Two Interaction Sites on Mammalian Adenylyl Cyclase Type I and II: modulation by calmodulin and Gβγ Susanne Diel, Michael Beyermann, Juana M Navarro Lloréns, Burghardt Wittig, Christiane Kleuss
To cite this version:
Susanne Diel, Michael Beyermann, Juana M Navarro Lloréns, Burghardt Wittig, Christiane Kleuss. Two Interaction Sites on Mammalian Adenylyl Cyclase Type I and II: modulation by calmodulin and Gβγ. Biochemical Journal, Portland Press, 2008, 411 (2), pp.449-456. 10.1042/BJ20071204. hal-00478881
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TWO INTERACTION SITES ON MAMMALIAN ADENYLYL CYCLASE TYPE I AND II:
Modulation by Calmodulin and G
¶
Susanne Diel*, Michael Beyermann*, Juana María Navarro Lloréns†,
Burghardt Wittig‡, Christiane Kleuss§¶
*Leibniz-Institut für Molekulare Pharmakologie, Dept. Peptide Chemistry & Biochemistry; †Universidad Complutense de Madrid, Departamento de Bioquímica y Biología Molecular I; ‡Charité – Universitaetsmedizin Berlin, Institut für Molekularbiologie UND Bioinformatik, 14195 Berlin, Arnimallee 22, Germany; §Mologen AG ¶correspondence: Tel.: +49-30-84451594, Fax: +49-30-84451516; [email protected]
PAGE HEADING TITLE: adenylyl cyclase regulation THIS IS NOT THE FINAL VERSION - see doi:10.1042/BJ20071204
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TWO INTERACTION SITES ON MAMMALIAN ADENYLYL CYCLASE TYPE I AND II
Modulation by Calmodulin and G
SYNOPSIS
Mammalian adenylyl cyclases are integrating effector molecules in signal transduction regulated by a plethora of molecules in either additive or synergistic or antagonistic manner. Out of nine different isoforms, each adenylyl cyclase subtype uses an individual set of regulators. Here, we use chimeric constructs, point mutations, and peptide competition studies with adenylyl cyclases to show for the regulatory molecules G and calmodulin a common mechanism of multiple contact sites. Despite their chemical, structural, and functional variety and different target motifs on adenylyl cyclase, G and calmodulin share a two-site-interaction mechanism with G s and forskolin to modulate adenylyl cyclase activity. Forskolin and G s are known to interact with both cytosolic domains of adenylyl cyclase – from
inside the catalytic cleft as well as at the periphery. An individual interaction site located at C1 of the specifically regulated adenylyl cyclase subtype had been ascribed for both G and calmodulin. We now show for these two regulators of adenylyl cyclase that a second isoform- and regulator-specific contact site in C2 is necessary to render enzyme activity susceptible to G or calmodulin modulation: In addition to the PFAHL-motif in C1b of ACII, G contacts the KF-loop in C2, while calmodulin requires not only the 2+ 2+ Ca -independent AC28-region in C1b but also a Ca -dependent domain in C2a of ACI with the VLG-loop to stimulate this adenylyl cyclase isoform.
