Changes in purine specificity in tandem GAF chimeras from cyanobacterial cyaB1 adenylate cyclase and rat phosphodiesterase 2 Ju ¨ rgen U. Linder, Sandra Bruder, Anita Schultz and Joachim E. Schultz Abteilung Pharmazeutische Biochemie, Fakulta ¨ tfu ¨ r Chemie und Pharmazie, Universita ¨ tTu ¨ bingen, Germany The second messengers cAMP and cGMP mediate many intracellular functions. Swift signal modulation requires highly regulated biosynthesis and degradation. In mammals, the latter is accomplished by a family of 11 phosphodiesterase (PDE) isoforms which possess similar C-terminal catalytic domains (20–45% identity [1]). Different regulatory features are imparted by the N-terminal domains. Five PDE families, 2, 5, 6, 10 and 11, have an N-terminal tandem GAF domain of about 500 amino acids, i.e. two GAF domains, termed A and B, are sequentially connected by a short linker. The acronym GAF is derived from the proteins of initial identification: mammalian cGMP-binding PDEs, Anabaena adenylate cyclases (ACs; EC 4.6.1.1), and Escherichia coli transcription factor F hlA. Meanwhile, GAF domains have been identified in more than 3000 proteins. They mediate protein dimerization and can allosterically regulate cognate enzymes [2,3]. The lig- and for the tandem GAF domains of PDE2, PDE5, PDE6, and PDE11 is cGMP and that for PDE10 is cAMP. Usually ligand-binding enhances catalytic activity [4–6]. Two ACs of the cyanobacterium Ana- baena sp. PCC 7120, cyaB1 and cyaB2, have N-ter- minal tandem GAF domains with sequence similarity Keywords adenylate cyclase; cAMP; cGMP; cyclic nucleotide phosphodiesterase; GAF domain Correspondence J. E. Schultz, Abteilung Pharmazeutische Biochemie, Fakulta ¨ tfu ¨ r Chemie und Pharmazie, Universita ¨ tTu ¨ bingen, Morgenstelle 8, 72076 Tu ¨ bingen, Germany Fax: +49 7071 295952 Tel: +49 7071 2974676 E-mail: joachim.schultz@uni-tuebingen.de (Received 7 November 2006, revised 18 December 2006, accepted 12 January 2007) doi:10.1111/j.1742-4658.2007.05700.x The C-terminal catalytic domains of the 11 mammalian phosphodiesterase families (PDEs) are important drug targets. Five of the 11 PDE families contain less well-characterized N-terminal GAF domains. cGMP is the lig- and for the GAF domains in PDEs 2, 5, 6 and 11, and cAMP is the ligand for PDE10. Structurally related tandem GAF domains signalling via cAMP are present in the cyanobacterial adenylate cyclases cyaB1 and cyaB2. Because current high-resolution crystal structures of the tandem GAF domains of PDE2 and cyaB2 do not reveal how cNMP specificity is enco- ded, we generated chimeras between the tandem GAF domains of cyaB1 and PDE2. Both bind the ligand in the GAF B subdomains. Segmental replacements in the highly divergent b1–b3 region of the GAF B sub- domain of cyaB1 by the corresponding PDE2 regions switched signalling from cAMP to cGMP. Using 10 chimeric constructs, we demonstrated that, for this switch in purine specificity, only 11% of the sequence of the cyanobacterial GAF B needs to be replaced by PDE2 sequences. We were unable, however, to switch the purine specificity of the PDE2 tandem GAF domain from cGMP to cAMP in reverse constructs, i.e. by replacement of PDE2 segments with those from the cyaB1 GAF tandem domain. The data provide a novel view on the structure–function relationships underlying the purine specificity of cNMP-binding GAF domains and indicate that, as potential drug targets, they must be characterized structurally and bio- chemically one by one. Abbreviations AC, adenylate cyclase; PDE, phosphodiesterase; mPDE2, mouse PDE2; rPDE2, rat PDE2. 