Structural characterization of protein kinase A as a function of nucleotide binding - Hydrogen-deuterium exchange studies using matrix-assisted laserdesorption ionization-time of flight mass spectrometry detection

Transient state kinetic studies indicate that substrate phosphorylation in protein kinase A is partially rate-limited by conformational changes, some of which may be associated with nucleotide binding (Shaffer, J,, and Adams, J. A. (1999) Biochemistry 38, 12072-12079). To assess whether specific str uctural changes are associated with the binding of nucleotides, hydrogen-de uterium exchange experiments were performed on the enzyme in the absence an d presence of ADP, Four regions of the protein are protected from exchange in the presence of ADP. Two regions encompass the catalytic and glycine-ric h loops and are integral parts of the active site. Conversely, protection o f probes in the C terminus is consistent with nucleotide-induced domain clo sure. One protected probe encompasses a portion of helix C, a secondary str uctural element that does not make any direct contacts with the nucleotide but has been reported to undergo segmental motion upon the activation of so me protein kinases. The combined data suggest that binding of the nucleotid e has distal structural effects that may include stabilizing the closed sta te of the enzyme and altering the position of a critical helix outside the active site. The latter represents the first evidence that the nucleotide a lone can induce changes in helix C in solution.