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

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.

Automated summary

The conserved GxGxxG motif in protein kinases plays a crucial role in the binding of inhibitors, with different mutants showing varied binding strengths. Surprisingly, the pan-kinome inhibitor staurosporine exhibits strengthened binding in mutants due to increased binding rates.