1ABBREVIATIONS: AA – alanyl-alanine; AC – adenylyl cyclase; AC28-region– sequence motif located in the
C1b domain of ACI; pAC28 – peptide comprising amino acids of the AC28-region; CaM – calmodulin; cAMP – THIS IS NOT THE FINAL VERSION - see doi:10.1042/BJ20071204
cyclic adenosine-3',5'-monophosphate; KF – sequence motif located in the C2a domain of ACII; NAAIRS –
asparagyl-alanyl-alanyl-isoleucyl-arginyl-serine; PFAHL – sequence motif located in the C1b domain of ACII;
VLG – sequence motif located in the C2a domain of ACI; wt – wild type
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INTRODUCTION
Adenylyl cyclases (AC1) catalyze the conversion of ATP into the universal second messenger cAMP. Class III ACs comprise a family of structurally similar enzymes (1) with a catalytic centre composed of
two pseudosymmetric domains, C1 and C2. Mammalian ACs contain those domains on a single polypeptide chain that is folded into two membrane regions each built up by six transmembrane helices; - C1 follows transmembrane region M1 and proceeds transmembrane region M2,C2 follows trans
membrane region M2 (Fig. 1A). On the basis of sequence similarity both cytosolic domains C1 and C2
comprise subdomains Ca and Cb. The C1a subdomain shares roughly 60% identity to C2a at amino acid level, and both subdomains heterodimerize to form the pseudosymmetrical catalytic core (2). Mammalian ACs are represented by at least nine different isoforms (ACI-ACIX) that have been cloned and analyzed (1). They are grouped into three subclasses (3) according to their regulatory molecules: ACI represents the prototype of calmodulin (CaM)-stimulated ACs, ACII belongs to the subgroup of ACs that are stimulated by G (G complex of the heterotrimeric G proteins), and ACV and ACVI 2+ are inhibited by submicromolar concentrations of Ca .G s ( subunit of the stimulatory heterotrimeric G protein) and forskolin are common stimulators of all those ACs, while inhibition by G i ( subunit of the inhibitory heterotrimeric G protein) is restricted to ACI as well as ACV and ACVI (4). Based on crystal structures of the catalytic domains of ACs, binding sites for forskolin and G s are
known. One molecule of forskolin binds to the C1+C2 heterodimer (5) at sites that are distinct to that for G s (6). Contacts between forskolin and both catalytic halves of AC occur at multiple amino acids
(K896, I940, G941, and S942 in C2 of ACII; F394, W507, V511, Y443 in C1 of ACV). When bound to
the domain interface, forskolin stabilizes the interaction between C1 and C2. Two structural elements of G s form the interface, primarily through contacts with C2. The most prominent interaction is the insertion of the G s switch II helix (residues 225 to 240) into the groove formed by 2' and the 3'- 4' loop of AC. The second contact surface is formed by the 3- 5 loop of G s, which interacts with both
C1 and C2 (6). THIS IS NOT THE FINAL VERSION - see doi:10.1042/BJ20071204
Obviously, domain association is a prerequisite for AC activity. However, C1+C2 assembly alone is clearly not sufficient to achieve the high level of AC activity displayed in the presence of forskolin, G s or both activators (7). Hence, it is assumed that the binding of forskolin or G s facilitates cAMP synthesis by altering the conformation of the active site. The 3D structure suggests one way such activation could occur: the 2'- 3' loop of C2 contacts K436 and L438 of the 2- 3 loop of C1, thereby linking residues that form the forskolin binding site with structural elements carrying residues important
for catalysis. In this model, the regulator contacts allosteric sites in C1 and C2 thereby rearranging
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residues at the C1/C2 cleft that positively impacts cAMP catalysis. Besides forskolin and G s, no other crystal structures are solved for AC complexed to regulatory proteins. Binding of G i was implicated to take place within the cleft formed by the 2and 3 helices of C1 (8), analogous but pseudosymmetric to the G s binding in C2. For both, CaM and G ,asingle
