Inhibitor binding to mutants of protein kinase A with GGGxxG and GxGxxA glycine‐rich loop motifs

Abstract

The conserved GxGxxG motif of protein kinases forms a beta turn at the tip of the flexible glycine‐rich loop and creates much of the ATP pocket binding surface. Notable exceptions to this sequence include GGGxxG in ABL kinase and GxGxxA in protein kinase C isoforms. We constructed the corresponding mutants of PKA, T51G, and G55A, and tested quinazoline inhibitors that were designed to bind via glycine‐rich loop interactions, testing also staurosporine for comparison. The quinazoline inhibitors have significantly reduced binding strengths in both mutants. In striking contrast to these results, the binding of the “pan‐kinome” inhibitor staurosporine is strengthened in the mutants. Surface plasmon resonance (SPR) shows that the tightened binding of staurosporine arises from increased kon rates, changes not offset by more moderately increased koff rates. The SPR results fit best to a two step binding process for staurosporine in wild type PKA, but not the mutants.

Drugging the Undruggable: How Isoquinolines and PKA Initiated the Era of Designed Protein Kinase Inhibitor Therapeutics

“…The conserved motif GxGxxG forms a β-hairpin turn at the tip of the G-loop. 23 The nucleotide-binding G-loops of protein kinases differ from the counterpart loops (P-loops) of other ATP-binding proteins such as hexokinase, hydrogenases, and ATPases. These contain a similar but contrasting motif GxxGxGK{S/T}, which is known as the Walker motif or Rossmann-fold.…”

“…The G-loop provides many of the ATP-binding interactions, anchoring the adenine ring and the nontransferable phosphate groups, i.e., the α- and β-phosphates, of ATP. The conserved motif GxGxxG forms a β-hairpin turn at the tip of the G-loop . The nucleotide-binding G-loops of protein kinases differ from the counterpart loops (P-loops) of other ATP-binding proteins such as hexokinase, hydrogenases, and ATPases.…”