests that a contribution from both N- and C-terminal domains of CaM are required for the activation of EF3.
Mutational Analysis to Probe the Residues That are Crucial for Catalysis of EF3
Mutational analysis of edema factor and related proteins has indicated several amino acid residues that are important in catalysis. The results are summarized in Fig.7. Single mutations at lysine 346 and 353 of edema factor and one of the homologous residue (Lys-65) of B. pertussis adenylyl cyclase significantly reduced catalytic activity in both enzymes (
). These two residues are hypothesized to bind nucleotide based on their fit to the consensus sequence GXXX(G/A)KS (fit in both forward and reverse directions) found generally in nucleotide triphosphate-binding proteins (
). However, in the homologous adenylyl cyclase from P. aeruginosa, this residue is lysine, casting doubt on this hypothesis. Single point mutations at the two conserved aspartates of both B. pertussis and P. aeruginosa AC (residues homologous to 491 and 493 of edema factor) reduced catalytic activity over 100-fold and therefore are also possibly involved in nucleotide binding (
Figure 7Sequence alignment of edema factor and its relatives. The residues important for catalysis are marked ∧ and * for those previously or currently reported in this paper, respectively.
In an attempt to coexpress the edema factor and CaM simultaneously inE. coli, we uncovered findings that allowed us to develop a genetic screen for residues critical for catalysis by the edema factor. The expression of edema factor alone did not complement the defect of cAMP in E. coli. Interestingly, we found that it was extremely difficult to transform the plasmid for the expression of edema factor into the cells that already harbor the compatible plasmid for CaM expression. We hypothesized that the expression of edema factor and CaM might be toxic because of cAMP overproduction. Similar toxicity was observed when adenylyl cyclase from E. coli was overexpressed (
). To test this hypothesis, we passed the plasmid that harbors the gene for the expression of edema factor through the mutator strain, XL-1 red (mutD5, mutS, mutT) to introduce random mutations. After the pool of the mutated plasmids was transformed into E. coli Δcya, a small number of colonies could be recovered. Two of such colonies were characterized further. Although they expressed normally, based on immunoblot analysis, the adenylyl cyclase activities were significantly reduced. Sequence analysis showed that both carried a single mutation resulting in the change of the highly conserved amino acid residue 491 from aspartate to asparagine (D491N) (Fig. 7).
The conserved aspartate pair is involved in “two-metal” mediated catalysis of nucleotidyltransferases, including DNA polymerases and mammalian adenylyl cyclases (
). Edema factor family has three conserved negatively charged residues, Glu-443, Asp-491, and Asp-493, and we made two additional mutants, E443Q and D493N to analyze the role of these three negatively charged residues (Fig. 7). An arginine in mammalian adenylyl cyclase is implicated in stabilizing the pentaphosphate intermediate (
). We thus made and analyzed a mutant that had a mutation at one of the two conserved arginine residues, R329M (Fig. 7). All four mutants, R329M, E443Q, D491N, and D493N were purified to homogeneity with the protein yields similar to wild-type EF3. All mutants had relatively normal EC50values for CaM activation (Fig. 8). All four mutants also had relatively normal Km for ATP (0.5 ± 0.1, 0.2 ± 0.02, 0.3 ± 0.1, 0.22 ± 0.07, and 0.27 ± 0.03 mm for wild type, R329M, E443Q, D491N, and D493N, respectively). However, D491N, D493N, and R329M had significantly reduced catalytic activity (10,000-, 5,000-, and 300-fold in Vmax values of CaM activation, respectively) whereas E443Q only had about a 10-fold reduction (Fig. 8). These results suggest that Asp-491, Asp-493, and Arg-329 residues are important for catalysis.
Figure 8The activation of edema factor and its mutants by CaM. The adenylyl cyclase assay was performed with EF3 (10 ng), EF3(R329M) (10 ng), EF3(E443Q) (1 μg), EF3(D491N) (50 μg), or EF3(D493N) (50 μg) at 30 °C for 5 min. Barsindicating mean ± S.E. are representative of at least two experiments.
