MAPPING SUBSTRATE-INDUCED CONFORMATIONAL-CHANGES IN CAMP-DEPENDENT PROTEIN-KINASE BY PROTEIN FOOTPRINTING

Upon binding of substrates the catalytic subunit (C) of cAMP-dependent protein kinase (cAPK) undergoes significant induced conformational ch anges that lead to catalysis. For the free apoenzyme equilibrium favor s a more open and malleable conformation while the ternary complex of C, MgATP, and a 20-residue inhibitor peptide [PKI (5-24)] adopts a tig ht and closed conformation [Zheng, J., et al. (1993) Protein Sci. 2, 1 559]. It is not clear that binding of either ligand alone is responsib le for this conformational switch or whether both are required. In add ition, the catalytic subunit binds MgATP and inhibitor peptide synergi stically. The structural basis for this synergism is also not defined at present. Using an Fe-EDTA-mediated protein footprinting technique, the conformational changes associated with the binding of MgATP and th e heat stable protein kinase inhibitor (PKI) were probed by mapping th e solvent-accessible surface and structural dynamics of C. The conform ation of the free enzyme was clearly distinguished from the ternary co mplex. Furthermore, binding of MgATP alone induced extensive conformat ional changes, both local and global, that include the glycine-rich lo op, the linker connecting the small and large lobes, the catalytic loo p, the Mg2+ positioning loop, the activation loop, and the F helix. Th ese changes, similar to those seen in the ternary complex, are consist ent with a transition from an open to a more closed conformation and l ikely reflect the motions that are associated with catalysis and produ ct release. In contrast, the footprinting pattern of C.PKT resembled f ree C, indicating minimal conformational changes. Binding of MgATP, by shifting the equilibrium to a more closed conformation, ''primes'' th e enzyme so that it is poised for the docking of PKI and provides an e xplanation for synergism between MgATP and PKI.