1514 FEBS Journal 274 (2007) 1514–1523 ª 2007 The Authors Journal compilation ª 2007 FEBS to the mammalian PDE GAF domains. cAMP is the ligand for these cyanobacterial tandem GAF domains, and cAMP binding results in stimulation of the C-terminal catalytic domain [7–9]. To date, two tandem GAF domains have been struc- turally elucidated. The mouse PDE2 (mPDE2) GAF tandem, resolved at 2.9 A ˚ , is a parallel dimer with two cGMP molecules bound to the respective GAF B sub- domains [4]. Two a-helices N-terminal to GAF A appear to be a major dimerization force. The tandem GAF domain of the cyaB2 AC, resolved at 1.9 A ˚ , turned out to be an antiparallel dimer with cAMP molecules bound to each subdomain, GAF A and B, i.e. it contains four cAMP molecules [9]. Here, the dimerizing a-helices N-terminal to GAF A are turned inwards and interact with each other and with the a-helical linkers between the GAF A and B subdomains. In both structures, the cAMP-binding and cGMP- binding pockets are similar. A neighbouring conserved NKFDE motif, which appears to be a sequence and structural hallmark of cyclic nucleotide-binding GAF domains, yet without direct contact to the respective ligand, can be superimposed almost seamlessly [6,8,10,11]. It remains to be seen whether both the parallel and antiparallel structures reflect disparate functional states, one in mPDE2 and possibly other members of mammalian PDEs, and the other in the cyanobacterial ACs. In this context, it is noteworthy that the rat PDE2 (rPDE2) GAF tandem, which is almost identical with the mouse sequence, can functionally replace the cyanobacterial one in the cyaB1 AC, converting it into a cGMP-activated enzyme [7]. Thus, the cyanobacterial AC is a powerful tool for biochemically characterizing the GAF domains of PDEs because, in the chimeras, cyclic nucleotides are not concomitantly substrate and allosteric activator as in PDE assays and product inhibition of the AC is absent [7]. A common feature of both GAF domain structures is that the bound cAMP or cGMP molecules are bur- ied, i.e. less than 10% of the surface of the bound cyc- lic nucleotide is solvent exposed [4,9]. Therefore, for entry and exit of the compounds, the structures must relax. The amino acids that contact the ligands in the crystal structures as determined by ligplots (Fig. 1C–E [4,9]) may be involved in the selectivity filter for cAMP and cGMP, respectively. We generated chimeras between the rPDE2 and cyaB1 tandem GAF domains to address the question of how cyclic nucleo- tide specificity is encoded and obtained proteins in which exclusively cGMP instead of cAMP activates the associated AC. Thus we defined a rather small pro- tein segment important in ligand specification. Results In the structure of the mPDE2A tandem GAF domain, 12 amino acids have been identified as inter- acting with bound cGMP [4]. The ribose phosphate moiety interacts with Ile453, Ala454, Tyr476, Asp480, T487 and Glu507 (rPDE numbering). The purine moi- ety is directly liganded by Ile417 (b1), Ser419 (b1), Phe433 (b2) and Asp434 (b2–b3 loop, Fig. 1B,C), and indirectly via water with Val479 and Thr483 (both in a4). Targeted mutations and cGMP-binding assays identified Phe433, Asp434 and Thr483 as important for specifying cGMP over cAMP as a ligand [12]. Taken together the b1–b3 region of the tandem GAF domain appears to be a major determinant of purine selection. The tandem GAF domain of the Anabaena AC cyaB2 is specific for cAMP [8,9]. Both GAF A and B bind cAMP (Fig. 1A,B,D,E). Again, the b1–b3 region is the major interaction site for the adenine moiety. In b1, an arginine residue (Ile417 in rPDE2) binds to N1 and a threonine (Ser419 in rPDE2) inter- acts with N7 and the N 6 amino group. The inter- actions of the b2–b3 element with the purine appear to be rather distinct in rPDE2 GAF B, cyaB2 GAF A and cyaB2 GAF B (Fig. 1C–E). This suggests not only that amino acid side chains determine ligand specificity, but the backbone conformation is also critical. Thus we reasoned that a switch from cAMP to cGMP bind- ing in the Anabaena tandem GAF domains probably requires structural changes in the b1–b3 region. Because in the tandem GAF domain of cyaB2 cAMP is bound by both subdomains, we used the tandem GAF domain of cyaB1 in which only GAF B appears to mediate signalling to investigate ligand specification. In fact, the interacting amino acids, Arg256 and Thr258, are conserved in the b1 sheet of the cyaB1 and cyaB2 GAF B subdomains. However, the b2–b3 elements of cyaB1 and cyaB2 are diverged to an extent that precludes prediction of a binding mode for cAMP in cyaB1. Initially, we replaced the entire b1–b3 region, Ile250 to Ile283, of the cyaB1 GAF B subdomain with Asn411 to Tyr443 of GAF B from rPDE2A (Fig. 1B, construct