Epac1 and cAMP-dependent protein kinase holoenzyme have similar cAMP affinity, but their cAMP domains have distinct structural features and cyclic nucleotide recognition.
The cAMP-dependent protein kinase (PKA I and II) and the cAMP-stimulated GDP exchange factors (Epac1 and -2) are major cAMP effectors. The cAMP affinity of the PKA holoenzyme has not been determined previously. We found that cAMP bound to PKA I with a K(d) value (2.9 microM) similar to that of Epac1. In contrast, the free regulatory subunit of PKA type I (RI) had K(d) values in the low nanomolar range. The cAMP sites of RI therefore appear engineered to respond to physiological cAMP concentrations only when in the holoenzyme form, whereas Epac can respond in its free form. Epac is phylogenetically younger than PKA, and its functional cAMP site has presumably evolved from site B of PKA. A striking feature is the replacement of a conserved Glu in PKA by Gln (Epac1) or Lys (Epac2). We found that such a switch (E326Q) in site B of human RIalpha led to a 280-fold decreased cAMP affinity. A similar single switch early in Epac evolution could therefore have decreased the high cAMP affinity of the free regulatory subunit sufficiently to allow Epac to respond to physiologically relevant cAMP levels. Molecular dynamics simulations and cAMP analog mapping indicated that the E326Q switch led to flipping of Tyr-373, which normally stacks with the adenine ring of cAMP. Combined molecular dynamics simulation, GRID analysis, and cAMP analog mapping of wild-type and mutated BI and Epac1 revealed additional differences, independent of the Glu/Gln switch, between the binding sites, regarding space (roominess), hydrophobicity/polarity, and side chain flexibility. This helped explain the specificity of current cAMP analogs and, more importantly, lays a foundation for the generation of even more discriminative analogs.
Dao
Khanh Kim
KK
Department of Biomedicine, University of Bergen, 5009 Bergen, Norway.
Teigen
Knut
K
Department of Biomedicine, University of Bergen, 5009 Bergen, Norway.
Kopperud
Reidun
R
Department of Biomedicine, University of Bergen, 5009 Bergen, Norway.
Hodneland
Erlend
E
Department of Biomedicine, University of Bergen, 5009 Bergen, Norway.
Schwede
Frank
F
BioLog Life Science Institute, Flughafendamm, D-28071 Bremen, Germany.
Christensen
Anne E
AE
Department of Biomedicine, University of Bergen, 5009 Bergen, Norway; Haukeland University Hospital, 5009 Bergen, Norway.
Martinez
Aurora
A
Department of Biomedicine, University of Bergen, 5009 Bergen, Norway.
Døskeland
Stein Ove
SO
Department of Biomedicine, University of Bergen, 5009 Bergen, Norway. Electronic address: stein.doskeland@biomed.uib.no.
D000595:Amino Acid Sequence; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry* / Q000378:metabolism; D020035:Holoenzymes / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010802:Phylogeny; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid
D020662:Guanine Nucleotide Exchange Factors; D020035:Holoenzymes; C116122:RAPGEF3 protein, human; D011994:Recombinant Proteins; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases