TI - Analysis of conformational changes during activation of protein kinase Pak2 by amide hydrogen/deuterium exchange . AB - During apoptotic stress , protein kinase Pak2 is cleaved by caspase 3 to form a heterotetramer that is constitutively activated following autophosphorylation . The active protein kinase migrates slightly slower than the inactive holoenzyme when analyzed by gel filtration , suggesting an expanded conformation . Activation of Pak2 comprises a series of structural changes resulting from caspase cleavage , ATP binding , and autophosphorylation of Pak2 . Changes at each step were individually analyzed by amide hydrogen/deuterium exchange coupled with mass spectrometry and compared with inactive Pak2 . The auto-inhibited form was shown to bind ATP in the active site , with minor changes in the glycine loop and the autoinhibitory domain ( AID ) . Caspase cleavage produced significant changes in solvent accessibility in the AID and upper lobe of the catalytic domain . Cleavage of ATP-bound Pak2 relaxes the allosteric inhibition , as shown by increased solvent accessibility in the upper and lower lobes , including the G-helix , facilitating the autophosphorylation of two sites required for activation , Ser-141 in the regulatory domain and Thr-402 in the catalytic domain . Autophosphorylation increased the amide hydrogen/deuterium exchange solvent accessibility of the contact region between the AID and the G-helix , the E-F loop , and the N terminus . Thus , activation of Pak2 via caspase cleavage is associated with structural relaxation of Pak2 that allows for complete auto-phosphorylation , resulting in a more comprehensive solvent-exposed and conformationally dynamic enzyme .