Effect of metal ions on high-affinity binding of pseudosubstrate inhibitors to PKA

Abstract

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 Mg 2+ with PKI, transient interaction kinetics with fast on-and off-rates were observed, whereas at high Mg 2+ 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 Mg 2+ . 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.

Dynamically committed, uncommitted, and quenched states encoded in protein kinase A revealed by NMR spectroscopy

“…This difference in affinity is amplified by ADP to 40 times, reflecting the strong cooperative effect between PKI and nucleotide (15,16). Remarkably, PKI [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24] and PLN [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] have different thermodynamics of binding (Fig. 1A).…”

“…To compare the effects of binding substrates and inhibitors to PKA-C, we synthesized two peptides: the first peptide corresponding to the cytoplasmic domain of phospholamban (PLN 1-20 ), an endogenous inhibitor for the sarcoplasmic reticulum Ca-ATPase and native substrate of PKA-C in cardiac muscle (14); and the second peptide corresponding to PKI [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24] (Fig. S1).…”

“…1A). PLN [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] binding to PKA-C is dominated by a favorable overall entropy to the binding free energy (ΔG). The presence of ADP increases the favorable entropy of binding, resulting in an enhanced binding affinity, revealing a positive cooperativity similar to that of Kemptide (17).…”

“…The combination of high Mg 2þ concentrations with PKI has implications for the cellular control of PKA-C activity to arrest transcription during mitosis (11,12). By comparing the results with those obtained in the presence of a peptide substrate (7) which competes with PKI 5-24 at the binding groove of PKA-C, we found that both PKI [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24] and excess Mg 2þ restrict the enzyme dynamics on a fast (picosecond to nanosecond) and slow (microsecond to millisecond) NMR timescale without drastically changing the conformation of the ternary complex. Inhibitor binding modifies the energy landscape by restricting the motions of the enzyme backbone (13).…”

PKA and the Structural Kinome

“…Embedded within each linker is an Inhibitor Site (IS) that docks into the active site cleft of the C-subunit in the holoenzyme and prevents binding of other substrates. Like PKI the RI subunits are pseudosubstrates; the P-site in the IS is replaced with Gly or Ala. C-subunits bound to both PKI and R-subunits bind ATP with high affinity (60 nM) when two metal ions are present (69,96,97). The synergistic high affinity binding of RI subunits and ATP is thus a conserved feature of the RI subunits and PKI.…”