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 HAL Id: hal-00478881 https://hal.archives-ouvertes.fr/hal-00478881 Submitted on 30 Apr 2010 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Biochemical Journal Immediate Publication. Published on 14 Jan 2008 as manuscript BJ20071204 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 Stage 2(a) POST-PRINT Licenced copy. Copying is not permitted, except with1 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 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 Stage 2(a) POST-PRINT1 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 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 Stage 2(a) POST-PRINT2 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 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 Stage 2(a) POST-PRINT3 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.