Analysis of conformational changes during activation of protein kinase Pak2 by amide hydrogen/deuterium exchange.
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.
Hsu
Yuan-Hao
YH
Department of Biochemistry and Division of Biomedical Sciences, University of California, Riverside, California 92521, USA.
Johnson
David A
DA
Traugh
Jolinda A
JA
D000595:Amino Acid Sequence; D000818:Animals; D053148:Caspase 3 / Q000378:metabolism; D002417:Cattle; D003903:Deuterium / Q000737:chemistry*; D004789:Enzyme Activation; D006859:Hydrogen / Q000737:chemistry*; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011817:Rabbits; D013917:Thrombin / Q000737:chemistry; D054462:p21-Activated Kinases / Q000378:metabolism*
D006859:Hydrogen; D003903:Deuterium; D054462:p21-Activated Kinases; D013917:Thrombin; D053148:Caspase 3