regulator-specific contact site was deduced so far: a) For CaM binding, an AC sequence in C1b (stretch of 28 amino acids forming the AC28-region) with high affinity for CaM had been identified (9). The AC28-region comprises a hydrophobic sequence containing basic amino acids as expected for CaM effector sites, and is located centrally in the cyclase. The corresponding peptide efficiently interferes with the stimulation of AC by CaM, but exhibits a higher affinity for CaM binding (2 nM) than does AC (Kd for CaM-mediated AC stimulation 15-20 nM; (9, 10)). b) G is a conditional regulator of ACs, i.e. both activation of ACII and inhibition of ACI are best observed at the pre-stimulated ACs (11). Recently, the PFAHL-motif in the variable C1b domain of ACII was shown to be indispensable for G - stimulation of the ACII (12). In the present work we show that G -, and also CaM-regulation of adenylyl cyclase isoforms required a second regulator-specific site in the other catalytic domain, C2.ForG the KF-loop in ACII between the 2' helix and the 2' sheet was identified, for CaM the VLG-loop at the C2a/C2b boundary of ACI. While the general adenylyl cyclase stimulators forskolin and G s contact conserved amino acids in C1 and C2, motifs of the isoform-specific regulators G and CaM were not conserved but rather showed subgroup specificity according to the different regulatory patterns of ACs. THIS IS NOT THE FINAL VERSION - see doi:10.1042/BJ20071204
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EXPERIMENTAL
Generation of AC constructs ACI with a N-terminal MYC-tag was generated by PCR using bovine cDNA (accession number P19754) encoding ACI as a template and primer myc-IM1C1 (5’-GGAGGAACTAGTACCATGGAA- CAAAAACTGATATCGGAAGAAGACCTCGCGGGGGCGCCGCGCGGCCGAGGC-3’). The PCR product was ligated into pBSKII+ (Stratagene) using SpeI and HindIII. Activity and regulation of this ACI (denoted ACI wt) was indistinguishable from untagged wild type ACI and was used as control throughout this work. The generation of constructs (Fig. 1B) encoding N- and C-terminal halves of ACI
(I-M1C1 and I-M2C2), ACII (II-M2C2), and ACV (V-M1C1) was performed by PCR-based mutagenesis
and has been described previously (13). Construction of ACII.IC1b was described (12). For generation of
ACI.IIC2 the oligonucleotides 5'- GCCGTCTCAGCAGAGTGAATATTACTGTAGGTTAG-3' and 5'- CCAAGCTTATTCAGGATGCCAAGTTGCTCTG-3' were applied in an analogous strategy using the endogenous restriction sites Bsu36I and HindIII. ACI-deletion mutant ACI.1057 was constructed using 5'-CTACACATCACCCGGGTCCAGTG-3' and 5'-CGAAAGCTTAGAAGTATGTCAGCATC- 3, and ACI. 1094 was constructed using 5'-CTACACATCACCCGGGTCCAGTG-3' and 5'-CGAAA- GCTTAGGGGTGACCCGC-3'; cloning was performed by the ACI-endogenous sites XmaI and
HindIII. Quick-Change Site-Directed Mutagenesis using PfuTurbo Cx Hotstart DNA Polymerase (Stra- tagene) was used according to manufacturer's instructions to generate NAAIRS1 and AA1 mutants. ACII.928 was generated analogously to the described procedure (12) using 5'-TGATGATCTGCTTT- CTAATGCTGCTATACGATCGGTTGAAAAGATCAAG-3' and the corresponding reverse comple- mentary oligonucleotide as primers. The ACII.AAxxx mutants were generated using primers: 5'-GCTG- ACTTTGATGATGCTGCTTCTAAGCCAAAGTTC-3' (ACII.AA925), 5'-TTTGATGATCTGCTTGC- TGCTCCAAAGTTCAGTGGT-3' (ACII.AA927), 5'- CTGCTTTCTAAGCCAGCTGCTAGTGGTGT- TGAAAAG-3' (ACII.AA930), and 5'-TCTAAGCCAAAGTTCGCTGCTGTTGAAAAGATCAAG-3' (ACII.AA932) in combination with their respective reverse complementary pendants. ACI-G mutants i THIS IS NOT THE FINAL VERSION - see doi:10.1042/BJ20071204 were generated using primers 5'-CCACGTTGCCCAGCACGCCCTAATGTCCAACCCTCG- 3'(ACI.F852A), 5'-GCACTTCCTAATG-TCCGCCCCTCGCAACATGGACC-3' (ACI.N856A), 5'-CC- TAATGTCCAACCCTGCCAACATGGA-CCTGTATTACC-3', (ACI.R858A), and 5'-CCTAATGTC- CAACCCTCGCGCCATGGACCTGTATT- ACC-3' (ACI.N859A).
