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 k_on rates, changes not offset by more moderately increased k_off rates. The SPR results fit best to a two step binding process for staurosporine in wild type PKA, but not the mutants.

Keywords: glycine-rich loop; inhibitor binding; point mutation; protein kinase; staurosporine.