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Mechanism of Epac Activation:Structural and Functional Analyses of Epac2 Hinge Mutants
Author(s) -
Tsalkova Tamara N,
Mei Fang C,
White Mark,
Cheng Xiaodong,
Blumenthal Donald
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.463.16
Subject(s) - hinge , chemistry , mutant , guanine nucleotide exchange factor , residue (chemistry) , phenylalanine , mutagenesis , biophysics , amino acid , biochemistry , biology , signal transduction , structural engineering , engineering , gene
Epac2 is a guanine nucleotide exchange factor which directly activated by cAMP. According to the model of Epac activation, a localized “hinge” motion is a major change in the Epac structure upon cAMP binding. In this study, we test the functional importance of hinge bending for Epac activation by targeted mutagenesis. We show that substitution of the conserved residue phenylalanine 435 by glycine facilitates the hinge bending. As a result, Epac2‐F435G mutant is constitutively active and stimulates nucleotide exchange in the absence of cAMP. In contrast, substitution of the same residue with a bulkier side chain, tryptophan, impedes the hinge motion and results in a dramatic decrease in Epac2 catalytic activity. Structural parameters for wild type Epac and two of its mutants determined by small‐angle X‐ray scattering (SAXS) further confirms the importance of hinge motion in Epac activation. Our study also suggests that the side‐chain size of the amino acid at the position 435 is a key to Epac functioning. It seems that phenylalanine at this position has the optimal size to prevent “hinge” bending and keep Epac closed and inactive in the absence of cAMP while still allowing the proper hinge motion for full Epac activation in the presence of cAMP.