Tailoring chimeric ligands for studying and biasing ErbB receptor family interactions

Abstract

Described is the development and application of a versatile semisynthetic strategy, based on a combination of sortase-mediated coupling and tetrazine ligation chemistry, which can be exploited for the efficient incorporation of tunable functionality into chimeric recombinant proteins. To demonstrate the scope of the method, the assembly of a set of bivalent ligands, which integrate members of the epidermal growth factor (EGF) ligand family, is described. By using a series of bivalent EGFs with variable intraligand spacing, the differences in structure were correlated with the ability to bias signaling in the ErbB receptor family in a cell motility assay. Biasing away from EGFR-HER2 dimerization with a bivalent EGF was observed to reduce cell motility in an intraligand distance-dependent fashion, thus demonstrating the utility of the approach for acutely perturbing receptor-mediated cell signaling pathways.

Keywords: biological activity; ligand design; protein modifications; receptors; structure-activity relationships.

Figure 1

Illustration of biased signaling in ErbB receptors with bivalent ligands (E = epidermal growth factor).

Figure 2

Semisynthetic bivalent linkage strategy assembles two protein ligands with tunable functionality. Protein ligand, e.g. = Epidermal Growth Factor (EGF), Neuregulin (NRG).

Figure 3

Representative SDS-PAGE analyses A) click ligation reaction progress and B) final purified bivalent ligand set (bivalent EGFs (EEs) shown, for bivalent NRG (NN) see SI).

Figure 3

Representative SDS-PAGE analyses A) click ligation reaction progress and B) final purified bivalent ligand set (bivalent EGFs (EEs) shown, for bivalent NRG (NN) see SI).

Figure 4

hTMSC migration response to 100 nM semisynthetic bivalent EGF ligand treatment as compared to the respective PEG_n-modified monovalent EGF. A) Mean and S.E. of the net displacement (μm) and total path length (μm) of hTMSCs upon treatment with monovalent and bivalent EGFs (65 Å and 90 Å); B) Fold-change in the random motility coefficient (RMC) (μm²/h) in response to various ligand treatments. See SI for statistical analyses.

Figure 4

hTMSC migration response to 100 nM semisynthetic bivalent EGF ligand treatment as compared to the respective PEG_n-modified monovalent EGF. A) Mean and S.E. of the net displacement (μm) and total path length (μm) of hTMSCs upon treatment with monovalent and bivalent EGFs (65 Å and 90 Å); B) Fold-change in the random motility coefficient (RMC) (μm²/h) in response to various ligand treatments. See SI for statistical analyses.

Figure 5

Frontal (left) and top (right) views of crystal structure representation of a homodimer of two extracellular EGFR domains with two bound EGFs, indicating approximate distance between C, N termini of EGF ligands. (PDB ID: 3NJP).