Entropy-driven cAMP-dependent allosteric control of inhibitory interactions in exchange proteins directly activated by cAMP.
Exchange proteins directly activated by cAMP (EPACs) are guanine nucleotide-exchange factors for the small GTPases Rap1 and Rap2 and represent a key receptor for the ubiquitous cAMP second messenger in eukaryotes. The cAMP-dependent activation of apoEPAC is typically rationalized in terms of a preexisting equilibrium between inactive and active states. Structural and mutagenesis analyses have shown that one of the critical determinants of the EPAC activation equilibrium is a cluster of salt bridges formed between the catalytic core and helices alpha1 and alpha2 at the N terminus of the cAMP binding domain and commonly referred to as ionic latch (IL). The IL stabilizes the inactive states in a closed topology in which access to the catalytic domain is sterically occluded by the regulatory moiety. However, it is currently not fully understood how the IL is allosterically controlled by cAMP. Chemical shift mapping studies consistently indicate that cAMP does not significantly perturb the structure of the IL spanning sites within the regulatory region, pointing to cAMP-dependent dynamic modulations as a key allosteric carrier of the cAMP-signal to the IL sites. Here, we have therefore investigated the dynamic profiles of the EPAC1 cAMP binding domain in its apo, cAMP-bound, and Rp-cAMPS phosphorothioate antagonist-bound forms using several 15N relaxation experiments. Based on the comparative analysis of dynamics in these three states, we have proposed a model of EPAC activation that incorporates the dynamic features allosterically modulated by cAMP and shows that cAMP binding weakens the IL by increasing its entropic penalty due to dynamic enhancements.
Das
Rahul
R
Department of Chemistry, Biochemistry, and Biomedical Sciences, McMaster University, Hamilton, Ontario L8S 4M1, Canada.
Mazhab-Jafari
Mohammad T
MT
Chowdhury
Somenath
S
SilDas
Soumita
S
Selvaratnam
Rajeevan
R
Melacini
Giuseppe
G
D000494:Allosteric Regulation / Q000502:physiology; D000242:Cyclic AMP / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019277:Entropy; D020662:Guanine Nucleotide Exchange Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D017433:Protein Structure, Secondary / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D015290:Second Messenger Systems / Q000502:physiology; D020668:rap GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020827:rap1 GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D020662:Guanine Nucleotide Exchange Factors; C116122:RAPGEF3 protein, human; D000242:Cyclic AMP; C116756:RAP2A protein, human; D020668:rap GTP-Binding Proteins; D020827:rap1 GTP-Binding Proteins