Exploring the interaction between the protein kinase A catalytic subunit and caveolin-1 scaffolding domain with shotgun scanning, oligomer complementation, NMR, and docking.
The techniques of phage-displayed homolog shotgun scanning, oligomer complementation, NMR secondary structure analysis, and computational docking provide a complementary suite of tools for dissecting protein-protein interactions. Focusing these tools on the interaction between the catalytic sub-unit of protein kinase A (PKAcat) and caveolin-1 scaffolding domain (CSD) reveals the first structural model for the interaction. Homolog shotgun scanning varied each CSD residue as either a wild-type or a homologous amino acid. Wild-type to homolog ratios from 116 different homologous CSD variants identified side-chain functional groups responsible for precise contacts with PKAcat. Structural analysis by NMR assigned an alpha-helical conformation to the central residues 84- 97 of CSD. The extensive mutagenesis data and NMR secondary structure information provided constraints for developing a model for the PKAcat-CSD interaction. Addition of synthetic CSD to phage-displayed CSD resulted in oligomer complementation, or enhanced binding to PKAcat. Together with previous experiments examining the interaction between CSD and endothelial nitric oxide synthase (eNOS), the results suggest a general oligomerization-dependent enhancement of binding between signal transducing enzymes and caveolin-1.
Levin
Aron M
AM
Department of Chemistry, University of California, Irvine, CA 92697, USA.
Coroneus
John G
JG
Cocco
Melanie J
MJ
Weiss
Gregory A
GA
D001665:Binding Sites; D020134:Catalytic Domain; D051242:Caveolin 1 / Q000737:chemistry* / Q000235:genetics; D019295:Computational Biology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D019151:Peptide Library; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry
D051242:Caveolin 1; D019151:Peptide Library; D021122:Protein Subunits; D017868:Cyclic AMP-Dependent Protein Kinases