Model for Protein Kinase A Type RIα Tetrameric Holoenzyme

Abstract

Protein kinase A holoenzyme contains two Catalytic (C) and two Regulatory (R) subunits, which dimerize through their N‐terminus Dimerization/Docking (D/D) domains. R‐subunit isoforms are highly conserved; the most divergent region lies at the linker connecting the D/D domain and the two tandem cAMP binding domains. Binding of four cAMP molecules to the R‐subunit dimer leads to significant structural changes that lead to the activation of the kinase. This process is highly cooperative in the tetrameric holoenzyme but the cooperativity is significantly reduced in the heterodimer which lacks the D/D domain and the linker. To understand the role of the linker region, we solved the structure of a PKA RIα(73–244)/C heterodimer, which contains the first cAMP binding domain and most of the linker. The overall structure is very similar to the previously solved RIα(91–244)/C heterodimer complex, however, the new structure allowed observation of 7 more residues (84–90) in the linker. Interestingly, this linker region docks to another symmetry related heterodimer, suggesting that one R‐subunit not only binds to one C‐subunit at multiple sites within the heterodimer but also cross‐talks with another heterodimer through the linker region when the tetrameric complex is formed. The β4‐β5 loop of the R‐subunit and the Y235PPPFF239 of the C‐subunit, which links αF and αG and is where PKI binds are also involved in this cross‐talk. By integrating small angle X‐ray/neutron scattering, molecular interface free‐energy calculations and other data, a model of RIα/C tetrameric complex is proposed.