I in Table 1). The purified recombinant pro- tein was stimulated 10.5-fold by cGMP with an EC 50 of 360 lm, similar to wild-type cyaB1, and, surpris- ingly, activation by cAMP was eliminated (Fig. 2A, Table 1). Obviously, exchange of this region switched the specificity of the cyaB1 tandem GAF domain from cAMP to cGMP, confirming the above considerations. Next we swapped the anterior part, Ile250 to Gly267, in cyaB1 GAF B with Asn411 to Asn426 of rPDE2A (construct II in Table 1). Construct II was stimulated J. U. Linder et al. Cyclic nucleotide specificity in GAF domains FEBS Journal 274 (2007) 1514–1523 ª 2007 The Authors Journal compilation ª 2007 FEBS 1515 by neither cAMP nor cGMP up to 10 mm (Fig. 2A). We were unable to determine whether cNMP access and binding was abrogated or whether intramolecular signalling was impaired because of incorrect folding of the GAF ensemble in spite of a robust basal activity of the C-terminal AC. Swapping the C-terminal W270 to I283 section of cyaB1 for Val429 to Tyr443 from rPDE2a (construct III in Table 1) yielded a protein that was highly stimulated by both cAMP and cGMP, i.e. purine specificity was lost (Fig. 2B). Stimulations by cAMP and cGMP were almost identical (11.3-fold for cAMP and 12.7-fold for cGMP; Table 1) as were maximal AC activities of 3.8 lmol cAMPÆmg )1 Æmin )1 . Likewise, the EC 50 values of 3.3 lm cAMP and 3.5 lm cGMP were identical and close to the value for cAMP stimulation of the parent cyaB1 AC (1 lm cAMP; Table 1) [7]. Thus in construct III, cGMP stimulation was gained and the cAMP response was retained. Taken together, this indicated that the section Asn411–Asn426 of rPDE2 was incompatible with sti- mulation by cAMP, whereas the C-terminally adjacent section, Val429 and Tyr443, was compatible with sti- mulation by both cNMPs. In the cyaB1 GAF B domain, however, the more N-terminal segment (Ile250–Gly267) was compatible with stimulation by both cAMP and cGMP, whereas the C-terminally located region (Trp270 to Ile283) excluded activation by cGMP. This suggested that, in cyaB1 and rPDE2, the determinants for cyclic nucleotide specificity are located in different regions. Therefore, continuing from construct III, we incre- mentally elongated the swapped stretch of amino acids towards the N-terminal, two amino acids at a time, to identify the amino acids that impeded cAMP stimula- CyaB2-GAFB-cAMP rPDE2A-GAF-B-cGMP CyaB2-GAF-A-cAMP CyaB2-GAF-B-cAMP A B C D E Fig. 1. Structural properties of the b1–b3 sheets and the interactions with cyclic nucleotide monophosphates. (A) GAF B–cAMP complex of cyaB2. The regions that correspond to the parts of cyaB1 exchanged in constructs II and III are shown in blue (A283-E303) and red (L304- P321). (B) Alignment of the respective sequence segments. (C) Interaction sites of cGMP in the structure of mPDE2 (amino acid numbering corresponds to rPDE2). (D) Interaction sites for cAMP in the structure of the cyaB2 GAF A domain. (E) Interaction sites for cAMP in the structure of the cyaB2 GAF B domain. Amino acids interacting with the purine moiety are colour-coded for clarity in (B–E). Hydrophobic inter- actions are depicted by grey ovals in (C)E). Cyclic nucleotide specificity in GAF domains J. U. Linder et al. 1516 FEBS Journal 274 (2007) 1514–1523 ª 2007 The Authors Journal compilation ª 2007 FEBS Table 1. Stimulation of cyaB1-rPDE2 chimeras by cAMP and cGMP. Assays were performed with 75 lM ATP as described in Experimental procedures. cyaB1-WT, Wild-type cyaB1 AC holoenzyme; rPDE2-cyaB1, chimera in which the cyaB1 AC tandem GAF domains (residues 50–385) are replaced by the rPDE2 tandem GAF domains. Sequences in bold indicate residues from rPDE2. N ¼ 2–8; usually recombinant protein from two expressions were used. Basal activity is expressed as nmol cAMPÆmg )1 Æmin )1 . Construct EC 50 (cAMP) (l M) EC 50 (cGMP) (l M) Ratio EC 50 (cAMP) to EC 50 (cGMP) Basal activity [nmol cAMPÆ mg )1 Æmin )1 ] Activation [X-fold] Sequence of b1–b 3 regioncAMP cGMP cyaB1-WT a 1 300 < 0.01 85 27 24 248 AR ILMQADRSTLFLYRKEMG EL WTKVAAAADTTQ-LI EIRIP 288 Construct I (> 3000) 360 ± 85 NA 3.5 ± 0.1 1.3 b 10.5 ± 0.4 AR NLSNAEICSVFLLDQ N EL VAKVFDGGVVDDESY