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

作者：Moorthy Balakrishnan Shenbaga; Badireddy Suguna; Anand Ganesh S^*

来源：International Journal of Mass Spectrometry, 2011, 302(1-3): 157-166.

DOI：10.1016/j.ijms.2010.09.012

• Allostery
• Intermediate
• cAMP
• Cooperativity
• Conformational change
• Dynamic

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 alpha C/C':A and alpha A: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.

• 出版日期2011-4-30