Journal
PROTEIN SCIENCE
Volume 26, Issue 9, Pages 1796-1806Publisher
WILEY
DOI: 10.1002/pro.3216
Keywords
HER2; ErbB receptors; protein engineering; conformational probe; DARPins
Categories
Funding
- Schweizerischer Nationalfonds [31003A_146278]
- ERC grant NEXTBINDERS
- Verband der Chemischen Industrie
- Swiss National Science Foundation (SNF) [31003A_146278] Funding Source: Swiss National Science Foundation (SNF)
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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.
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