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Synthetic peptides
pAC28: NH2-IKPAKRMKFKTVCYLLVQLMHCRKMFKA-COOH, derived from sequences located
in IC1b; pVLG: NH2-TEEVHRLLRRGSYRFVCRGKV-COOH, derived from sequences located in IC2a;
pAAG: NH2-TEEAHRLARRGSYRFVCRGKV-COOH, identical to pVLG except for two alanine
mutations at the CaM-critical residues V and L (underlined); pTT: NH2-TQPKTDHAHCCVEMGLDM-
IDT-COOH, derived from sequences located in IC1a.
Preparation of AC All AC constructs were expressed in Sf9 cells using the baculovirus expression system; purified plasma membranes were the sources for AC assays. Baculovirus encoding the AC was generated from the pFastBac1-AC construct in Sf9 insect cells (Invitrogen). Cells (106/ml) were then infected with the baculovirus (1 plaque-forming unit/cell), harvested 48-52 h later, and lysed by nitrogen cavitation. After removal of nuclei by centrifugation, membranes were collected, washed, and resuspended.
Regulators of AC G was expressed in Sf9 insect cells using baculoviruses encoding G 1 and G 2, detergent extracted and purified by affinity chromatography as described using the G -attached N-terminal His-tag (14). G s was expressed in bacteria and purified using the C-terminal attached His-tag (16). Purified G s was activated in vitro with guanosine 5´-[ thio]triphosphate (GTP S) by incubation for 30 min at 30 °C and 1 h on ice. The activated G s-GTP S was purified by subsequent size exclusion chromatography to remove the free nucleotide. The concentration of the activated G s was determined by inclusion of radiolabeled [35S]-GTP S during activation. CaM was purchased from Calbiochem.
Dansyl-CaM
Equimolar amounts of dansylchloride and N-hydroxysuccinimide were mixed in dimethylformamide THIS IS NOT THE FINAL VERSION - see doi:10.1042/BJ20071204 and activated by addition of 1 equivalent of triethylamine. Activated dansylester was incubated with 1/4 equivalent CaM at pH 7.5 over night at room temperature before the formed dansyl-CaM was purified by size exclusion chromatography. This procedure was dissimilar to protocols used by others and Sigma in the past, but allowed the preferred dansylation of just the N-terminal amino acid of CaM, rather than an uncontrolled number of internal hydroxyl or amino groups.
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Dansyl-CaM binding A volume of 800 µl Dansyl-CaM (80 nM) was adjusted to the indicated concentrations of Ca2+ ions, peptides and/or EGTA; dilution effects were maintained below 3%. Fluorescence emission spectra were recorded from =450 nm to =550 nm ( ex=334 nm, 20 nm/min) at 27 °C with the Luminescence Spectrometer LS50B (Perkin Elmer).
Enzyme assays AC activity was determined based on the conversion of [32P]-ATP to 32P-cAMP with subsequent purification of cAMP by sequential chromatography using cation exchange (Dowex 50) and neutral alumina resins. All samples contained pyruvate kinase and phosphoenol pyruvate as ATP generating system and Ro 20-1724 as inhibitor of cAMP-specific phosphodiesterases. Assays were performed for 7-10 min at 30 °C in a final volume of 100 µl with the indicated amounts of recombinant Sf9
membranes in the presence of 10 mM MgCl2 and 0.5 mM ATP. To determine CaM-regulation, AC containing membranes were washed with 1 mM EGTA to remove
Sf9-endogenous CaM, then AC activity was determined in the presence of 100 µM CaCl2. After a 2-min pre-incubation at 30 °C the AC assay was started by addition of the substrate and stopped after another 7 min incubation period. For peptide competition studies, peptides were pre-incubated for 60 min on ice in the presence of CaM. CaMkinase assays were performed according to the manufacturer's instructions (SignaTECT from Promega).
