Abstract
The members of the human epidermal growth factor receptor (HER) family are among the most intensely studied oncological targets. HER3 (ErbB3), which had long been neglected, has emerged as a key oncogene, regulating the activity of other receptors and being involved in progression and tumor escape in multiple types of cancer. Designed ankyrin-repeat proteins (DARPins) serve as antibody mimetics that have proven to be useful in the clinic, in diagnostics and in research. DARPins have previously been selected against EGFR (HER1), HER2 and HER4. In particular, their combination into bivalent binders that separate or lock receptors in their inactive conformation has proved to be a promising strategy for the design of potent anticancer therapeutics. Here, the selection of DARPins targeting extracellular domain 4 of HER3 (HER3d4) is described. One of the selected DARPins, D5, in complex with HER3d4 crystallized in two closely related crystal forms that diffracted to 2.3 and 2.0 Å resolution, respectively. The DARPin D5 epitope comprises HER3d4 residues 568-577. These residues also contribute to interactions within the tethered (inactive) and extended (active) conformations of the extracellular domain of HER3.
Highlights
The human members of the epidermal growth factor receptor (HER, ErbB) family are membrane receptors that are involved in cell division, survival and migration (Yarden & Sliwkowski, 2001)
We report the selection and structural characterization of Designed ankyrin-repeat proteins (DARPins) binding to HER3 extracellular domain 4 (HER3d4), because these DARPins could be used to generate bispecific constructs with diverse functions, for example molecules that are able to lock the receptor into an inactive conformation, in analogy to the strategy reported previously (Jost et al, 2013)
The sequence alignment of monomeric clones showed that all N2C binders were derivatives of one clone (DARPins D1–D5, with 1–4 amino-acid differences between them), but the N3C binders fell into two different subpopulations (DARPins D6 and D8, with one amino-acid difference between them, and DARPin D7) (Supplementary Fig. S1)
Summary
The human members of the epidermal growth factor receptor (HER, ErbB) family are membrane receptors that are involved in cell division, survival and migration (Yarden & Sliwkowski, 2001). HER proteins comprise four extracellular domains, a transmembrane domain, an intracellular kinase domain and a long unstructured C-terminal tail that carries phosphorylation sites which can be bound by adaptor proteins. Homodimerization and heterodimerization, usually induced by ligand binding, promote mutual phosphorylation of the kinase domain and the C-terminal tail, which in turn activates a variety of signaling cascades (Hynes & MacDonald, 2009). The extracellular domain of HER3 (UniProt entry P21860) is subdivided into four domains, namely domain 1 (residues 1–183; numbering of the mature protein, not counting the 19 residues of the signal sequence), domain 2 (residues 184–308), domain 3 (residues 309–480) and domain 4 (residues 481–611). In the absence of its ligand, HER3 exists predominantly in a tethered conformation in which extracellular domains 2 and 4 form a contact (Cho & Leahy, 2002). Upon binding its natural ligand, such as neuregulin-1 or neuregulin-2 ( known as heregulins), via extracellular domains 1 and 3, the extracellular domains are structurally rearranged into an upright
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