EIRIP Construct II (> 3000) (> 3000) NA 4.2 ± 0.1 1.3 b 1.2 b AR NLSNAEICSVFLLDQ N EL WTKVAAAADTTQ-LI EIRIP Construct III 3.3 ± 0.3 3.5 ± 0.3 0.94 363 ± 21 11.3 ± 0.2 12.7 ± 0.5 AR ILMQADRSTLFLYRKEMG EL VAKVFDGGVVDDESY EIRI Construct IV 8.2 ± 0.4 13.7 ± 1.0 0.60 7.2 ± 0.1 10.9 ± 0.3 12.8 ± 0.5 AR ILMQADRSTLFLYRQ N EL VAKVFDGGVVDDESY EIRIP Construct V 5.5 ± 0.5 6.8 ± 0.2 0.81 56.5 ± 7.3 15.4 ± 5.4 18.1 ± 6.3 AR ILMQADRSTLFLYRQEMN EL VAKVFDGGVVDDESY EIRIP Construct VI 7.3 ± 2.4 19.9 ± 0.8 0.37 24.3 ± 3.3 46.1 ± 7.2 54.2 ± 6.6 AR ILMQADRSTLFLLDQ N EL VAKVFDGGVVDDESY EIRIP Construct VII 78.9 ± 30.9 9.9 ± 0.3 7.97 7.8 ± 0.7 35.6 ± 2.3 394 ± 30 AR ILMQADRSSVFLLDQ N EL VAKVFDGGVVDDESY EIRIP Construct VIII 56.9 ± 32.6 4.6 ± 0.6 12.4 9.9 ± 1.0 27.1 ± 4.8 96.4 ± 5.3 AR ILMQADRSSVFLYRKEMG EL VAKVFDGGVVDDESY EIRIP Construct IX 1.4 ± 0.1 0.04 ± 0.01 35.0 79.7 ± 7.4 5.4 ± 0.3 7.1 ± 0.4 AR ILMQADRSSLFLYRKEMG EL VAKVFDGGVVDDESY EIRIP Construct X 72.8 ± 21.8 814 ± 122 0.09 3.8 ± 0.2 34.3 ± 6.7 38.5 ± 7.6 AR ILMQADRSTVFLYRKEMG EL VAKVFDGGVVDDESY EIRIP Construct XI 11.7 ± 0.5 > 3000 < 0.01 1.6 ± 0.1 48.9 ± 2.5 7.9 b AR ILMQADRSSVFLYRKEMG EL WTKVAAAADTTQ-LI EIRIP rPDE2-cyaB1 a (> 3000) 3 NA 2.4 2 b 10 409 AR NLSNAEICSVFLLDQ N EL VAKVFDGGVVDDESY EIRIP 448 Construct XII (> 3000) (> 3000) NA 0.6 ± 0.1 1.1 b 1.3 b AR ILMQADRSTLFLYRKEMG EL WTKVAAAADTTQ-LI EIRIP Construct XIII (> 3000) (> 3000) NA 0.6 ± 0.1 1.4 b 1.2 b AR ILMQADRSTLFLYRKEMG EL VAKVFDGGVVDDESY EIRIP Construct XIV NA NA NA 0.5 ± 0.1 1.0 b 1.0 b AR NLSNAEICSVFLLDQ N EL WTKVAAAADTTQ-LI EIRIP a Data taken from [7]; b measured at 3–10 mM cNMP. J. U. Linder et al. Cyclic nucleotide specificity in GAF domains FEBS Journal 274 (2007) 1514–1523 ª 2007 The Authors Journal compilation ª 2007 FEBS 1517 tion (Table 1). A length variation of two amino acids was taken into account to avoid missing an essential function (constructs IV and V in Table 1). All con- structs were expressed in E. coli, affinity-purified, and assayed for activation via cAMP and cGMP (Table 1). In constructs IV, V and VI, the ratio of EC 50 concen- trations for cAMP and cGMP barely changed com- pared with construct III. Throughout, cAMP was slightly more effective than cGMP. When Thr258⁄ Leu259 were included in the domain swapping and replaced by Ser and Val, however, cNMP specificity was inverted, i.e. the efficacy of cAMP was almost lost, whereas cGMP stimulated cyaB1 AC potently (construct VII in Table 1; Fig. 3A). This phenotype differed from construct I, where the cAMP response was abolished and stimulation by cGMP was un- affected. Next we examined whether a T258S ⁄ L259V double mutation in the background of construct III was suffi- cient to change cNMP specificity (Table 1, con- structs VIII–XI). Corresponding assays demonstrated that this was the case (Table 1, Fig. 3B). Properties of constructs VII and VIII were comparable, i.e. cGMP was 12-fold more potent in stimulating the associated AC activity than cAMP (Table 1). A notable difference between the exchange of the N-terminal region Ile250 to Gly267 (construct I) and the T258 ⁄ L259 couple (construct VIII) was the complete abrogation of cAMP stimulation in the former versus the remaining cAMP response in the latter (compare Fig. 2A and Fig. 3B). However, the loss of cAMP stimulation in construct I was achieved at the expense of reduced efficacy for cGMP (compare constructs I and VIII in Table 1). Next, we asked whether the switch in cNMP specificity was attributable to either T258S or L259V (con- structs IX and X, Fig. 3C, Table 1). In construct IX (T258S) the preference for cGMP over cAMP was 35-fold (Table 1), and the affinity for both cyclic nuc- leotides was enhanced by almost two orders of mag- nitude compared with construct VIII (Table 1). The EC 50 for cGMP was 40 nm and that for cAMP 1.4 lm. Therefore construct IX differed from con- struct I and VII by an increase in cGMP sensitivity without affecting the EC 50 for cAMP. Going from construct III to IX is tantamount to removing a single methyl group (replacement of Thr by Ser). This may cause subtle structural changes in the b1–b3 region which affect the carbon backbone and thus impact nucleotide