Miscellaneous Membrane proteins were quantified by dye binding using acidic Coomassie Brilliant Blue (BioRad) with bovine serum albumin as standard. Immunodetection of AC constructs was performed with commercially available antibodies anti-c-MYC: 9E10 (Santa Cruz), anti-ACII: C20 (Santa Cruz) and
anti-HA: 12CA5 (Roche). THIS IS NOT THE FINAL VERSION - see doi:10.1042/BJ20071204
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Results and Discussion
A second CaM site in ACI
Amino acids 495-522 (denoted as AC28-region) in the C1b domain of ACI were shown to be necessary for stimulation of this AC subtype by CaM and were represented by the synthetic peptide pAC28 (9). To
test if the AC28-region in the context of the intact C1b domain of ACI was able to transfer CaM-
stimulation to the CaM-insensitive ACII, we generated the ACII.IC1b chimera (see Fig. 1). The data shown in Fig. 2 revealed that the presence of the AC28-region was not sufficient to turn ACII into a
CaM-stimulated ACII.IC1b. Another chimera indicated a second site located in C2 necessary for CaM to
stimulate the catalytic activity of type I AC. This second chimera, ACI.IIC2, was a type I like AC with
IC2 substituted by the homologous C2 domain of the CaM-insensitive ACII. ACI.IIC2 was no longer regulated by CaM, although it was still stimulated by G s and forskolin; indeed, G s- and forskolin
stimulated activities of ACI.IIC2 by far exceeded those of parent wild type ACs, type I and type II (data not shown).
In order to localize the missing CaM motif in either C2a or C2b of ACI, two deletion mutants of ACI were generated (see Fig. 1). In ACI. 1094 the 44 C-terminal residues of the C2b domain were removed; ACI. 1057 was devoid of all amino acids assigned to C2b. Both deletion mutants were active and
stimulated by Ca/CaM as well as ACI (Fig. 3). These results provided evidence that the C2a of ACI domain was necessary for CaM to stimulate ACI.
In order to define the amino acids in C2a responsible for mediating the CaM-stimulatory signal to ACI,
the catalytic C2a subdomain was checked for regions matching the rules for putative CaM interaction sites described by Rhoads et al. (17). Although no universal CaM binding motif had been defined and CaM interaction sites were difficult to predict, we could identify one stretch of 14 amino acids in the C-
terminus of the C2a subdomain of ACI that obeys the 1-5-8 rule (Fig. 4A): This domain contained hydrophobic residues at positions 1, 5, and 8 (valine V, leucine L, glycine G) and was therefore called the VLG-loop. The VLG-homologous region in the ACII crystal showed a helix-loop-helix structure THIS IS NOT THE FINAL VERSION - see doi:10.1042/BJ20071204 and was located in the periphery of the catalytic heterodimer (Fig. 4B). To show the involvement of the VLG-loop in CaM-stimulation of ACI, two amino acids at the key positions 1 and 5 of the motif, V1027 and L1031, were mutated to alanine. The resulting mutant was not catalytically active under basal, G s- or forskolin-stimulated conditions (not shown). The fact that point mutations within the VLG-region were sufficient to completely abolish the catalytic activity of the enzyme lead to the conclusion that this region was crucial for the integrity of the catalytic core. Its impact on AC's enzymatic function already became obvious when truncation immediately C-terminal to
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the VLG-region resulted in a mutant (ACI.1057) with reduced catalytic activity (see Fig. 3). To date
the crystal structure of the IC2a domain is not known. All structural data rely on sequence alignments of
IC2a with the IIC2a subdomain the structure of which had been resolved in complex with VC1a (6). In
ACII, lacking a C2b subdomain, the VLG-homologous sequence is assigned to the C-terminal part of the enzyme (see Fig. 4B) and has not been reported to be involved in catalysis. In contrast, the VLG-motif
