%0 Journal Article %1 lorenzmutations %A Lorenz, R. %A Moon, E. %A Kim, J.J. %A Schmidt, S.H. %A Sankaran, B. %A Pavlidis, I.V. %A Kim, C. %A Herberg, F.W. %D 2017 %J Biochemical Journal %K herberg myown %N 14 %P 2389-2403 %R 10.1042/BCJ20160969 %T Mutations of PKA cyclic nucleotide binding domains reveal novel aspects of cyclic nucleotide selectivity %U http://www.biochemj.org/content/474/14/2389.long %V 474 %X Cyclic AMP and cyclic GMP are ubiquitous second messengers that regulate the activity of effector proteins in all forms of life. The main effector proteins, the cAMP-dependent protein kinase (PKA) and the cGMP-dependent protein kinase (PKG), are preferentially activated by cAMP and cGMP, respectively. However, the molecular basis of this cyclic nucleotide is still not fully understood. Analysis of isolated cyclic nucleotide binding (CNB) domains of PKA RIα reveals that the C-terminal CNB-B has a higher cAMP affinity and selectivity than the N-terminal CNB-A. Here we show that introducing cGMP-specific residues using site-directed mutagenesis reduces the selectivity of CNB-B, while the combination of two mutations (G316R/A336T) results in a cGMP-selective binding domain. Furthermore, introducing the corresponding mutations (T192R/A212T) into the PKA RIα CNB-A turns this domain into a highly cGMP-selective domain, underlining the importance of these contacts for achieving cGMP-specificity. Binding data with the generic purine nucleotide cIMP reveal that introduced arginine residues interact with the position 6 oxygen of the nucleobase. Co-crystal structures of an isolated CNB-B G316R/A336T double mutant with either cAMP or cGMP reveal that the introduced threonine and arginine residues maintain their conserved contacts as seen in PKG I CNB-B. These results improve our understanding of cyclic nucleotide binding as well as the molecular basis of cyclic nucleotide specificity.

@article{lorenzmutations, abstract = {Cyclic AMP and cyclic GMP are ubiquitous second messengers that regulate the activity of effector proteins in all forms of life. The main effector proteins, the cAMP-dependent protein kinase (PKA) and the cGMP-dependent protein kinase (PKG), are preferentially activated by cAMP and cGMP, respectively. However, the molecular basis of this cyclic nucleotide is still not fully understood. Analysis of isolated cyclic nucleotide binding (CNB) domains of PKA RIα reveals that the C-terminal CNB-B has a higher cAMP affinity and selectivity than the N-terminal CNB-A. Here we show that introducing cGMP-specific residues using site-directed mutagenesis reduces the selectivity of CNB-B, while the combination of two mutations (G316R/A336T) results in a cGMP-selective binding domain. Furthermore, introducing the corresponding mutations (T192R/A212T) into the PKA RIα CNB-A turns this domain into a highly cGMP-selective domain, underlining the importance of these contacts for achieving cGMP-specificity. Binding data with the generic purine nucleotide cIMP reveal that introduced arginine residues interact with the position 6 oxygen of the nucleobase. Co-crystal structures of an isolated CNB-B G316R/A336T double mutant with either cAMP or cGMP reveal that the introduced threonine and arginine residues maintain their conserved contacts as seen in PKG I CNB-B. These results improve our understanding of cyclic nucleotide binding as well as the molecular basis of cyclic nucleotide specificity.}, added-at = {2017-06-07T13:49:53.000+0200}, author = {Lorenz, R. and Moon, E. and Kim, J.J. and Schmidt, S.H. and Sankaran, B. and Pavlidis, I.V. and Kim, C. and Herberg, F.W.}, biburl = {https://www.bibsonomy.org/bibtex/261550655a06390adfce9e48293603abc/biochemie}, description = {Mutations of PKA cyclic nucleotide binding domains reveal novel aspects of cyclic nucleotide selectivity | Biochemical Journal}, doi = {10.1042/BCJ20160969}, interhash = {b54977cefb127c88a647ecd421046bdf}, intrahash = {61550655a06390adfce9e48293603abc}, journal = {Biochemical Journal}, keywords = {herberg myown}, number = 14, pages = {2389-2403}, timestamp = {2017-10-15T11:44:26.000+0200}, title = {Mutations of PKA cyclic nucleotide binding domains reveal novel aspects of cyclic nucleotide selectivity}, url = {http://www.biochemj.org/content/474/14/2389.long}, volume = 474, year = 2017 }