• Matthew G Gold, Douglas M Fowler, Christopher K Means, Catherine T Pawson, Jason J Stephany, Lorene K Langeberg, Stanley Fields, and John D Scott.
• Howard Hughes Medical Institute, University of Washington School of Medicine, Seattle, Washington 98195, USA. m.gold@ucl.ac.uk
• J. Biol. Chem. 2013 Jun 14; 288 (24): 17111-21.

AbstractPKA is retained within distinct subcellular environments by the association of its regulatory type II (RII) subunits with A-kinase anchoring proteins (AKAPs). Conventional reagents that universally disrupt PKA anchoring are patterned after a conserved AKAP motif. We introduce a phage selection procedure that exploits high-resolution structural information to engineer RII mutants that are selective for a particular AKAP. Selective RII (RSelect) sequences were obtained for eight AKAPs following competitive selection screening. Biochemical and cell-based experiments validated the efficacy of RSelect proteins for AKAP2 and AKAP18. These engineered proteins represent a new class of reagents that can be used to dissect the contributions of different AKAP-targeted pools of PKA. Molecular modeling and high-throughput sequencing analyses revealed the molecular basis of AKAP-selective interactions and shed new light on native RII-AKAP interactions. We propose that this structure-directed evolution strategy might be generally applicable for the investigation of other protein interaction surfaces.

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