Amide H/2H exchange reveals communication between the cAMP and catalytic subunit-binding sites in the R(I)alpha subunit of protein kinase A

J Mol Biol. 2002 Oct 18;323(2):377-86. doi: 10.1016/s0022-2836(02)00919-1.

Abstract

The changes in backbone hydrogen/deuterium (H/2H) exchange in the regulatory subunit (R(I)alpha(94-244)) of cyclic AMP-dependent protein kinase A (PKA) were probed by MALDI-TOF mass spectrometry. The three naturally occurring states of the regulatory subunit were studied: (1) free R(I)alpha(94-244), which likely represents newly synthesized protein, (2) R(I)alpha(94-244) bound to the catalytic (C) subunit, or holoenzyme, and (3) R(I)alpha(94-244) bound to cAMP. Protection from amide exchange upon C-subunit binding was observed for the helical subdomain, including the A-helix and B-helix, pointing to regions adjacent to those shown to be important by mutagenesis. In addition, C-subunit binding caused changes in observed amide exchange in the distal cAMP-binding pocket. Conversely, cAMP binding caused protection in the cAMP-binding pocket and increased exchange in the helical subdomain. These results suggest that the mutually exclusive binding of either cAMP or C-subunit is controlled by binding at one site transmitting long distance changes to the other site.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amides / chemistry
  • Amino Acid Sequence
  • Binding Sites
  • Catalytic Domain
  • Cyclic AMP / chemistry
  • Cyclic AMP / metabolism*
  • Cyclic AMP-Dependent Protein Kinases / chemistry
  • Cyclic AMP-Dependent Protein Kinases / metabolism*
  • Hydrogen / chemistry*
  • Models, Molecular
  • Molecular Sequence Data
  • Protein Binding
  • Protein Structure, Tertiary*
  • Protein Subunits / chemistry
  • Protein Subunits / metabolism*
  • Solvents
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Substances

  • Amides
  • Protein Subunits
  • Solvents
  • Hydrogen
  • Cyclic AMP
  • Cyclic AMP-Dependent Protein Kinases