Conformational control of protein kinases is an important way of modulating catalytic activity. Crystal structures of the C (catalytic) subunit of PKA (protein kinase A) in complex with physiological inhibitors and/or nucleotides suggest a highly dynamic process switching between open and more closed conformations. To investigate the underlying molecular mechanisms, SPR (surface plasmon resonance) was used for detailed binding analyses of two physiological PKA inhibitors, PKI (heat-stable protein kinase inhibitor) and a truncated form of the R (regulatory) subunit (RIα 92–260), in the presence of various concentrations of metals and nucleotides. Interestingly, it could be demonstrated that high-affinity binding of each pseudosubstrate inhibitor was dependent only on the concentration of divalent metal ions. At low micromolar concentrations of Mg2+ with PKI, transient interaction kinetics with fast on- and off-rates were observed, whereas at high Mg2+ concentrations the off-rate was slowed down by a factor of 200. This effect could be attributed to the second, low-affinity metal-binding site in the C subunit. In contrast, when investigating the interaction of RIα 92–260 with the C subunit under the same conditions, it was shown that the association rate rather than the dissociation rate was influenced by the presence of high concentrations of Mg2+. A model is presented, where the high-affinity interaction of the C subunit with pseudosubstrate inhibitors (RIα and PKI) is dependent on the closed, catalytically inactive conformation induced by the binding of a nucleotide complex where both of the metal-binding sites are occupied.
Analysis of High-Affinity Binding of Protein Kinase R to Double-Stranded RNA