Dissecting the mechanism of Epac activation via hydrogen-deuterium exchange FT-IR and structural modeling.
Exchange proteins directly activated by cAMP (Epac) make up a family of cAMP binding domain-containing proteins that play important roles in mediating the effects of cAMP through the activation of downstream small GTPases, Ras-proximate proteins. To delineate the mechanism of Epac activation, we probed the conformation and structural dynamics of Epac using amide hydrogen-deuterium (H-D) exchange coupled with Fourier transform infrared spectroscopy (FT-IR) and structural modeling. Our studies show that unlike that of cAMP-dependent protein kinase (PKA), the classic intracellular cAMP receptor, binding of cAMP to Epac does not induce significant changes in overall secondary structure and structural dynamics, as measured by FT-IR and the rate of H-D exchange, respectively. These results suggest that Epac activation does not involve significant changes in the amount of exposed surface areas as in the case of PKA activation, and conformational changes induced by cAMP in Epac are most likely confined to small local regions. Homology modeling and comparative structural analyses of the CBDs of Epac and PKA lead us to propose a model of Epac activation. On the basis of our model, Epac activation by cAMP employs the same underlying structural principal utilized by PKA, although the detailed structural and conformational changes associated with Epac and PKA activation are significantly different. In addition, we predict that during Epac activation the first beta-strand of the switchboard switches its conformation to a alpha-helix, which folds back to the beta-barrel core of the CBD and interacts directly with cAMP to form the base of the cAMP-binding pocket.
Yu
Shaoning
S
Sealy Center for Structural Biology, The University of Texas Medical Branch, Galveston, Texas 77555-1031, USA.
Fan
Fenghui
F
Flores
Samuel C
SC
Mei
Fang
F
Cheng
Xiaodong
X
D000595:Amino Acid Sequence; D017362:Cyclic AMP Response Element-Binding Protein / Q000737:chemistry / Q000378:metabolism; D041961:Deuterium Exchange Measurement; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017550:Spectroscopy, Fourier Transform Infrared
D017362:Cyclic AMP Response Element-Binding Protein; D020662:Guanine Nucleotide Exchange Factors; C116122:RAPGEF3 protein, human; C442864:RAPGEF4 protein, human