Rigidity of the extracellular part of HER2: Evidence from engineering subdomain interfaces and shared-helix DARPin-DARPin fusions

Abstract

The second member of the human ErbB family of receptor tyrosine kinases, HER2/hErbB2, is regarded as an exceptional case: The four extracellular subdomains could so far only be found in one fixed overall conformation, designated "open" and resembling the ligand-bound form of the other ErbB receptors. It thus appears to be different from the extracellular domains of the other family members that show inter-subdomain flexibility and exist in a "tethered" form in the absence of ligand. For HER2, there was so far no direct evidence for such a tethered conformation on the cell surface. Nonetheless, alternative conformations of HER2 in vivo could so far not be excluded. We now demonstrate the rigidity of HER2 on the surface of tumor cells by employing two orthogonal approaches of protein engineering: To directly test the potential of the extracellular domain of HER2 to adopt a pseudo-tethered conformation on the cell surface, we first designed HER2 variants with a destabilized interface between extracellular subdomains I and III that would favor deviation from the "open" conformation. Secondly, we used differently shaped versions of a Designed Ankyrin Repeat Protein (DARPin) fusion, recognizing subdomain I of HER2, devised to work as probes for a putative pseudo-tethered extracellular domain of HER2. Combining our approaches, we exclude, on live cells and in vitro, that significant proportions of HER2 deviate from the "open" conformation.

Keywords: DARPins; ErbB receptors; HER2; conformational probe; protein engineering.

Figure 1

Conformational changes and domain interactions in EGFR (PDB ID 1YY9 and 3NJP) and HER2 (PDB ID 1N8Z and 3N85). (A) Tethered conformation of the EGFR ECD (PDB ID 1YY9). Contacts between the tethering loops in subdomain II and IV, stabilizing the tethered conformation, are highlighted in red (van‐der‐Waals contacts ≤3.6 Å) and orange (solvent‐excluding contacts ≤5 Å), non‐contact atoms in contact residues are shown in yellow. (B) Comparison of the tethered and extended conformations of EGFR. The hinge motion relating the two conformations is predominantly due to a change of the main‐chain torsion angles of Lys 335, located at the boundary between subdomains II and III and corresponding to Arg 340 in HER2. (C) Extended conformation of EGFR (PDB ID 3NJP) in the EGFR homodimer. The tethering loops in subdomains II and IV interact with the corresponding loops of the second molecule in the dimer. (D) The open conformation of EGFR is stabilized by the ligand EGF (dark blue) binding to both subdomain I (white) and III (cyan). The two subdomains barely touch. (E) The extended conformation of HER2 is stabilized by direct interactions of subdomains I and III.

Figure 2

Mutations specifically destabilizing the open conformation of full‐length HER2 (flHER2) result in little functional protein at the cell surface. Binding of DARPin 929 to HEK293T/17 cells transiently transfected with wild‐type HER2 or mutants (see Table I for list of mutations) was detected in flow cytometry using anti‐DARPin serum. Note that despite at least equal overall protein production (monitored by the signal from the C‐terminally fused GFP), the surface binding signal of DARPin 929 for the HER2 mutants (green, red, blue bars) is maximally about 3‐fold over background (corresponding to endogenous HER2 of HEK293 cells, as seen in non‐transfected cells, gray bars). Error bars represent 1 SD of technical triplicates. The GFP signal was recorded in a separate channel and scaled for display.

Figure 3

Models of shared helix constructs in complex with HER2 ECD. (A) Shared helix constructs (DD) are rigid fusions of two DARPins, in which the C‐terminal helix of one DARPin continues into the N‐terminal helix of a second DARPin. The length of this shared helix determines the relative orientation of the two DARPins and the overall shape of the construct, and is indicated by Hnn. The N‐terminal DARPin (tan or yellow) is a non‐binding DARPin (N3C), the C‐terminal DARPin 929 (orange) recognizes HER2 subdomain I. (B) Five out of nine designed DD constructs display van‐der‐Waals clashes with HER2 subdomain I (shown as red spheres), as shown for the example of N3C_H06_929. (C) Three constructs are predicted to bind to both isolated HER2_I and the full HER2 ECD without any clashes. One of these, N3C_H12_929, was used as positive control in binding experiments. (D) One construct, N3C_H09_929, is predicted to bind to the isolated HER2_I, but is prevented from binding to the full HER2 ECD in extended conformation by clashes with subdomain III. (E) A hinge motion similar to the one relating the extended to the closed conformation in EGFR would allow the HER2 ECD to escape this clash and allow N3C_H09_929 to bind. The models of the extended and of a hypothetical pseudo‐tethered HER2 ECD were aligned by a least‐squares fit of the Cα atoms of subdomain I (residues 24–212).

Figure 4

Binding experiments with the engineered conformational probe. (A) DD constructs and controls (100 nM; detection antibody background, gray; 929, red; N3C_H09_929, blue; and N3C_H12_929, green bars) binding to immobilized HER2_I or HER2 ECD in ELISA; error bars represent 1 SD of technical triplicates. (B) Binding of DD constructs and controls (100 nM; detection antibody background, gray; 929, red; N3C_H09_929, blue; and N3C_H12_929, green solid line) to live BT474 cells in flow cytometry. Bound DARPin constructs were detected using rabbit anti‐DARPin serum and fluorescently labeled secondary antibody.