specificity (see above and the Discussion). Similarly, in construct X (L259V), a single methylene group is removed. The EC 50 for cGMP ( 820 lm, Table 1) could only be estimated because, even at 10 mm, cGMP saturation was not reached (not shown). Remarkably, the affinity for cAMP was at least 11-fold higher than for cGMP (Table 1). Yet, with an EC 50 of 72.8 lm, cAMP affinity was low com- pared with that of the cyaB1 holoenzyme (1 lm) [7] and construct III (3.3 lm ). This indicated that the L259V mutation not only reduced cGMP potency but also interfered with cNMP binding and signalling in general. Apparently, this amino-acid side chain makes Fig. 2. Dose–response curves for cAMP and cGMP stimulation of cyaB1 AC in constructs I–III. The nature of the chimeras is depicted in Table 1. (A) Curves for construct I and II. Note that construct I is stimulated by cGMP, whereas cAMP is inactive. Construct II is sti- mulated by neither cAMP nor by cGMP. (B) cNMP dose–response curve for construct III. cAMP and cGMP are equally potent at sti- mulating cAMP formation by cyaB1 AC. The insets show western blots of the affinity-purified proteins indicating the presence of undegraded proteins. Cyclic nucleotide specificity in GAF domains J. U. Linder et al. 1518 FEBS Journal 274 (2007) 1514–1523 ª 2007 The Authors Journal compilation ª 2007 FEBS important contributions to functionally important features. Next we examined whether the effect of the double mutation T258S ⁄ L259V would also materialize in the cyaB1 AC parent protein, i.e. a reduction in cAMP efficacy and a persistent discrimination against cGMP. In the T258S ⁄ L259V-cyaB1 AC (construct XI, Fig. 4, Table 1), the EC 50 for cAMP was 12 lm and maximal stimulation was about 50-fold. The response to cGMP was greatly diminished (Fig. 4). The data corresponded to the high preference for cAMP over cGMP of the parent cyaB1 protein [7], with a 10-fold increased EC 50 for cAMP. Further the specific activities of basal and 1mm cAMP-stimulated T258S ⁄ L259V-cyaB1 AC were reduced approximately 45-fold (1.4 and 84 (basal), 67.5 and 2244 nmol cAMPÆmg )1 Æmin )1 (activated with 1mm cAMP)). We conclude that to some extent Thr258 ⁄ Leu259 in cyaB1 govern cAMP signalling; however, they do not alone define purine specificity of the regulatory domain. Only together with other regio- nal changes, such as in construct VIII, does the T258S ⁄ 1259V double mutation assist in switching pur- ine recognition from adenine to guanine. So far, the cyaB1 tandem GAF domain has been modified by swapping the b1–b3 region with the cor- responding region from rPDE2. Can we observe sim- ilar effects when we transfer the corresponding region of cyaB1 to the rPDE2 tandem GAF domain? We pre- viously showed that the exchange of the cyaB1 rPDE2 tandem GAF domains generated a cGMP-activated AC [7]. In analogy with construct I, we now replaced Asn411–Tyr443 in the rPDE2 tandem GAF domain with Ile250–Ile283 from cyaB1 (construct XII, Table 1). Fig. 4. Dose–response curves for cAMP and cGMP stimulation of cyaB1 AC in construct XI (see Table 1 for composition of the chi- mera). Western blot of affinity-purified protein is shown as an inset. Fig. 3. Dose–response curves for cAMP and cGMP stimulation of cyaB1 AC in constructs VII (A), VIII (B) and IX (C). The chimera compositions are delineated in Table 1. Western blots of affinity purified proteins are shown as insets in the respective figures. J. U. Linder et al. Cyclic nucleotide specificity in GAF domains FEBS Journal 274 (2007) 1514–1523 ª 2007 The Authors Journal compilation ª 2007 FEBS 1519 Similarly to constructs II and III, the N-terminal and C-terminal segments of these regions in rPDE2 were replaced by the corresponding cyaB1 sequences (con- structs XIII and XIV, respectively). All three con- structs, XII to XIV, were expressed in E. coli, albeit rather poorly. Western blots of the partially purified proteins showed that they were intact (not shown). Basal AC activity was 0.5–0.6 nmol cAMPÆmg )1 Æmin )1 . Addition of cAMP or cGMP up to 10 mm slightly and dose-dependently activated AC activity (maximal acti- vation 1.4-fold, Table 1). Clearly, we could not repeat the