We have combined biochemical, spectroscopic, and mutational approaches to analyze the mechanism of edema factor activation by CaM. Based on the above results, we have developed a hypothetical “low resolution” model. Our model is derived from the following observed constraints. (i) The 60-kDa monomeric EF3 fragment of edema factor consists of a highly structured core domain and of a loosely structured, approximately 160-residue long, C-terminal domain. (ii) The C-terminal region acquires a well defined structure (becomes resistant to proteolysis) upon CaM binding to EF3. (iii) The C-terminal region is essential for full CaM activation of edema factor. (iv) CaM has an extended conformation while bound to EF3. (v) Whereas both domains of CaM are required for the activation of edema factor, calcium binding in the C-terminal domain plays a privileged role in activation of edema factor when compared with calcium binding in the N-terminal domain. We infer from these empirical constraints that the CaM-dependent activation of edema factor involves a change in relative position of a part of or the entire C-terminal domain with respect to the N-terminal core domain. This type of reorientation could result in the stabilization of a catalytically active state of the N-terminal region or even directly contribute residues to aid in catalysis.
Because the functional unit of EF3 is a monomer and because of the expected large conformational change in the C-terminal domain of EF3 induced by CaM, we predict that the mechanism of activation of EF3 by CaM is very different from that seen in mammalian adenylyl cyclases. Mammalian membrane-bound adenylyl cyclases (AC1–AC9) have two homologous cyclase domains (C1 and C2) punctuated by two transmembrane domains (M1 and M2) (
). They are diversely regulated by intracellular proteins, i.e. G-proteins, protein kinases, and CaM. Molecular structures of a catalytically inactive C2a dimer and catalytically active C1a·C2a·Gsα complex have recently been determined (
). The catalytic site occurs at the interface of the two homologous cyclase domains and includes residues from both regions. The structural mode of activation of adenylyl cyclase does not involve a major conformational change in either C1a or C2a domains upon binding of the stimulatory G-protein. Instead, catalysis is activated by an induced juxtaposition of these two domains to properly form the catalytic cleft at their interface. With the lack of any internal homologous repeat within EF3 and a different potency order for the importance of calcium binding sites in CaM, our model predicts that the structural events leading to EF3 activation by CaM will have little relation to those responsible for mammalian adenylyl cyclase activation by CaM.
The homologous internally repeated cyclase domains of mammalian adenylyl cyclase contain substructures that resemble the palm domains of the polymerase I family of prokaryotic DNA polymerases, includingE. coli DNA polymerase and Thermus aquaticus(Taq) polymerase. More strikingly, DNA polymerase and adenylyl cyclase also share the same catalytic mechanism, using two metal ions to polarize the 3′-hydroxyl of the nucleotide and thus stabilize a pentavalent intermediate (
). Two metal ions are coordinated by a pair of aspartates that are spatially close, commonly seen as a DXD sequence in DNA polymerases. Interestingly, edema factor has a single DXD motif and we have shown that mutations at these two aspartates can drastically reduce catalysis (
). We have found that arginine 329 of edema factor is crucial for catalysis and may play a similar role in stabilizing the catalytic intermediate. With no other structural data available on the catalytic site of edema factor, the toxin's structural solution for the performance of a two-metal-mediated catalysis remains unclear.
CaM adopts both extended and compact structures. It is well established that the compact conformation of CaM can bind an amphipathic peptide that leads to the activation of numerous CaM-regulated enzymes. There is significant evidence for other entirely different types of CaM target complexes and therefore possibly different mechanisms of target activation. For example, low angle x-ray scattering data shows that whereas one of the two CaM-binding peptides from phosphorylase kinase (PhK5) induces a compact conformation in CaM, the other (PhK13) induces an extended conformation (
), have identified an amphipathic helix derived from residues 499–532 of edema factor that binds CaM, thus possibly localizing a site for CaM interaction. In addition, Munier et al. (
), have performed a cross-linking study that localized CaM binding to residues 613–767. Whereas distances in primary sequence are poor predictors of spatial distances within folded proteins, the differing results for the location of a single CaM-binding site are commensurate with two binding sites interacting with an extended CaM. Additional spectroscopic and mutational analyses in combination with molecular structure determination of edema factor alone and edema factor/CaM complex are currently in progress, and the solutions should yield valuable insight into how CaM activates the catalytic activity of edema factor.
Acknowledgments
We are grateful to P. Gardner (Howard Hughes Medical Institute, University of Chicago) for help in oligonucleotide synthesis and DNA sequencing and superb technical assistance from Z.-H. Huang.
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