Electrostatic Interactions as Mediators in the Allosteric Activation of Protein Kinase A RIα

Biochemistry. 2017 Mar 14;56(10):1536-1545. doi: 10.1021/acs.biochem.6b01152. Epub 2017 Mar 6.

Abstract

Close-range electrostatic interactions that form salt bridges are key components of protein stability. Here we investigate the role of these charged interactions in modulating the allosteric activation of protein kinase A (PKA) via computational and experimental mutational studies of a conserved basic patch located in the regulatory subunit's B/C helix. Molecular dynamics simulations evidenced the presence of an extended network of fluctuating salt bridges spanning the helix and connecting the two cAMP binding domains in its extremities. Distinct changes in the flexibility and conformational free energy landscape induced by the separate mutations of Arg239 and Arg241 suggested alteration of cAMP-induced allosteric activation and were verified through in vitro fluorescence polarization assays. These observations suggest a mechanical aspect to the allosteric transition of PKA, with Arg239 and Arg241 acting in competition to promote the transition between the two protein functional states. The simulations also provide a molecular explanation for the essential role of Arg241 in allowing cooperative activation, by evidencing the existence of a stable interdomain salt bridge with Asp267. Our integrated approach points to the role of salt bridges not only in protein stability but also in promoting conformational transition and function.

Publication types

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

MeSH terms

  • Allosteric Regulation
  • Allosteric Site
  • Amino Acid Sequence
  • Arginine / chemistry*
  • Arginine / metabolism
  • Aspartic Acid / chemistry*
  • Aspartic Acid / metabolism
  • Catalytic Domain
  • Cloning, Molecular
  • Cyclic AMP / chemistry*
  • 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 Activation
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Gene Expression
  • Humans
  • Kinetics
  • Molecular Docking Simulation
  • Molecular Dynamics Simulation
  • Mutation
  • Protein Structure, Secondary
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Salts / chemistry
  • Sequence Alignment
  • Static Electricity
  • Thermodynamics

Substances

  • Cyclic AMP-Dependent Protein Kinase RIalpha Subunit
  • Recombinant Proteins
  • Salts
  • Aspartic Acid
  • Arginine
  • Cyclic AMP