regional exchange of the b1–b3 region with the rPDE2 tandem GAF domain serving as the receiver domain. Apart from the problem of poor expression and perhaps partial misfolding of constructs XII–XIV, it is obvious that the data with the cyaB1 GAF domain cannot be generalized, i.e. a free exchange of corresponding GAF domain regions between different cNMP-binding tandem GAF domains is obviously impossible. This means that these GAF domains, in spite of the high similarity between them, have prob- ably retained considerable individuality in ligand binding and intramolecular signalling. Discussion So far, the structures of two tandem GAF domains are known. The mPDE2 one contains two cGMP moi- eties bound to each GAF B in a parallel dimer [4]. The other structurally elucidated tandem GAF domain is from the cyanobacterial AC cyaB2 [9]. It signals via cAMP which binds to GAF A and ⁄ or GAF B. Hence, the structure contains four cAMP molecules in an anti- parallel dimer. For construction of chimeras, we used a rat PDE2, which has an amino-acid sequence identi- cal with the mouse isoform in this region, and the GAF tandem domain from the cyaB1 AC because this domain, despite its sequence identity with that of cyaB2 (45% identity, 59% similarity), signals only via GAF B [7]. All residue swapping procedures therefore involved the GAF B regions. We hoped to prototypi- cally identify individual residues or narrow regions in the cyaB1 and rPDE2 tandem GAF domains that dis- criminate between the adenine and guanine of cAMP and cGMP as primary ligands. Previous experiments investigating cGMP binding to GAF domains of mammalian PDEs were designed on the basis of the mPDE2 GAF domain crystal structure [12]. Of the 12 amino acids identified as interaction partners for cGMP, only four bind directly to the pur- ine moiety and are thus potentially involved in defining purine specificity (see above). These residues are located in the b1–b3 region studied here. In earlier studies, three of these amino acids were mutated in the tandem GAF domains of PDE2A, PDE5 and PDE6 [12–14] to assess their contribution to cNMP binding. In mPDE2, mutation of Ser424 (Ser419 in rPDE2), which interacts with the imidazole ring, to alanine abolished cNMP binding. Further, replacement of the Phe ⁄ Asp couple in mPDE2 (Phe433 ⁄ Asp434 in rPDE2) that interacts with the pyrimidine ring of the purine, either individu- ally or in various combinations in 12 different con- structs, enhanced cAMP binding but had little effect on cGMP binding. Taken together, ligand specificity was more or less lost, and a switch of purine specificity was not accomplished [12]. In analogy, in PDE5 GAF A, a F205A mutation (Phe433 in rPDE2) abro- gated cGMP binding [13], and, in PDE6, both of the amino acids corresponding to Phe433 ⁄ Asp434 in rPDE2A are required for cGMP binding [14]. Our initial mutations in cyaB1 GAF B also involved individual replacement of the residues positionally cor- responding to Phe433 and Asp434 in rPDE2. Neither an A275D mutation [7] nor a 274 AAA 276 to FDG triple exchange affected cAMP specificity of the cyaB1 tandem GAF domain (T. Kanacher, unpublished results). Nei- ther was cAMP stimulation lost nor cGMP activation gained. While these experiments were in progress, the crystal structure of the cAMP-binding GAF tandem domain of cyaB2 became available [9]. It showed that the backbone conformations of the b1–b3 regions are par- ticularly divergent, as is the primary sequence. This was not surprising because the corresponding segments A250 to I283 of cyaB1 and N411 to Y443 in the rPDE2 tandem GAF domains are only 24% identical. Therefore, we hypothesized that regional chimeras instead of point mutants might yield clues about which structural elements are involved in coding purine spe- cificity. The expectation of this hypothesis was fulfilled by construct I. A cyaB1 tandem GAF domain with replacement of this segment by the rPDE2 region, which comprised only 23% of the residues of the cyaB1 GAF B domain, was exclusively, yet weakly, sti- mulated by cGMP, whereas the activation by cAMP was lost. Accordingly, using additional constructs, we narrowed the region responsible