of ACI is followed by the variable C2b subdomain, thereby forming the C2a/C2b boundary that might be crucial for the correct orientation of the two subdomains and thereby for the integrity of the catalytic core. As we had identified the key amino acids of the VLG-loop, V1027 and L1031, to be indispensable for the catalytic activity of ACI, peptide studies were applied to show indirect evidence for the involvement of that loop in CaM regulation. The peptide pVLG, comprising all amino acids of the VLG-loop, was synthesized and analyzed for direct CaM interaction and for competition with the CaM effectors ACI and the cyclase-unrelated CaM-dependent kinase II. The peptide corresponding to the VLG-loop did bind CaM as shown by fluorescence-changes of dansyl- CaM (Fig. 5A). Whereas the fluorescence intensity stayed unaltered in the presence of a non-binding peptide (pTT), increasing concentrations of pVLG lead to increasing intensity of the dansyl-CaM fluorescence. Similar fluorescence-changes were obtained using the peptide pAC28 covering the amino
acids of C1b, already known to be necessary for ACI stimulation by CaM. Obviously, the concentration
of 80 nM dansyl-CaM used in this experiment exceeded the Kd of pAC28 and pVLG for CaM as shown by the linear relation between fluorescence-change and peptide concentration below the equimolar ratio peptide/CaM indicating a 1:1 binding stochiometry. A minimum of 80 nM dansyl-CaM was necessary to obtain precise fluorescence data. When the key amino acids V and L of pVLG were changed to alanine (pAAG), fluorescence changes caused by the peptide were reduced, pointing to a sequence dependence of the VLG-region for binding to CaM. The peptide pVLG did not only bind CaM, but obviously occupied the effector-interacting epitope of CaM, as the CaM-pVLG complex could no longer stimulate the CaM effectors ACI (Fig. 5C ) and THIS IS NOT THE FINAL VERSION - see doi:10.1042/BJ20071204 CaM-dependent kinase II (Fig. 5D). Again, the importance of amino acids V1027 and L1031 in ACI (position 1 and 5 in the VLG-motif, respectively) for CaM binding became evident as pAAG competed less efficiently than pVLG with both effectors for CaM. Surprisingly, ACI inhibition was not exclusively mediated by peptide binding to CaM, but also by a direct peptide effect on ACI as depicted in the insert of Fig. 5C. Nevertheless, the calculated peptide net effect on CaM stimulation – corrected by the peptide effect on basal AC activity - was still more pronounced for pVLG than for pAAG. The phenomenon that pVLG and pAAG also inhibited the basal
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activity of ACI corroborated our earlier hypothesis evolved from the inactive point mutations in ACI that integrity of the VLG-region was a prerequisite for overall enzymatic activity of ACI. Due to read-out dependent differences in assay sensitivity, higher CaM concentrations were applied for peptide competitions with ACI than for dansyl-CaM binding (200 nM versus 80 nM; see Fig. 5C versus 5A). No differences between pAC28 and pVLG were detected in the fluorescence assay as 80 nM
dansyl-CaM was well above the Kd of pAC28 binding to CaM (9). In contrast, the AC readout with the coupled reaction scheme, involving CaM binding and AC interaction at two sites (see below), revealed
for pAC28 (IC50 500 nM) higher affinity in binding CaM than for pVLG (IC50 10 µM; not shown). This would point to a minor role of the VLG-motif in transmitting CaM-stimulation to ACI compared to the
AC28-region. However, despite its relatively high affinity for CaM, C1b harbouring the AC28-region could not be the relevant CaM-sensitive region of ACI for the following reasons: a) In the context of
chimera ACII.IC1b the AC28-region did not transmit the stimulatory Ca/CaM signal onto a Ca/CaM- insensitive AC isoform; b) while ACI activity modulation by CaM is known to be strongly Ca2+- dependent (10), pAC28 bound to CaM in a Ca2+-independent manner: Figure 5B shows that pAC28 binding was detected also in the absence of free calcium ions (“+EGTA”), a hitherto unappreciated phenomenon. In contrast, pVLG derived from the newly detected second CaM-site in ACI exhibited Ca2+-dependent binding to CaM (see Fig. 5B). Thereby, CaM-regulation became both highly potent by the AC28-region and Ca2+-sensitive by the VLG-region, establishing a molecular basis by which ACI activity can be changed during cytosolic calcium oscillations (24). A similar scenario was already described for the second CaM-stimulated AC isoform, ACVIII, with the
