Importance of the A-helix of the catalytic subunit of cAMP-dependent protein kinase for stability and for orienting subdomains at the cleft interface.

All eukaryotic protein kinases share a conserved catalytic core. In the catalytic (C) subunit of cAMP-dependent protein kinase (cAPK) this core is preceded by a myristylation motif followed by a long helix with Trp 30 at the end of this A-helix filling a hydrophobic cavity between the two lobes of the core. To understand the importance of the A-helix, the myristylation motif (delta 1-14) as well as the entire N-terminal segment (delta 1 -39) were deleted. In addition, Trp 30 was replaced with both Tyr and Ala. All proteins were overexpressed in E. coli and purified to homogeneity. rC(delta 1-14), rC(W30Y), and rC(W30A) all had reduced thermostability, but were catalytically indistinguishable from wild-type C. Based on Surface Plasmon Resonance, all three also formed stable holoenzyme complexes with the RI-subunit, although the appKds were reduced by more than 10-fold due to decrease in the association rate. Surprisingly, however, the holoenzymes were even more thermostable than wild-type holoenzyme. To obtain active enzyme, it was necessary to purify rC(delta 1-39) as a fusion protein with glutathione-S-transferase (GST-rC(delta 1-39), although its thermostability (Tm) was decreased by 12.5 degrees C, was catalytically similar to wild-type C and was inhibited by both the type I and II R-subunits and the heat-stable protein kinase inhibitor (PKI). The Tm for holoenzyme II formed with GST-rC(delta 1-39) was 16.5 degrees C greater than the Tm for free GST-rC(delta 1-39), and the Ka(cAMP) was increased nearly 10-fold. These mutants point out striking and unanticipated differences in how the RI and RII subunits associate with the C-subunit to form a stable holoenzyme and indicate, furthermore, that this N-terminal segment, far from the active site cleft, influences those interactions. The importance of the A-helix and Trp 30 for stability correlates with its location at the cleft interface where it orients the C-helix in the small lobe and the activation loop in the large so that these subdomains are aligned in a way that allows for correct configuration of residues at the active site. This extensive network of contacts that links the A-helix directly to the active site in cAPK is compared to other kinases whose crystal structures have been solved.