Site-directed mutagenesis of the glycine-rich loop of death associated protein kinase (DAPK) identifies it as a key structure for catalytic activity.
Death associated protein kinase (DAPK) is a calmodulin (CaM)-regulated protein kinase that is a therapeutic target for central nervous system (CNS) disorders. We report here the results of studies that test the hypothesis of McNamara et al. (2009) that conformational selection in DAPK's glycine-rich region is key for catalytic activity. The hypothesis was tested by site-directed mutagenesis of glutamine-23 (Q23) in the middle of this loop. The glycine-rich loop exhibits localized differences in structure among DAPK conformations that correlate with different stages of the catalytic cycle. Changing the Q23 to a Valine (V23), found at the corresponding position in another CaM regulated protein kinase, results in a reduced catalytic efficiency. High resolution X-ray crystal structures of various conformations of the Q23V mutant DAPK and their superimposition with the corresponding conformations from wild type catalytic domain reveal localized changes in the glycine-rich region. The effect of the mutation on DAPK catalytic activity and the finding of only localized changes in the DAPK structure provide experimental evidence implicating conformational selection in this domain with activity. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.
Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA.
Laurie K
LK
McNamara
Joseph S
JS
Brunzelle
James P
JP
Schavocky
D Martin
DM
Watterson
Valerie
V
Grum-Tokars
D000244:Adenosine Diphosphate / Q000378:metabolism; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D051017:Apoptosis Regulatory Proteins / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D055162:Biocatalysis; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000737:chemistry* / Q000378:metabolism*; D018360:Crystallography, X-Ray; D064550:Death-Associated Protein Kinases; D057075:Enzyme Assays; D005973:Glutamine / Q000235:genetics; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D014633:Valine / Q000235:genetics
D051017:Apoptosis Regulatory Proteins; D008024:Ligands; D050505:Mutant Proteins; D005973:Glutamine; D000266:Adenylyl Imidodiphosphate; D000244:Adenosine Diphosphate; D064550:Death-Associated Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D014633:Valine