for this specificity switch. In summary, in the cyaB1 GAF domain, it is sufficient to replace T258 ⁄ L259 and residues W270 to I283 (11% of GAF B) by the corresponding residues of rPDE2 to change the specificity for the allosteric regulator from cAMP to cGMP. Thus the assumption that subtle changes in regional structural details are required for switching specificity appears to be correct. The next question was how generalized is this find- ing. This was investigated by producing the reverse Cyclic nucleotide specificity in GAF domains J. U. Linder et al. 1520 FEBS Journal 274 (2007) 1514–1523 ª 2007 The Authors Journal compilation ª 2007 FEBS chimeras, i.e. in the rPDE2 GAF B domains identical sequence stretches were replaced by those from the cyaB1 GAF B domain (construct XII to XIV). Most disappointingly, stimulation by cNMPs was basically lost, and no switch in nucleotide specificity was obtained (Table 1). This is reminiscent of similar stud- ies of ATP ⁄ GTP substrate preference in mammalian adenylate and guanylate cyclases. Whereas point muta- tions converted a guanylate cyclase protein to an AC, the reverse was not achieved. Therefore, we conclude that, not withstanding considerable sequence and structural similarities, cNMP-binding GAF tandem domains have evolved to the extent that structurally homologous regions cannot be functionally exchanged between members of this tandem GAF domain family. The structural differences affect coding for purine spe- cificity and possibly also signalling to the associated catalytic output. Furthermore, considering that cAMP in the cyaB2 and cGMP in the mPDE2 GAF tandem domain structures are less than 10% exposed to sol- vent, amino acids additional to those identified as binding to and interacting with cNMPs in the crystal structures may be required. For cNMPs to access the binding pocket, the structures undoubtedly must open up. Therefore, a structure without bound ligand would be helpful in this context to elucidate the structural transitions upon cNMP binding. When one compares the sequences of the cAMP- binding GAF tandem domain of PDE10, the PDE from Trypanosoma brucei [15] and cyaB1 and cyaB2 ACs with the cGMP-binding tandem GAF domains from mammalian PDEs in conjunction with the avail- able biochemical and structural data, it is impossible to predict which amino acids or sequences encode pur- ine specificity in each case. Our data contribute to the complex structure–function relationships underlying purine specificity of cNMP binding GAF domains. In addition, they highlight that, in order to potentially use the tandem GAF domains as potential drug tar- gets, we must characterize GAF domains structurally and biochemically one by one. Experimental procedures Recombinant DNAs The cyaB1 holoenzyme and a chimera consisting of the rPDE2A GAF tandem domain (207–546) in the cyaB1 AC background (replacing cyanobacterial residues 50–385) have been described [7]. They served as PCR templates in this study. All numbering refers to either rPDE2A (GenBank accession number NM_031079) or cyaB1 AC (GenBank accession number D89623), as applicable. Construct III (Table 1) was prepared by introduction of silent SacI and EcoRI sites at E268 ⁄ L269 and R286 ⁄ I287 ⁄ P288 into the cyaB1 AC gene. rPDE2A V429–Y443 was inserted via these SacI ⁄ EcoRI sites, replacing the cy- anobacterial sequence W270–I283. Construct I (Table 1) was created via a introduced silent BssHII site at A248 ⁄ R249 in the cyaB1 AC and the EcoRI site. Thus, amino acids N411–Y443 of rPDE2A replaced the cyano- bacterial amino acids I250–I283. For construct II (Table 1), the region between BssHII and SacI (I250–G267 of cyaB1) was replaced by N411–N426 of rPDE2A. By transfer into the pQE30 expression plasmid, all constructs were fitted with an N-terminal MRGSH 6 GS affinity tag. For replacement of incremental segments (constructs IV– X, Table 1) specific SacI-containing antisense primers were used. The PCR products were inserted into construct III in pQE30 between a newly generated silent PinAI site at T181 ⁄ G182 in cyaB1 and the above SacI site. To generate T258S ⁄ L259V-cyaB1 AC (construct XI in Table 1), con- structs II