title: Dynamics of phosphodiesterase-induced cAMP dissociation from protein kinase A: capturing transient ternary complexes by HDXMS.
abstract: cAMP signaling is a fundamental cellular process necessary for mediating responses to hormonal stimuli. In contrast to cAMP-dependent activation of protein kinase A (PKA), an important cellular target, far less is known on termination in cAMP signaling, specifically how phosphodiesterases (PDEs) facilitate dissociation and hydrolysis of bound cAMP. In this study, we have probed the dynamics of a ternary complex of PKA and a PDE-RegA with an excess of a PDE-nonhydrolyzable cAMP analog, Sp-cAMPS by amide hydrogen/deuterium exchange mass spectrometry (HDXMS). Our results highlight how HDXMS can be used to monitor reactions together with mapping conformational dynamics of transient signaling complexes. Our results confirm a two-state model for active RegA-mediated dissociation of bound cAMP. Further, our results reveal that Sp-cAMPS and RegA mediate mutually exclusive interactions with the same region of PKA and at specific concentrations of Sp-cAMPS, RegA is capable of blocking Sp-cAMPS reassociation to PKA. This provides a molecular basis for how PDEs modulate levels of intracellular cAMP so that PKA is better suited to responding to fluxes rather than constant levels of cAMP. This study underscores how HDXMS can be a powerful tool for monitoring reactions together with mapping conformational dynamics in signaling proteins. This article is part of a Special Issue entitled: Mass spectrometry in structural biology.
authors.affiliation: Department of Biological Sciences, National University of Singapore, Singapore.
authors.forename: Srinath
authors.initials: S
authors.lastname: Krishnamurthy
authors.forename: Balakrishnan Shenbaga
authors.initials: BS
authors.lastname: Moorthy
authors.forename: Lin
authors.initials: L
authors.lastname: Liqin
authors.forename: Ganesh S
authors.initials: GS
authors.lastname: Anand
mesh_terms: D000577:Amides / Q000737:chemistry; D000242:Cyclic AMP / Q000737:chemistry* / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000378:metabolism; D003903:Deuterium / Q000737:chemistry; D041961:Deuterium Exchange Measurement / Q000379:methods; D006859:Hydrogen / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods; D056004:Molecular Dynamics Simulation; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D015398:Signal Transduction; D050801:Ternary Complex Factors / Q000737:chemistry*
chemical_list: D000577:Amides; D050801:Ternary Complex Factors; D006859:Hydrogen; D003903:Deuterium; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D010727:Phosphoric Diester Hydrolases