ABSTRACT
Epac1 and Epac2, the most recently discovered cAMP receptors,1,2 are guanine nucleotide exchange factors (GEFs) for the small G protein Rap. Small G proteins are GTPases that cycle between an inactive GDP and an active GTP-bound state,3 and GEFs such as Epac bind cAMP so that they may catalyze the release of GDP from Rap, after which GTP may then bind to Rap.4 The ability of cAMP to signal
6.1 Introduction ....................................................................................................99 6.2 The CNB Domain as a Sensor of Cyclic Nucleotides .................................. 101 6.3 Catalytic Mechanism of Guanine Nucleotide Exchange .............................. 101 6.4 Methods for Characterization of Epac2 by X-Ray Crystallography ............. 102
6.4.1 Protein Expression and Purication ................................................. 102 6.4.1.1 Epac2 .................................................................................. 102 6.4.1.2 Rap ..................................................................................... 104
6.4.2 Crystallization .................................................................................. 106 6.4.2.1 Crystallization of Epac21-463 .............................................. 106 6.4.2.2 Cryoprotection of Epac21-463 Crystals ................................ 106 6.4.2.3 Crystal Properties of Epac21-463 ......................................... 106 6.4.2.4 Crystallization of Epac21-993 .............................................. 107 6.4.2.5 Cryoprotection of Epac21-993 Crystals ................................ 107 6.4.2.6 Crystal Properties of Epac21-993 ......................................... 107 6.4.2.7 Crystallization of Epac2306-993 in Complex with
Rap1b and Cyclic Nucleotide ............................................. 107 6.4.2.8 Cryoprotection of Crystals of Epac2306-993 in
Complex with Rap1b and Cyclic Nucleotide ..................... 108 6.4.2.9 Crystal Properties of Epac2306-993 in Complex with
Rap1b and Cyclic Nucleotide ............................................. 108 6.4.3 Structure Solution and Model Building ............................................ 110
6.5 Summary and Open Questions ..................................................................... 110 References .............................................................................................................. 110
through Epac allows the GTP-bound form of Rap to interact with its downstream effector proteins and thereby to transduce the cAMP signal so that cellular functions will be altered.
