Structural characterization of protein kinase A as a function of nucleotide binding. Hydrogen-deuterium exchange studies using matrix-assisted laser desorption 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 structural changes are associated with the binding of nucleotides, hydrogen-deuterium exchange experiments were performed on the enzyme in the absence and presence of ADP. Four regions of the protein are protected from exchange in the presence of ADP. Two regions encompass the catalytic and glycine-rich loops and are integral parts of the active site. Conversely, protection of probes in the C terminus is consistent with nucleotide-induced domain closure. One protected probe encompasses a portion of helix C, a secondary structural element that does not make any direct contacts with the nucleotide but has been reported to undergo segmental motion upon the activation of some protein kinases. The combined data suggest that binding of the nucleotide has distal structural effects that may include stabilizing the closed state of the enzyme and altering the position of a critical helix outside the active site. The latter represents the first evidence that the nucleotide alone can induce changes in helix C in solution.