and VII in pQE30 were digested with SacI and EcoRV, and the appropriate fragments were ligated. Chimeras between the C-terminal cyaB1 AC and the N-terminal rPDE2A tandem GAF domain, which carried corresponding inserts of the cyaB1 tandem GAF domain (constructs XII–XIV) were generated by introduction of a silent BssHII at A409 ⁄ R410 (numbering of rPDE2A), a SacI site at E427 ⁄ L428, and an EcoRI restriction site at R446 ⁄ I447 ⁄ P448, and employing the same cloning strategy as for constructs I–III. Constructs I–XI were expressed from pQE30. For expres- sion of the inverse chimeras (constructs XII to XIV), a pET16b vector with the multiple cloning site of pQE30 (bp267–431 of pET16b were replaced by bp199–90 of pQE30) was used retaining the N-terminal MRGSH 6 GS tag. The fidelity of all constructs was verified by double- stranded sequencing. Primer sequences and additional clo- ning details are available on request. Expression and purification of bacterially expressed proteins Chimeric constructs based on the cyanobacterial GAF tan- dem domain were transformed into E. coli BL21(DE3)- [pRep4] and those based on the rPDE2 GAF tandem domain into E. coli BL21(DE3)[pLysS]. Cultures were grown in Lennox L broth at 30 °C containing 100 mgÆL )1 ampicillin and 50 mgÆL )1 kanamycin. Expression was induced at an A 600 of 0.5 with 15–50 (for pQE30) or 75 lm isopropyl thio b-d-galactoside (for the pET16b derivative) for 5–20 h at 16–20 °C. Bacteria were collected by centrifu- gation at 2600 g for 15 minutes in a Hermle A6.14 rotor (Gosheim, Germany), rinsed with 50 mm Tris ⁄ HCl, pH 8.5, and stored at )80 °C. For purification, cells from 600 mL cultures were suspended in 20–40 mL lysis buffer [50 mm Tris ⁄ HCl, pH 8.5, 50 mm NaCl, 20% glycerol, 7.5 mm J. U. Linder et al. Cyclic nucleotide specificity in GAF domains FEBS Journal 274 (2007) 1514–1523 ª 2007 The Authors Journal compilation ª 2007 FEBS 1521 imidazole and CompleteÒ protease inhibitor mix (EDTA- free; Roche Diagnostics, Mannheim, Germany)] and passed through a French Press at 1000 psi (6894.75 kPa). Cell deb- ris was removed by centrifugation (45 min, 48 000 g,4°C) using a Sorvall 5534 rotor. Then 50–250 lLNi 2+ ⁄ nitrilotri- acetate slurry (Qiagen, Hilden, Germany) was added to the supernatants, and binding was for 2.5 h on ice. The resin was washed sequentially (2 mL per wash) with buffer A (50 mm Tris ⁄ HCl, pH 8.5, 2 mm MgCl 2, 400 mm NaCl, 7.5 mm imi- dazole, 20% glycerol), buffer B (buffer A +15 mm imidaz- ole) and buffer C (buffer A + 25 mm imidazole, 10 mm NaCl). Proteins were eluted with 0.3 mL buffer C containing 300 mm imidazole. The eluates were dialysed for 2 h against buffer (50 mm Tris ⁄ HCl, pH 8.5, 2 mm MgCl 2, 10 mm NaCl, 35% glycerol) and stored at )20 °C. Purity of recombinant proteins The purity and integrity of expression products was exam- ined by SDS ⁄ PAGE and western blotting. For western blots, proteins were blotted on to poly(vinylidene difluo- ride) membranes and sequentially probed with an antibody to RGSH 4 (Qiagen) and with a peroxidase-conjugated goat anti-(mouse IgG-F c ) secondary antibody (Dianova, Ham- burg, Germany). Detection was carried out with the ECL Plus kit (GE Health Care, Munich, Germany). Western blots are included in the figures to demonstrate the absence of proteolytic degradation products. Because fragments that lost the His tag do not bind to the Ni 2+ resin used for purification, they do not constitute a purity problem. AC assay Enzyme activity was assayed for 4 min (cyaB1-based constructs) or 10 min (rPDE2a-GAF-based constructs) at 37 °C in 100 lL containing 22% glycerol, 50 mm Tris ⁄ HCl, pH 7.5, 10 mm MgCl 2 ,10lg BSA and 75 lm [a- 32 P]ATP (25 kBq). [2,8- 3 H]cAMP (2 mm; 150 Bq) was added when the reaction was stopped to monitor yield during product isola- tion [16]. Substrate conversion was limited to < 10%. Values are means ± SEM from two to eight experiments mostly carried out with proteins from two individual expressions. Acknowledgements This work was supported by grants from the Deutsche Forschungsgemeinschaft. We thank Dr T. Kanacher for initial experiments. 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