Changes in purine specificity in tandem GAF chimeras from cyanobacterial cyaB1 adenylate cyclase and rat phosphodiesterase 2.
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 ligand 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 encoded, 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 beta1-beta3 region of the GAF B subdomain 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 biochemically one by one.
Linder
Jürgen U
JU
Abteilung Pharmazeutische Biochemie, Fakultät für Chemie und Pharmazie, Universität Tübingen, Morganstelle 8, 72076 Tübingen, Germany.
Bruder
Sandra
S
Schultz
Anita
A
Schultz
Joachim E
JE
D000262:Adenylyl Cyclases / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D020134:Catalytic Domain; D002678:Chimera; D000458:Cyanobacteria / Q000201:enzymology*; D006152:Cyclic GMP / Q000378:metabolism; D005092:Exonucleases / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D011687:Purines / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D011687:Purines; D011994:Recombinant Proteins; D005092:Exonucleases; C022650:spleen exonuclease; D000262:Adenylyl Cyclases; D006152:Cyclic GMP