Cooperativity and allostery in cAMP-dependent activation of Protein Kinase A: Monitoring conformations of intermediates by amide hydrogen/deuterium exchange

Balakrishnan Shenbaga Moorthy, Suguna Badireddy, Ganesh S. Anand

Research output: Contribution to journalArticlepeer-review

6 Scopus citations

Abstract

Amide hydrogen/deuterium exchange mass spectrometry is a powerful method both for mapping protein-protein interactions and measuring conformational dynamics of protein complexes. In this study we report its application to monitoring the stepwise process governing cAMP-dependent activation of Protein Kinase A (PKA). In the absence of cAMP, PKA exists in an inactive complex of catalytic (C) and regulatory (R) subunits. cAMP binding induces large conformational changes within the R-subunit leading to dissociation of the active C-subunit. Although crystal structures of end-point, inactive and active states are available, the molecular basis for cooperativity in cAMP-dependent activation of PKA is not clear. In this study we report application of amide hydrogen/deuterium exchange mass spectrometry on tracking the stepwise cAMP-induced conformational changes using a single point mutant (R209K) at the cyclic nucleotide binding domain (CNB)-A site. Our amide exchange results reveal that binding of one molecule of cAMP increases amide exchange in important regions within the second CNB-B domain. Increased exchange was also seen at the interface between CNB-B and the C-subunit suggesting weakening of the R-C interface without dissociation. Importantly, binding of the first molecule of cAMP greatly increases the conformational mobility/dynamics of two key regions coupling the two CNBs, the αC/C′:A and αA:B helix. We believe that the enhanced dynamics of these regions forms the basis for the positive cooperativity in the cAMP-dependent activation of PKA. In summary, our results reveal the close allosteric coupling between CNB-A and CNB-B with the C-subunit providing important molecular insights into the function of CNB-B domain.

Original languageEnglish (US)
Pages (from-to)157-166
Number of pages10
JournalInternational Journal of Mass Spectrometry
Volume302
Issue number1-3
DOIs
StatePublished - Apr 30 2011

All Science Journal Classification (ASJC) codes

  • Instrumentation
  • Condensed Matter Physics
  • Spectroscopy
  • Physical and Theoretical Chemistry

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