Double barrel shotgun scanning of the caveolin-1 scaffolding domain

Abstract

In the postgenomic era, a major challenge remains, elucidating the thermodynamic forces governing receptor-ligand specificity and promiscuity. We report a straightforward approach for mapping side-chain contributions to binding for the multipartner interactions characteristic of the human proteome. Double barrel shotgun scanning dissects binding to two or more targets through combinatorial mutagenesis of one protein binding to multiple targets. Examined here, the caveolin-1 scaffolding domain (CSD) binds to and inhibits both endothelial nitric oxide synthase (eNOS) and protein kinase A (PKA). Homolog shotgun scanning of CSD highlights residues responsible for CSD oligomerization and binding to eNOS and PKA. The experiments uncover a general mechanism in which CSD oligomerizes and deoligomerizes to modulate binding affinity to partner proteins. The results provide a detailed look at a multipartner protein interaction, uncovering strategies for one protein binding to multiple partners.

Figures

Figure 1

Selectants from double barrel shotgun scanning. a) Binding to eNOS by phage-displayed CSD homologs from shotgun scanning. Serial dilutions of phage-displayed CSD derivatives selected from a homolog shotgun scanning library were incubated in eNOS-coated microtiter wells. Binding phage were quantified by anti-phage antibody ELISA conjugated to HRP. Each data point represents the average of three experiments, and error bars indicate standard deviation. b) CSD homolog variants binding to PKA. In this phage ELISA, PKA bound to microtiter plates was exposed to phage-displayed CSD and CSD variants (5 nM) before developing the ELISA as usual. The depicted CSD variants represent the strongest and weakest 12 variants, with wild-type CSD in the middle. Bar heights represent the average of three ELISAs, and error bars indicate standard deviation.

Figure 2

CSD binding and oligomerization. a) Helical wheel of CSD. The seven residues in bold italics correspond to the residues highlighted green in Table 1. These residues are on the face of the CSD helix most likely to bind both eNOS and PKA. b) Oligomer complementation. In this ELISA, a constant concentration of phage-displayed CSD (10 nM) was evaluated for binding to eNOS in the presence of the indicated concentration of chemically synthesized CSD. Error bars represent the standard error for the average of three experiments. An analogous experiment with PKA generated similar results (15). c) Oligomer complementation of truncated CSD variants binding to eNOS. Phage-displayed truncated CSD variants were incubated in eNOS coated microtiter wells with the indicated concentrations of chemically synthesized CSD. Affinity enhancement indicates the difference in HRP activity upon addition of CSD peptide. d) Binding to PKA by truncated CSD variants. CSD variants were derived from truncation of C-terminal or N-terminal residues, as described in the text. The white bars represent ELISA binding by phage-displayed CSD variants with the addition of 5 μM chemically synthesized CSD.

Figure 2

CSD binding and oligomerization. a) Helical wheel of CSD. The seven residues in bold italics correspond to the residues highlighted green in Table 1. These residues are on the face of the CSD helix most likely to bind both eNOS and PKA. b) Oligomer complementation. In this ELISA, a constant concentration of phage-displayed CSD (10 nM) was evaluated for binding to eNOS in the presence of the indicated concentration of chemically synthesized CSD. Error bars represent the standard error for the average of three experiments. An analogous experiment with PKA generated similar results (15). c) Oligomer complementation of truncated CSD variants binding to eNOS. Phage-displayed truncated CSD variants were incubated in eNOS coated microtiter wells with the indicated concentrations of chemically synthesized CSD. Affinity enhancement indicates the difference in HRP activity upon addition of CSD peptide. d) Binding to PKA by truncated CSD variants. CSD variants were derived from truncation of C-terminal or N-terminal residues, as described in the text. The white bars represent ELISA binding by phage-displayed CSD variants with the addition of 5 μM chemically synthesized CSD.

Figure 3

Schematic diagram illustrating how CSD can bind tightly to PKA and eNOS as oligomers, and then release the proteins upon de-oligomerization.