Dynamics of phosphodiesterase-induced cAMP dissociation from protein kinase A: Capturing transient ternary complexes by HDXMS

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• 作者：Srinath Krishnamurthy¹ ; Balakrishnan Shenbaga Moorthy¹ ; Lin Liqin ; Ganesh S. Anand ; ^{dbsgsa@nus.edu.sg}
• 关键词：BME ; ¦Â-mercaptoethanol ; CNB ; cyclic nucleotide binding ; cAMP ; cyclic adenosine 3&prime ; 5&prime ; -monophosphate ; D2O ; deuterium oxide ; EDTA ; ethylenediaminetetraacetic acid ; FPLC ; fast protein liquid chromatography ; HDXMS ; amide hydrogen/deuterium exchang
• 刊名：Biochimica et Biophysica Acta - Proteins and Proteomics
• 出版年：2013
• 出版时间：June, 2013
• 年：2013
• 卷：1834
• 期：6
• 页码：1215-1221
• 全文大小：1055 K

文摘

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.