Measurement of State-Specific Association Constants in Allosteric Sensors through Molecular Stapling and NMR

J Am Chem Soc. 2015 Aug 26;137(33):10777-85. doi: 10.1021/jacs.5b06557. Epub 2015 Aug 17.

Abstract

Allostery is a ubiquitous mechanism to control biological function and arises from the coupling of inhibitory and binding equilibria. The extent of coupling reflects the inactive vs active state selectivity of the allosteric effector. Hence, dissecting allosteric determinants requires quantification of state-specific association constants. However, observed association constants are typically population-averages, reporting on overall affinities but not on allosteric coupling. Here we propose a general method to measure state-specific association constants in allosteric sensors based on three key elements, i.e., state-selective molecular stapling through disulfide bridges, competition binding saturation transfer experiments and chemical shift correlation analyses to gauge state populations. The proposed approach was applied to the prototypical cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA-RIα), for which the structures of the inactive and active states are available, as needed to design the state-selective disulfide bridges. Surprisingly, the PKA-RIα state-specific association constants are comparable to those of a structurally homologous domain with ∼10(3)-fold lower cAMP-affinity, suggesting that the affinity difference arises primarily from changes in the position of the dynamic apo inhibitory equilibrium.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Allosteric Regulation
  • Apoenzymes / chemistry
  • Apoenzymes / genetics
  • Apoenzymes / metabolism
  • Cyclic AMP / metabolism
  • Cyclic AMP-Dependent Protein Kinase RIalpha Subunit / chemistry*
  • Cyclic AMP-Dependent Protein Kinase RIalpha Subunit / genetics
  • Cyclic AMP-Dependent Protein Kinase RIalpha Subunit / metabolism*
  • Enzyme Stability
  • Magnetic Resonance Spectroscopy
  • Models, Molecular
  • Mutation
  • Protein Structure, Tertiary
  • Sulfides / chemistry

Substances

  • Apoenzymes
  • Cyclic AMP-Dependent Protein Kinase RIalpha Subunit
  • Sulfides
  • Cyclic AMP