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. 2011 Jun 29;133(25):9855-62.
doi: 10.1021/ja201792q. Epub 2011 Jun 7.

In vitro evolution of ligands to the membrane protein caveolin

Sudipta Majumdar  1 , Agnes Hajduczki, Rosemarie Vithayathil, Tivoli J Olsen, Ryan M Spitler, Aaron S Mendez, Travis D Thompson, Gregory A Weiss
Affiliations

In vitro evolution of ligands to the membrane protein caveolin

Sudipta Majumdar et al. J Am Chem Soc. .

Abstract

Membrane proteins comprise a third of the human genome, yet present challenging targets for reverse chemical genetics. For example, although implicated in numerous diseases including multiple myeloma, the membrane protein caveolin-1 appears to offer a poor target for the discovery of synthetic ligands due to its largely unknown structure and insolubility. To break this impasse and identify new classes of caveolae controlling lead compounds, we applied phage-based, reverse chemical genetics for the discovery of caveolin-1 ligands derived from the anti-HIV therapeutic T20. Substitution of homologous residues into the T20 sequence used a process analogous to medicinal chemistry for the affinity maturation to bind caveolin. The resultant caveolin-1 ligands bound with >1000-fold higher affinity than wild-type T20. Two types of ELISAs and isothermal titration calorimetry (ITC) measurements demonstrated high affinity binding to caveolin by the T20 variants with K(d) values in the 150 nM range. Microscopy experiments with the highest affinity caveolin ligands confirmed colocalization of the ligands with endogenous caveolin in NIH 3T3 cells. The results establish the foundation for targeting caveolin and caveolae formation in living cells.

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Figures

Figure 1
Figure 1
Direct assay of phage-displayed full-length caveolin binding to gp41 and T20. Serial dilutions of phage-displayed, full-length caveolin (Cav-1) and KO7+ phage not displaying a protein, as a negative control, were incubated with gp41, gp41-T20 (1:1 molar ratio), or T20 coated in microtiter plate wells. The relative levels of bound proteins were quantified by anti-M13 HRP-conjugated antibody. Caveolin binds with low affinity to T20, but T20 enhances the caveolin-gp41 interaction. In ELISAs throughout this report, each data point represents the average of three experiments, and error bars indicate standard error.
Figure 2
Figure 2
Phage-based ELISAs of representative T20 variants selected from the libraries. (A, B) In these experiments, the target cav(1-104) was coated on microtiter plates, and the ELISA developed as before. The selectants bound to cav(1-104) with much higher relative affinity than the wild-type T20 interaction with cav(1-104). (C) To demonstrate specificity for caveolin, ligands 4 and 5 (10 nM) displayed on KO7+ phage were assayed for binding to various negative control proteins coated on the plate and a positive control anti-FLAG antibody (α–FLAG), which can recognize a FLAG epitope fused to the N-terminus of the ligands. In this experiment, nonfat milk was used as a blocking agent. KO7+ without a displayed peptide provided a negative control for the assays.
Figure 3
Figure 3
ELISAs with over-expressed T20 variants removed from the phage surface. (A, B) Selected from phage-based affinity maturation, ligands 4 and 5 fused to MBP bound much more strongly to phage-displayed full-lenth caveolin than wild-type T20-MBP. (C) The ligands also bound to cav(1-104). MBP provided a negative control for the assay.
Figure 4
Figure 4
ITC measurements of caveolin ligands 4 and 5 binding to cav(1-104). (A) Ligand 4, or (B) 5 was injected into a solution of cav(1-104). The top graph depicts the calorimetric output for the interaction, which has been integrated in the lower graph. The molar ratio of the x-axis indicates the ratio of ligand to cav(1-104). The solid line represents the best least squares fit for a two binding site model. Table 2 lists the thermodynamic and kinetic parameters derived from these data.
Figure 5
Figure 5
Colocalization of caveolin-binding ligands 4 and 5 with endogenous caveolin in NIH 3T3 cells. Endogenously expressed caveolin is designated by the red signal. (A) MBP-fused ligand 4 or (B) the negative control, MBP, is labeled green and in the merged image, the yellow regions highlight areas of colocalization. (C) Comparable experiment showing the results with MBP-fused caveolin ligand 5 with the corresponding MBP control in panel D.
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