2+ N-terminus pre-assembling CaM and the C2 domain enabling both Ca -sensitivity and the stimulatory action of CaM (25). ACI and ACVIII do not provide identical signal transfer motifs for CaM, but their mechanistic modulation is based on the same two-site scheme. The pre-recruitment of CaM by the AC28-region and the N-terminus of ACI and ACVIII, respectively, is highly beneficial - if not essential - in the intact cell. In living cells, the majority of the total of 10 µM CaM is sequestered, tethered or compartmentalized, so the concentration of potential CaM binding proteins in the cytosol or cytoplasma THIS IS NOT THE FINAL VERSION - see doi:10.1042/BJ20071204 facing membranes exceeds that of the free, available CaM (ca. 45 nM) about 2-fold (26). In the resting cell, CaM-tethering is important to advance complex formation between the three partners Ca2+,CaM, and effector. Following an increase in intracellular calcium concentration, the AC-pre-associated CaM
might change its conformation and activate the AC involving the catalytic AC-domain C2a, supposingly by stabilizing structural elements like the VLG-loop. Similar activation schemes have already been described for the two AC exotoxins of Bordetella pertussis and Bacillus anthracis. CaM in an extended conformation binds to several loops close to the catalytic centre of the bacterial AC and activates the enzyme through subtle changes in the surroundings of the active cleft (27). Stage 2(a) POST-PRINT9
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A second G -site in ACII The structural basis of ACII regulation by G showed strong analogy to stimulation of ACI by CaM. Also for G , an obligatory motif in C1b had been described to observe ACII stimulation. Like the
AC28-region in ACI, amino acids of the PFAHL-motif in C1b of ACII had been identified as an essential G signal transfer site of ACII because substitution of IIC1b or just the PFAHL-motif by IC1b or irrelevant amino acids, respectively, completely abolished G -stimulation (12). Here, we showed that besides the PFAHL-motif on C1b, also domain C2 of ACII was important to mediate G -stimulation to sensitive ACs.
In a first step, AC was cut into halves comprising M1C1 and M2C2 (see Fig. 1). Both halves together
lined up to the bisected ACI (AChalvesI+I) and ACII (AChalvesII+II), and - after coexpression - functionally rebuilt the G -inhibited ACI (Fig. 6) and G -stimulated ACII (14). This proved that the G -
regulatory pattern was preserved even in the bisected AC. However - in a second step - IIM2C2 was co- expressed with IM1C1, the N-terminal half of the G -inhibited AC type I, or VM1C1, the N-terminal half of the G -insensitive canine AC V (18). Both resulting bisected chimeras AChalvesI+II and AChalvesV+II, respectively, were still stimulated by G (1.5-2-fold) indicating a G -stimulating feature
of IIM2C2. This stimulatory effect was even observed when only IIC2 was introduced into an ACI background (ACI.IIC2) pointing to a second G -site located in C2 of ACII. The inverse chimera ACII.IC2 was catalytically inactive under basal, G s- and forskolin-stimulated conditions (not shown). To define amino acids in C2 responsible for mediating the G -stimulatory signal to ACII, the catalytic
C2 domain was screened by NAAIRS-substitutions. The hexapeptide NAAIRS is a flexible linker adopting various secondary structures in different proteins. It therefore had been applied in different
substitution experiments, for example leading to the identification of the PFAHL-motif in the C1b domain of the ACII (12). Although all amino acids that were known from the literature to be involved in catalysis, in G s or in forskolin interaction were excluded from this screen, 50% of the generated mutants were not catalytically active; the remaining eight mutants that functionally could be tested were
