Transforming PKA into PKG – a structure-function approach to understand cyclic nucleotide selectivity

Robin Lorenz,Jeong J Kim,Albert S Reger,Eui-Whan Moon,Daniela Bertinetti,Eugen Franz,Choel Kim,Friedrich W Herberg,Gilbert Y Huang

doi:10.1186/2050-6511-14-s1-p41

Abstract

BackgroundcAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG) are the main effectors ofdistinct cyclic nucleotide pathways and are preferentiallyactivated by cAMP or cGMP, respectively.We recently characterized the isolated C-terminal cyc-lic nucleotide binding domain (CNB-B) of the humanPKG Ib as highly cGMP-selective (manuscript in pre-paration). In a crystal structure of the CNB-B two novelcGMP-specific interaction sites were identified in addi-tion to the previously described threonine residue (T317)in the phosphate binding cassette [1]. Mutation of eachindividual site resulted in reduced cGMP-selectivity andinterfered with cGMP-dependent activation of PKG Ib.To gain further insight into the molecular basis of cyc-lic nucleotide selectivity, we inserted two cGMP-specificinteraction sites into the CNB-B of human PKA RIa bymutating corresponding residues. We hypothesize thatthis way cGMP-specific interaction contacts can be cre-ated in PKA and thereby modulate cAMP-selectivity[1,2].ResultsWe characterized a deletion construct of the PKA hRIaCNB-B as cAMP-selective using fluorescence polarization(FP) and surface plasmon resonance (SPR).In comparison to the wildtype PKA hRIa CNB-B, singlemutant constructs showed similar affinities for cAMP-and cGMP-analogs, revealing a loss of selectivity. Thecombination of two mutations led to a construct withhigher affinity for cGMP compared to cAMP.Co-crystal structures of this double mutant with cAMPor cGMP, respectively, showed that the cGMP-specificinteraction contacts retained their function in the contextof the PKA hRIa CNB-B.ConclusionThegeneralstructureofcyclicnucleotidebindingdomains is conserved. However, varying amino acids inthe binding pocket enable the distinction between cAMPand cGMP. Here we show that cGMP interaction sitesfound in PKG do restore their specific binding mechan-isms when introduced into PKA.The results underline the relevance of the describednovel binding sites in mediating cGMP-selectivity. Still,other features of CNB domains involved in the specificbinding mechanism as well as the detailed mechanism ofkinase activation need to be investigated.

Highlights

CAMP-dependent protein kinase (PKA) and cGMPdependent protein kinase (PKG) are the main effectors of distinct cyclic nucleotide pathways and are preferentially activated by cAMP or cGMP, respectively.We recently characterized the isolated C-terminal cyclic nucleotide binding domain (CNB-B) of the human PKG Ib as highly cGMP-selective
In a crystal structure of the CNB-B two novel cGMP-specific interaction sites were identified in addition to the previously described threonine residue (T317) in the phosphate binding cassette [1]
To gain further insight into the molecular basis of cyclic nucleotide selectivity, we inserted two cGMP-specific interaction sites into the CNB-B of human PKA RIa by mutating corresponding residues. We hypothesize that this way cGMP-specific interaction contacts can be created in PKA and thereby modulate cAMP-selectivity [1,2]

Summary

Introduction

We recently characterized the isolated C-terminal cyclic nucleotide binding domain (CNB-B) of the human PKG Ib as highly cGMP-selective (manuscript in preparation). In a crystal structure of the CNB-B two novel cGMP-specific interaction sites were identified in addition to the previously described threonine residue (T317) in the phosphate binding cassette [1]. Mutation of each individual site resulted in reduced cGMP-selectivity and interfered with cGMP-dependent activation of PKG Ib. To gain further insight into the molecular basis of cyclic nucleotide selectivity, we inserted two cGMP-specific interaction sites into the CNB-B of human PKA RIa by mutating corresponding residues.

Results

Conclusion