21
Modeling of Biomolecular Systems Interactions, Dynamics, and Allostery Session II Abstracts
How Enzymes Access Caged Substrates? Phosphodiesterase-Protein Kinase A Interactions
Mediate Hydrolysis of PKA Receptor Bound Cyclic AMP
Srinath Krishnamurthy
1
, Nikhil K. Tulsian
1
, Xin Xiang Lim
1
, Arun Chandramohan
1
, Kavitha
Bharatham
3
, Ivana Mihalek
3
, Ganesh S. Anand
1,2
.
1
National University of Singapore, Singapore,
3
A*Star Institutes, Singapore,
2
National
University of Singapore, Singapore.
CAMP dependent-Protein Kinase (PKA) signaling is a fundamental regulatory pathway for
mediating cellular responses to hormonal stimuli. The pathway is activated by association of
cAMP with regulatory subunit of PKA and signal termination is achieved upon cAMP
dissociation from PKA. While steps in the activation phase are well understood, little is known
on how signal termination/resetting occurs. Due to the high affinity of cAMP to PKA (KD ~ low
nM), bound cAMP does not readily dissociate from PKA, thus begging the question of how
bound cAMP dissociates from PKA to reset its signaling state to respond to subsequent stimuli.
We specifically set out to determine how cAMP-bound to the regulatory subunit is hydrolyzed to
return PKA to an inactive state and the role of phosphodiesterases in resetting of the system. We
report discovery of a novel signaling complex between phosphodiesterase (PDE8) and PKA
Regulatory subunit (RIα) in mammalian cAMP signaling by a combination of Structural Mass
spectrometry, specifically Amide hydrogen/deuterium exchange mass spectrometry (HDXMS),
peptide arrays and computational docking. Using experimental data as input, a computational
model for the complex was derived. This model reveals the phosphodiesterase active site in close
proximity to the cAMP binding site on PKA and highlights a role for substrate channeling in the
PDE-dependent dissociation and hydrolysis of cAMP bound to PKA. Real time reaction
monitoring by Structural Mass spectrometry and fluorescence polarization assays provides
further evidence for substrate channeling. This is the first instance of PDEs directly interacting
with a cAMP-receptor protein in mammalian systems and highlights an entirely new class of
binding partners for RIα. Furthermore, this introduces molecular channeling as a new paradigm
for macromolecular assemblies in microdomains and localized stimulus-response cycles in cell
signaling.
