Exploring the interaction between the protein kinase A catalytic subunit and caveolin-1 scaffolding domain with shotgun scanning, oligomer complementation, NMR, and docking

Abstract

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.

Figures

Figure 1.

Oligomer complementation of the PKAcat–CSD interaction demonstrated by phage ELISA. (A) Phage-displayed CSD solutions (1 nM) were mixed with the indicated concentrations of synthetic CSD, and incubated with immobilized PKAcat. (B) Serial dilutions of phage-displayed CSD were assayed with or without the addition of 5 μM synthetic CSD, and incubated in wells containing immobilized PKAcat. Anti-phage antibody conjugated to HRP was used for quantification as usual. Error bars depict standard deviation.

Figure 2.

NMR analysis of CSD. This downfield section of a CSD NOESY map (τ m = 400 msec; 20° C, 33% d-TFE, 25 mM d-sodium acetate) shows cross-peaks between back bone amide protons that are consistent with α-helical secondary structure. The major species was found to populate a helical conformation between residues Ile84CSD and Tyr97CSD.

Figure 3.

Model of the PKAcat–CSD interaction from computational modeling, mutagenesis, and NMR analysis. (A) Docking structure of CSD (red) spanning across PKAcat sites 1 (yellow), 2 (green), and 3 (orange). The N and C termini of CSD are labeled. (B) Residues Trp98CSD and Phe99CSD before (yellow) and after (red) side-chain refinement. (C) Strongly conserved CSD residue K86 in close contact with PKAcat residues D166, G200, and T201. An arginine substitution in this position could disrupt hydrogen bonds or salt bridges. (D) Strongly conserved CSD residue F89 pointing into PKAcat hydrophobic residues P243, I244, and Y247. Substitution of a hydroxyl at the position marked with an * (resulting from a F89Y mutation) could prevent this close interaction. (E) CSD residue W85 in close proximity to Pro243PKA and Phe89CSD. (F) CSD residue W98 packing against hydrophobic PKAcat residues K86, I85, and possibly hydrogen bonding to E86. CSD residues are labeled in bold. PKAcat residues are labeled in italics.

Figure 4.

Interactions between CSD (left) and PKAcat (right) from protein–protein docking. Hydrogen bonds are denoted as arrows, and van der Waals interactions are denoted as circles.