Mechanism of Epac activation. Structural and functional analyses of Epac2 hinge mutants with constitutive and reduced activities

Tamara Tsalkova, Donald K. Blumenthal, Fang C. Mei, Mark White, Xiaodong Cheng

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

Epac2 is a member of the family of exchange proteins directly activated by cAMP (Epac). Our previous studies suggest a model of Epac activation in which cAMP binding to the enzyme induces a localized "hinge" motion that reorients the regulatory lobe relative to the catalytic lobe without inducing large conformational changes within individual lobes. In this study, we identified the location of the major hinge in Epac2 by normal mode motion correlation and structural alignment analyses. Targeted mutagenesis was then performed to test the functional importance of hinge bending for Epac activation. We show that substitution of the conserved residue phenylalanine 435 with glycine (F435G) facilitates the hinge bending and leads to a constitutively active Epac2 capable of stimulating nucleotide exchange in the absence of cAMP. In contrast, substitution of the same residue with a bulkier side chain, tryptophan (F435W), impedes the hinge motion and results in a dramatic decrease in Epac2 catalytic activity. Structural parameters determined by small angle x-ray scattering further reveal that whereas the F435G mutant assumes a more extended conformation in the absence of cAMP, the F435W mutant is incapable of adopting the fully extended and active conformation in the presence of cAMP. These findings demonstrate the importance of hinge motion in Epac activation. Our study also suggests that phenylalanine at position 435 is the optimal size side chain to keep Epac closed and inactive in the absence of cAMP while still allowing the proper hinge motion for full Epac extension and activation in the presence of cAMP.

Original languageEnglish (US)
Pages (from-to)23644-23651
Number of pages8
JournalJournal of Biological Chemistry
Volume284
Issue number35
DOIs
StatePublished - Aug 28 2009

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Hinges
Chemical activation
Proteins
Phenylalanine
Conformations
Substitution reactions
Mutagenesis
Tryptophan
Glycine
Nucleotides
X-Rays
Catalyst activity
Scattering
X rays
Enzymes

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

Mechanism of Epac activation. Structural and functional analyses of Epac2 hinge mutants with constitutive and reduced activities. / Tsalkova, Tamara; Blumenthal, Donald K.; Mei, Fang C.; White, Mark; Cheng, Xiaodong.

In: Journal of Biological Chemistry, Vol. 284, No. 35, 28.08.2009, p. 23644-23651.

Research output: Contribution to journalArticle

Tsalkova, Tamara ; Blumenthal, Donald K. ; Mei, Fang C. ; White, Mark ; Cheng, Xiaodong. / Mechanism of Epac activation. Structural and functional analyses of Epac2 hinge mutants with constitutive and reduced activities. In: Journal of Biological Chemistry. 2009 ; Vol. 284, No. 35. pp. 23644-23651.
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