all stimulated by G (not shown). THIS IS NOT THE FINAL VERSION - see doi:10.1042/BJ20071204 In a next screen we substituted only two instead of six amino acids. The alanyl-alanine substitutions
were introduced into a short loop (named KF-loop according to its central amino acids) in IIC2 that showed significant similarity for all three G -stimulated ACs (type II, IV, and VII; Fig. 7A), but none for the G -inhibited ACI or the G -insensitive canine ACV. Additionally the loop appeared an ideal target for regulators like G as it is located dorsal to the catalytic cleft, thereby supposed to be easily accessible for regulators (Fig. 7B). Moreover, the KF-loop connects the two major structural elements of C2a that form the interface sites with C1a,i.e.the 2' sheet of C2a that stacks on top of the 4- 5 loop
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of C1a, and the 2' helix of C2a that contacts the N-terminal segment of 2 and the C-terminal end of 4 of C1a (6). Consequently, a conformational change of the KF-loop - caused by the docking of G - should have a direct effect on the AC activity. The consecutive substitution of two amino acids from this nine-residue motif resulted in four mutants (Fig. 7C) that were catalytically active (Fig. 7D). In contrast, NAAIRS-substitution in this region starting at amino acid residue 928 had generated the catalytically inactive ACII.928 mutant. One reason might be the detrimental substitution of P929 as a structurally relevant amino acid in the NAAIRS screen, while P929 was not exchanged in the alanyl-alanine screen. The potential impact of P929 on intrinsic enzyme activity was underlined by the diminished activity of ACII.AA930 with the two amino acids substituted adjacent to P929. Fig. 8 depicts the G -responses of the four ACII alanyl-alanine mutants. Substitution of the KF-pair by two alanine residues resulted in ACII.AA930 that was no longer stimulated by G . This mutant defined K930 and F931 of ACII as key residues for G -stimulation. Alanyl-alanine substitutions preceding the central KF-spot and adjacent to P929 (ACII.AA927) showed diminished G -stimulation while substitutions more distant to the KF-pair did not significantly change G -regulation of the resulting mutants ACII.AA925 or ACII.AA932. These data provided clear evidence that the KF-loop was an essential motif in ACII to observe G -mediated stimulation.
The QEHA-motif (amino acids 956-982) was another region in IIC2 that had been described in the past to interact with the G -complex (28); however, domain swaps and substitutions in that region revealed that - in contrast to the PFAHL-motif and KF-loop - the QEHA-motif was not necessary as a mediator of the stimulatory effect of G to ACII (14). Hence, we assumed that the QEHA-motif as a neutral G -tether provided local concentration of G , without playing a role in the regulatory signal transfer to the AC. During the course of manuscript preparation Gao et al. published solid data about the G -stimulation of the human ACV and ACVI (18) while canine ACV was G -insensitive. Interestingly, the human
ACV amino acid sequence is identical to canine ACV in the PFAHL-motif as well as the KF-loop and THIS IS NOT THE FINAL VERSION - see doi:10.1042/BJ20071204 dissimilar to the orthologous ACVI as well as ACII sequences. Furthermore, Gao et al. identified a G interaction site at the N-terminus of human ACV and human ACVI. We concluded that the PFAHL- and the KF-motif were specific G -regulatory hot spots for type II-like ACs (including ACIV and ACVII, all providing identical motifs), while other AC subgroups provided different motifs to result in analogous modulation.
In summary, the presented data show a minimum of two sites being necessary for AC isoforms to be
Stage 2(a) POST-PRINT11
Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Biochemical Society Biochemical Journal Immediate Publication. Published on 14 Jan 2008 as manuscript BJ20071204